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Structural and functional characterization of a glycoside hydrolase family 3 β-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7 (2019)

Itoh, Takafumi ; Araki, Tomomitsu ; Nishiyama, Tomohiro ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 503-515. Published 2019 Sep 10. doi: 10.1093/jb/mvz072.

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Publication Date: 2019-09-10

Description: Chitin, a β-1,4-linked homopolysaccharide of N-acetyl-d-glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N,N′-diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc)2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus β-N-acetyl-d-glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)2–4, 4-nitrophenyl N-acetyl β-d-glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)2–6, but has no detectable activity against 4-nitrophenyl β-d-glucose, 4-nitrophenyl β-d-galactosamine and colloidal chitin. In this study, we present a 1.9 Å crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 β-N-acetyl-d-glucosaminidase from Paenibacillus sp.

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Observation of unexpected molecular binding activity for Mu phage tail fibre chaperones (2019)

Sakai, Kohei ; Iwazaki, Takuma ; Yamashita, Eiki ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 529-535. Published 2019 Aug 30. doi: 10.1093/jb/mvz068.

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Publication Date: 2019-08-30

Description: In the history of viral research, one of the important biological features of bacteriophage Mu is the ability to expand its host range. For extending the host range, the Mu phage encodes two alternate tail fibre genes. Classical amber mutation experiments and genome sequence analysis of Mu phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S’ (gpS’ = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU’ (gpU’ = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu phage to recognize different host cell surface. Since several fibrous proteins including some phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.

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Persulphide-responsive transcriptional regulation and metabolism in bacteria (2019)

Shimizu, Takayuki ; Masuda, Shinji

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Aug 06. doi: 10.1093/jb/mvz063. [early online release]

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Publication Date: 2019-08-06

Description: Hydrogen sulphide (H2S) impacts on bacterial growth both positively and negatively; it is utilized as an electron donor for photosynthesis and respiration, and it inactivates terminal oxidases and iron-sulphur clusters. Therefore, bacteria have evolved H2S-responsive detoxification mechanisms for survival. Sulphur assimilation in bacteria has been well studied, and sulphide:quinone oxidoreductase, persulphide dioxygenase, rhodanese and sulphite oxidase were reported as major sulphide-oxidizing enzymes of sulphide assimilation and detoxification pathways. However, how bacteria sense sulphide availability to control H2S and sulphide metabolism remains largely unknown. Recent studies have identified several bacterial (per)sulphide-sensitive transcription factors that change DNA-binding affinity through persulphidation of specific cysteine residues in response to highly reactive sulphur-containing chemicals and reactive sulphur species (RSS). This review focuses on current understanding of the persulphide-responsive transcription factors and RSS metabolism regulated by RSS sensory proteins.

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Metal binding to cutinase-like enzyme from Saccharomonospora viridis AHK190 and its effects on enzyme activity and stability (2019)

Senga, Akane ; Hantani, Yoshiji ; Bekker, Gert-Jan ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Mar 02. doi: 10.1093/jb/mvz020. [early online release]

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Publication Date: 2019-03-02

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Distinct molecular assembly of homologous peroxiredoxins from Pyrococcus horikoshii and Thermococcus kodakaraensis (2019)

Himiyama, Tomoki ; Oshima, Maki ; Uegaki, Koichi ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 89-95. Published 2019 Feb 22. doi: 10.1093/jb/mvz013.

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Publication Date: 2019-02-22

Description: Peroxiredoxins from Pyrococcus horikoshii (PhPrx) and Thermococcus kodakaraensis (TkPrx) are highly homologous proteins sharing 196 of the 216 residues. We previously reported a pentagonal ring-type decameric structure of PhPrx. Here, we present the crystal structure of TkPrx. Despite their homology, unlike PhPrx, the quaternary structure of TkPrx was found to be a dodecamer comprised of six homodimers arranged in a hexagonal ring-type assembly. The possibility of the redox-dependent conversion of the molecular assembly, which had been observed in PhPrx, was excluded for TkPrx based on the crystal structure of a mutant in which all of the cysteine residues were substituted with serine. The monomer structures of the dodecameric TkPrx and decameric PhPrx coincided well, but there was a slight difference in the relative orientation of the two domains. Molecular assembly of PhPrx and TkPrx in solution evaluated by gel-filtration chromatography was consistent with the crystallographic results. For both PhPrx and TkPrx, the gel-filtration elution volume slightly increased with a decrease in the protein concentration, suggesting the existence of an equilibrium state between the decameric/dodecameric ring and lower-order assembly. This structural assembly difference between highly homologous Prxs suggests a significant influence of quaternary structure on function, worthy of further exploration.

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Gene expression profiling of the bone trabecula in patients with osteonecrosis of the femoral head by RNA sequencing (2019)

Bai, Haobo ; Chen, Tingmei ; Lu, Qian ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 475-484. Published 2019 Sep 13. doi: 10.1093/jb/mvz060.

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Publication Date: 2019-09-13

Description: Early diagnosis and treatment of osteonecrosis of the femoral head (ONFH) is challenging. Bone trabecula play a vital role in the severity and progression of ONFH. In the present study, the investigators used gene expression profiling of bone trabecula to investigate gene alterations in ONFH patients. Osteonecrotic bone trabecula (ONBT) such as necrosis, fibrosis, and lacuna were confirmed by histological examination in the patients. The adjacent ‘normal’ bone trabecula (ANBT) did not show any pathological changes. Gene sequencing data revealed that although ANBT showed no significant histological changes, alteration of mRNA profiling in ANBT was observed, similar to that in ONBT. Our results indicated that the alteration of mRNA profiling in ANBT may cause normal bone tissue to develop into necrotic bone. RNA-seq data indicated that 2,297 differentially abundant mRNAs were found in the ONBT group (1,032 upregulated and 1,265 downregulated) and 1,523 differentially abundant mRNAs in the ANBT group (744 upregulated and 799 downregulated) compared with the healthy control group. Gene ontology (GO) enrichment analysis suggested that fatty acid metabolism and degradation were the main zones enriched with differentially expressed genes (DEG). Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis indicated that peroxisome proliferator-activated receptor γ (PPAR-γ) pathway was the most significantly regulated pathway. Lipocalin-2 (LCN2), an osteoblast-enriched secreted protein, was significantly decreased in ONBT suggesting that downregulation of LCN2 might affect lipid metabolism and lead to hyperlipidemia, and thus promote pathogenesis of ONFH.

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Phosphorylation of repressive histone code readers by casein kinase 2 plays diverse roles in heterochromatin regulation (2019)

Murakami, Yota

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 3-6. Published 2019 Jun 13. doi: 10.1093/jb/mvz045.

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Publication Date: 2019-06-13

Description: Heterochromatin is a condensed and transcriptionally silent chromatin structure and that plays important roles in epigenetic regulation of the genome. Two types of heterochromatin exist: constitutive heterochromatin is primarily associated with trimethylation of histone H3 at lysine 9 (H3K9me3), and facultative heterochromatin with trimethylation of H3 at lysine 27 (H3K27me3). The methylated histones are bound by the chromodomain of histone code ‘reader’ proteins: HP1 family proteins for H3K9me3 and Polycomb family proteins for H3K27me3. Each repressive reader associates with various ‘effector’ proteins that provide the functional basis of heterochromatin. Heterochromatin regulation is primarily achieved by controlling histone modifications. However, recent studies have revealed that the repressive readers are phosphorylated, like other regulatory proteins, suggesting that phosphorylation also participates in heterochromatin regulation. Detailed studies have shown that phosphorylation of readers affects the binding specificities of chromodomains for methylated histone H3, as well as the binding of effector proteins. Thus, phosphorylation adds another layer to heterochromatin regulation. Interestingly, casein kinase 2, a strong and predominant kinase within the cell, is responsible for phosphorylation of repressive readers. In this commentary, I summarize the regulation of repressive readers by casein kinase 2-dependent phosphorylation and discuss the functional meaning of this modification.

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Identification and biochemical characterization of a second zebrafish autotaxin gene (2019)

Kise, Ryoji ; Okasato, Ryohei ; Kano, Kuniyuki ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 165(3): 269-275. Published 2019 Jan 10. doi: 10.1093/jb/mvy114.

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Publication Date: 2019-01-10

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GRWD1 directly interacts with p53 and negatively regulates p53 transcriptional activity (2019)

Fujiyama, Hiroki ; Tsuji, Takahiro ; Hironaka, Kensuke ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 15-24. Published 2019 Sep 23. doi: 10.1093/jb/mvz075.

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Publication Date: 2019-09-23

Description: Glutamate-rich WD40 repeat containing 1 (GRWD1) functions as a histone chaperone to promote loading of the MCM replication helicase at replication origins. GRWD1 is overexpressed in several cancer cell lines, and GRWD1 overexpression confers tumorigenic potential in human cells. However, less is known concerning its oncogenic activity. Our previous analysis showed that GRWD1 negatively regulates the tumour suppressor p53 via the RPL11-MDM2-p53 and RPL23-MDM2-p53 axes. Here, we demonstrate that GRWD1 directly interacts with p53 via the p53 DNA-binding domain. Upon DNA damage, GRWD1 downregulation resulted in increased p21 expression. Conversely, GRWD1 co-expression suppressed several p53-regulated promoters. GRWD1 interacted with the p21 and MDM2 promoters, and these interactions required p53. By using the Human Cancer Genome Atlas database, we found that GRWD1 expression levels are inversely correlated with the expression levels of some p53-target genes. Interestingly, high GRWD1 expression in combination with low expression levels of some p53-target genes was significantly correlated with poor prognosis in skin melanoma patients with wild-type p53. Taken together, our findings suggest a novel oncogenic function of GRWD1 as a transcriptional regulator of p53 and that GRWD1 might be an attractive therapeutic target and prognostic marker in cancer therapy.

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PINK1 import regulation at a crossroad of mitochondrial fate: the molecular mechanisms of PINK1 import (2019)

Sekine, Shiori

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Sep 04. doi: 10.1093/jb/mvz069. [early online release]

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Publication Date: 2019-09-04

Description: PTEN-induced kinase 1 (PINK1) is a mitochondrial kinase whose activity is tightly regulated by the mitochondrial health status. In response to mitochondrial damage, activated PINK1 can promote mitophagy, an autophagic elimination of damaged mitochondria, by cooperating with Parkin ubiquitin ligase. Loss-of-function of PINK1/Parkin-mediated mitophagy results in the accumulation of dysfunctional mitochondria, which could be one aetiology of Parkinson’s disease (PD). Within step-by-step signalling cascades of PINK1/Parkin-mediated mitophagy, mitochondrial damage-dependent PINK1 kinase activation is a critical step to trigger the mitophagy signal. Recent investigation of this process reveals that this stress-dependent PINK1 kinase activation is achieved by its regulated import into different mitochondrial compartments. Thus, PINK1 import regulation stands at an important crossroad to determine the mitochondrial fate—‘keep’ or ‘remove’? In this review, we will summarize how the PINK1 import is regulated in a mitochondrial health status-dependent manner and how this process could be pharmacologically modulated to activate the PINK1/Parkin pathway.

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Temperature-sensitive mutants of MscL mechanosensitive channel (2019)

Owada, Naoto ; Yoshida, Megumi ; Morita, Kohei ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(3): 281-288. Published 2019 May 07. doi: 10.1093/jb/mvz035.

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Publication Date: 2019-05-07

Description: MscL is a mechanosensitive channel that undergoes a global conformational change upon application of membrane stretching. To elucidate how the structural stability and flexibility occur, we isolated temperature-sensitive (Ts) mutants of Escherichia coli MscL that allowed cell growth at 32°C but not at 42°C. Two Ts mutants, L86P and D127V, were identified. The L86P mutation occurred in the second transmembrane helix, TM2. Substitution of residues neighbouring L86 with proline also led to a Ts mutation, but the substitution of L86 with other amino acids did not result in a Ts phenotype, indicating that the Ts phenotype was due to a structural change of TM2 helix by the introduction of a proline residue. The D127V mutation was localized in the electrostatic belt of the bundle of cytoplasmic helices, indicating that stability of the pentameric bundle of the cytoplasmic helix affects MscL structure. Together, this study described a novel class of MscL mutations that were correlated with the thermodynamic stability of the MscL structure.

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Effect of redox imbalance on protein modifications in lymphocytes of psoriatic patients (2019)

Wójcik, Piotr ; Gęgotek, Agnieszka ; Wroński, Adam ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 323-331. Published 2019 Nov 09. doi: 10.1093/jb/mvz096.

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Publication Date: 2019-11-09

Description: Lymphocytes are one of the most important cells involved in the pathophysiology of psoriasis; therefore, the aim of this study was to assess the redox imbalance and protein modifications in the lymphocytes of patients with psoriasis vulgaris (PsV) or psoriatic arthritis (PsA). The results show a stronger shift in redox status to pro-oxidative conditions (observed as an increased reactive oxygen species level, a decrease in catalase activity and lower levels of glutathione peroxidase and vitamin E compared with healthy controls) in the lymphocytes of PsA than PsV patients. It is also favoured by the enhanced level of activators of the Nrf2 transcription factor in lymphocytes of PsV compared with decreased of these proteins level in PsA. Moreover, the differential modifications of proteins by lipid peroxidation products 4-oxononenal (mainly binding proteins) and malondialdehyde (mainly catalytic proteins with redox activity), promoted a pro-apoptotic pathway in lymphocytes of PsV, which was manifested by enhanced expression of pro-apoptotic caspases, particularly caspase 3. Taken together, differences in Nrf2 pathway activation may be responsible for the differential level of redox imbalance in lymphocytes of patients with PsV and PsA. This finding may enable identification of a targeted therapy to modify the metabolic pathways disturbed in psoriasis.

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MicroRNA-1271-5p inhibits cell proliferation and enhances radiosensitivity by targeting CDK1 in hepatocellular carcinoma (2019)

Liu, Hong-Mei ; Tan, Hua-Yan ; Lin, Yue ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(5): 513-524. Published 2019 Dec 27. doi: 10.1093/jb/mvz114.

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Publication Date: 2019-12-27

Description: This study aims to determine whether miR-1271-5p inhibits cell proliferation and enhances the radiosensitivity by targeting cyclin-dependent kinase 1 (CDK1) in hepatocellular carcinoma (HCC). Its expression levels in the HCC cell lines were significantly lower than those in normal human liver cell line. Bioinformatics analysis indicated CDK1 was a potential target of miR-1271-5p. Dual-Luciferase Reporter Assay confirmed that CDK1 is a direct target gene of miR-1271-5p. With overexpression of miR-1271-5p in SMMC-7721 and HuH-7 cells, cell proliferation was decreased, radiosensitivity was enhanced, cell cycle distribution was altered and the growth of transplanted tumours in nude mice was significantly reduced. miR-1271-5p overexpression enhanced radiosensitivity, which could be reduced by CDK1 overexpression. Overall, our findings suggested that miR-1271-5p inhibits cell proliferation and enhances the radiosensitivity of HCC cell lines by targeting CDK1.

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Impaired plasmalogen synthesis dysregulates liver X receptor-dependent transcription in cerebellum (2019)

Honsho, Masanori ; Dorninger, Fabian ; Abe, Yuichi ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 353-361. Published 2019 May 28. doi: 10.1093/jb/mvz043.

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Publication Date: 2019-05-28

Description: Synthesis of ethanolamine plasmalogen (PlsEtn) is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) on peroxisomal membrane, a rate-limiting enzyme in plasmalogen synthesis. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis in cultured cells by altering the stability of squalene epoxidase (SQLE). However, regulation of PlsEtn synthesis and physiological consequences of plasmalogen homeostasis in tissues remain unknown. In the present study, we found that the protein but not the transcription level of Far1 in the cerebellum of the Pex14 mutant mouse expressing Pex14p lacking its C-terminal region (Pex14ΔC/ΔC) is higher than that from wild-type mouse, suggesting that Far1 is stabilized by the lowered level of PlsEtn. The protein level of SQLE was increased, whereas the transcriptional activity of the liver X receptors (LXRs), ligand-activated transcription factors of the nuclear receptor superfamily, is lowered in the cerebellum of Pex14ΔC/ΔC and the mice deficient in dihydroxyacetonephosphate acyltransferase, the initial enzyme for the synthesis of PlsEtn. These results suggest that the reduction of plasmalogens in the cerebellum more likely compromises the cholesterol homeostasis, thereby reducing the transcriptional activities of LXRs, master regulators of cholesterol homeostasis.

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miR-324-5p upregulation potentiates resistance to cisplatin by targeting FBXO11 signalling in non-small cell lung cancer cells (2019)

Ba, Zhichang ; Zhou, Yufei ; Yang, Zhaoyang ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 517-527. Published 2019 Sep 16. doi: 10.1093/jb/mvz066.

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Publication Date: 2019-09-16

Description: Dysregulation of microRNAs (miRNAs) plays a key role during the pathogenesis of chemoresistance in lung cancer (LCa). Previous study suggests that miR-324-5p may serve as a unique miRNA signature for LCa, but its role and the corresponding molecular basis remain largely explored. Herein, we report that miR-324-5p expression was significantly increased in cisplatin (CDDP)-resistant LCa tissues and cells, and this upregulation predicted a poor post-chemotherapy prognosis in LCa patients. miR-324-5p was further shown to impact CDDP response: Ectopic miR-324-5p expression in drug-naïve LCa cells was sufficient to attenuate sensitivity to CDDP and to confer more robust tumour growth in CDDP-challenged nude mice. Conversely, ablation of miR-324-5p expression in resistant cells effectively potentiated CDDP-suppressed cell growth in vitro and in vivo. Using multiple approaches, we further identified the tumour suppressor FBXO11 as the direct down-stream target of miR-324-5p. Stable expression of FBXO11 could abrogate the pro-survival effects of miR-324-5p in CDDP-challenged LCa cells. Together, these findings suggest that miR-324-5p upregulation mediates, at least partially, the CDDP resistance by directly targeting FBXO11 signalling in LCa cells. In-depth elucidation of the molecular basis underpinning miR-324-5p action bears potential implications for mechanism-based strategies to improve CDDP responses in LCa.

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The ‘mitochondrial contact site and cristae organising system’ (MICOS) in health and human disease (2019)

Eramo, Matthew J ; Lisnyak, Valerie ; Formosa, Luke E ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 243-255. Published 2019 Dec 11. doi: 10.1093/jb/mvz111.

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Publication Date: 2019-12-11

Description: The ‘mitochondrial contact site and cristae organising system’ (MICOS) is an essential protein complex that promotes the formation, maintenance and stability of mitochondrial cristae. As such, loss of core MICOS components disrupts cristae structure and impairs mitochondrial function. Aberrant mitochondrial cristae morphology and diminished mitochondrial function is a pathological hallmark observed across many human diseases such as neurodegenerative conditions, obesity and diabetes mellitus, cardiomyopathy, and in muscular dystrophies and myopathies. While mitochondrial abnormalities are often an associated secondary effect to the pathological disease process, a direct role for the MICOS in health and human disease is emerging. This review describes the role of MICOS in the maintenance of mitochondrial architecture and summarizes both the direct and associated roles of the MICOS in human disease.

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Phosphorylation by protein kinase C stabilizes ABCG1 and increases cholesterol efflux (2019)

Watanabe, Taro ; Kioka, Noriyuki ; Ueda, Kazumitsu ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 309-315. Published 2019 May 21. doi: 10.1093/jb/mvz039.

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Publication Date: 2019-05-21

Description: ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/βI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

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Calcium signalling: a key regulator of neuronal migration (2019)

Horigane, Shin-ichiro ; Ozawa, Yukihiro ; Yamada, Hirokazu ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 165(5): 401-409. Published 2019 Feb 12. doi: 10.1093/jb/mvz012.

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Publication Date: 2019-02-12

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Photoactive yellow protein and its chemical probes: an approach to protein labelling in living cells (2019)

Kumar, Naresh ; Hori, Yuichiro ; Kikuchi, Kazuya

Oxford University Press

In: Journal of Biochemistry. 2019; 166(2): 121-127. Published 2019 Jul 29. doi: 10.1093/jb/mvz051.

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Publication Date: 2019-07-29

Description: Labelling technologies developed over the past few years have changed the way of looking at biomolecules and have made a considerable contribution to our understanding of the functions and regulation of dynamic biological processes. One of the robust technologies employed to image proteins in a cellular environment is based on the use of chemical tags and their fluorescent probes, which provides flexibility in developing probes with a wide range of synthetic fluorophores. A variety of chemical tags, ranging from short amino acid sequences to small proteins, have been employed to generate protein-labelling systems. One such chemical tag is the photoactive yellow protein (PYP)-tag, which is a small bacterial protein, developed for the selective labelling and imaging of proteins. Herein, we briefly discuss the protein-labelling system developed based on PYP-tag technology, with a focus on the design strategy for PYP-tag labelling probes and their applications in protein imaging.

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Glycoengineering of HEK293 cells to produce high-mannose-type N-glycan structures (2019)

Ren, Wei-Wei ; Jin, Ze-Cheng ; Dong, Weijie ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(3): 245-258. Published 2019 May 07. doi: 10.1093/jb/mvz032.

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Publication Date: 2019-05-07

Description: Therapeutic proteins are a developing part of the modern biopharmaceutical industry, providing novel therapies to intractable diseases including cancers and autoimmune diseases. The human embryonic kidney 293 (HEK293) cell line has been widely used to produce recombinant proteins in both basic science and industry. The heterogeneity of glycan structures is one of the most challenging issues in the production of therapeutic proteins. Previously, we knocked out genes encoding α1,2-mannosidase-Is, MAN1A1, MAN1A2 and MAN1B1, in HEK293 cells, establishing a triple-knockout (T-KO) cell line, which produced recombinant protein with mainly high-mannose-type N-glycans. Here, we further knocked out MAN1C1 and MGAT1 encoding another Golgi α1,2-mannosidase-I and N-acetylglucosaminyltransferase-I, respectively, based on the T-KO cells. Two recombinant proteins, lysosomal acid lipase (LIPA) and immunoglobulin G1 (IgG1), were expressed in the quadruple-KO (QD-KO) and quintuple-KO (QT-KO) cell lines. Glycan structural analysis revealed that all the hybrid-type and complex-type N-glycans were eliminated, and only the high-mannose-type N-glycans were detected among the recombinant proteins prepared from the QD-KO and QT-KO cells. Overexpression of the oncogenes MYC and MYCN recovered the slow growth in QD-KO and QT-KO without changing the glycan structures. Our results suggest that these cell lines could be suitable platforms to produce homogeneous therapeutic proteins.

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Selenoprotein P as a significant regulator of pancreatic β cell function (2019)

Saito, Yoshiro

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Aug 22. doi: 10.1093/jb/mvz061. [early online release]

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Publication Date: 2019-08-22

Description: Selenoprotein P (SeP; encoded by SELENOP) is selenium (Se)-rich plasma protein that is mainly produced in the liver. SeP functions as a Se-transport protein to deliver Se from the liver to other tissues, such as the brain and testis. The protein plays a pivotal role in Se metabolism and antioxidative defense, and it has been identified as a ‘hepatokine’ that causes insulin resistance in type 2 diabetes. SeP levels are increased in type 2 diabetes patients, and excess SeP impairs insulin signalling, promoting insulin resistance. Furthermore, increased levels of SeP disturb the functioning of pancreatic β cells and inhibit insulin secretion. This review focuses on the biological function of SeP and the molecular mechanisms associated with the adverse effects of excess SeP on pancreatic β cells’ function, particularly with respect to redox reactions. Interactions between the liver and pancreas are also discussed.

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How to rebuild the kidney: recent advances in kidney organoids (2019)

Islam, Mazharul ; Nishinakamura, Ryuichi

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 7-12. Published 2019 Mar 07. doi: 10.1093/jb/mvz021.

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Publication Date: 2019-03-07

Description: It is difficult to restore kidney function once it has become severely impaired. Although kidney transplantation is a curative therapy, donor numbers remain limited. Thus, the generation of kidney organoids (mainly comprising glomeruli and renal tubules) from multipotent stem cells represents an important advance in regenerative medicine of the kidney. Recently, a protocol that can generate the higher-order structure of the mouse embryonic kidney was reported. Kidney organoids are now being used for disease modelling, and may eventually be applicable for clinical transplantation. In this review, we summarize the recent advances in kidney organoid research, and discuss the issues to be resolved toward kidney reconstruction.

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Effect of PlzD, a YcgR homologue of c-di-GMP-binding protein, on polar flagellar motility in Vibrio alginolyticus (2019)

Kojima, Seiji ; Yoneda, Takuro ; Morimoto, Wakako ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 77-88. Published 2019 Feb 18. doi: 10.1093/jb/mvz014.

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Publication Date: 2019-02-18

Description: YcgR, a cyclic diguanylate (c-di-GMP)-binding protein expressed in Escherichia coli, brakes flagellar rotation by binding to the motor in a c-di-GMP dependent manner and has been implicated in triggering biofilm formation. Vibrio alginolyticus has a single polar flagellum and encodes YcgR homologue, PlzD. When PlzD or PlzD-GFP was highly over-produced in nutrient-poor condition, the polar flagellar motility of V. alginolyticus was reduced. This inhibitory effect is c-di-GMP independent as mutants substituting putative c-di-GMP-binding residues retain the effect. Moderate over-expression of PlzD-GFP allowed its localization at the flagellated cell pole. Truncation of the N-terminal 12 or 35 residues of PlzD abolished the inhibitory effect and polar localization, and no inhibitory effect was observed by deleting plzD or expressing an endogenous level of PlzD-GFP. Subcellular fractionation showed that PlzD, but not its N-terminally truncated variants, was precipitated when over-produced. Moreover, immunoblotting and N-terminal sequencing revealed that endogenous PlzD is synthesized from Met33. These results suggest that an N-terminal extension allows PlzD to localize at the cell pole but causes aggregation and leads to inhibition of motility. In V. alginolyticus, PlzD has a potential property to associate with the polar flagellar motor but this interaction is too weak to inhibit rotation.

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Phosphorylation of PDE4A5 by MAPKAPK2 attenuates fibrin degradation via p75 signalling (2019)

Houslay, K F ; Fertig, B A ; Christian, F ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 97-106. Published 2019 Mar 11. doi: 10.1093/jb/mvz016.

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Publication Date: 2019-03-11

Description: Phosphodiesterases (PDEs) shape local cAMP gradients to underpin the specificity of receptor function. Key to this process is the highly defined nature of the intra-cellular location of PDEs in the cell. PDE4A5 is a PDE isoform that specifically degrades cAMP and is known to associate with the p75 neurotrophin receptor (p75NTR) where it modulates cAMP signalling cascades that regulate extracellular matrix remodelling in the lungs. Here we map and validate novel protein–protein interaction sites that are important for formation of the PDE4A5–p75NTR complex and show, for the first time, that phosphorylation of PDE4A5 by MAPKAPK2 enhances PDE4A5 interaction with p75NTR and that this, in turn, serves to attenuate fibrin degradation.

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Characterization of monoclonal antibodies recognizing each extracellular loop domain of occludin (2019)

Shimizu, Yoshimi ; Shirasago, Yoshitaka ; Suzuki, Takeru ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 297-308. Published 2019 May 11. doi: 10.1093/jb/mvz037.

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Publication Date: 2019-05-11

Description: The tight junction protein occludin (OCLN) is a four-pass transmembrane protein with two extracellular loops (ELs), and also functions as a co-receptor for hepatitis C virus (HCV). Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo, and these mAbs were applicable for flow cytometric (FCM) analysis, immunocytochemistry (ICC) and cell-based enzyme-linked immunosorbent assay. In the present study, we further examined the application of these anti-OCLN mAbs and characterized their binding properties. All four mAbs were available for immunoprecipitation. The three first EL (EL1)-recognizing mAbs were applicable for immunoblotting, but the second EL (EL2)-recognizing one was not. Using site-directed mutagenesis, we also determined residues of OCLN critical for recognition by each mAb. Our findings showed that the small loop between two cysteines of the EL2 domain is essential for the binding to one EL2-recognizing mAb and that the recognition regions by three EL1-recognizing mAbs overlap, but are not the same sites of EL1. To obtain a deeper understanding of OCLN biology and its potential as a therapeutic target, specific mAbs to detect or target OCLN in intact cells should be powerful tools for future studies.

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Effect of sodium ions on conformations of the cytoplasmic loop of the PomA stator protein of Vibrio alginolyticus (2019)

Mino, Taira ; Nishikino, Tatsuro ; Iwatsuki, Hiroto ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 331-341. Published 2019 May 30. doi: 10.1093/jb/mvz040.

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Publication Date: 2019-05-30

Description: The sodium driven flagellar stator of Vibrio alginolyticus is a hetero-hexamer membrane complex composed of PomA and PomB, and acts as a sodium ion channel. The conformational change in the cytoplasmic region of PomA for the flagellar torque generation, which interacts directly with a rotor protein, FliG, remains a mystery. In this study, we introduced cysteine mutations into cytoplasmic charged residues of PomA, which are highly conserved and interact with FliG, to detect the conformational change by the reactivity of biotin maleimide. In vivo labelling experiments of the PomA mutants revealed that the accessibility of biotin maleimide at position of E96 was reduced with sodium ions. Such a reduction was also seen in the D24N and the plug deletion mutants of PomB, and the phenomenon was independent in the presence of FliG. This sodium ions specific reduction was also detected in Escherichia coli that produced PomA and PomB from a plasmid, but not in the purified stator complex. These results demonstrated that sodium ions cause a conformational change around the E96 residue of loop2–3 in the biological membrane.

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A unique mechanism for thiolation of serum albumins by disulphide molecules (2019)

Nakashima, Fumie ; Shibata, Takahiro ; Uchida, Koji

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Oct 09. doi: 10.1093/jb/mvz084. [early online release]

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Publication Date: 2019-10-09

Description: Protein S-thiolation is a reversible oxidative modification that serves as an oxidative regulatory mechanism for certain enzymes and binding proteins with reactive cysteine residues. It is generally believed that the thiolation occurs at free sulphydryl group of cysteine residues. Meanwhile, despite the fact that disulphide linkages, serving structural and energetic roles in proteins, are stable and inert to oxidative modification, a recent study shows that the thiolation could also occur at protein disulphide linkages when human serum albumin (HSA) was treated with disulphide molecules, such as cystine and homocystine. A chain reaction mechanism has been proposed for the thiolation at disulphide linkages, in which free cysteine (Cys34) is involved in the reaction with disulphide molecules to form free thiols (cysteine or homocysteine) that further react with protein disulphide linkages to form the thiolated cysteine residues in the protein. This review focuses on the recent finding of this unique chain reaction mechanism of protein thiolation.

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Deciphering the mechanism for induction of senescence-associated secretory phenotype (SASP) and its role in ageing and cancer development (2019)

Ohtani, Naoko

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 289-295. Published 2019 Jul 12. doi: 10.1093/jb/mvz055.

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Publication Date: 2019-07-12

Description: Cellular senescence is an irreversible form of cell cycle arrest that can be induced by persistent DNA damage, and is well known to function as an important tumour suppression mechanism. Cellular senescence is detected in aged organisms; thus, it is also recognized as a hallmark of organismal ageing. Unlike apoptotic cells, senescent cells can survive for long periods of time. Recently, it has been shown that the late stage of senescent cells are capable of expressing a variety of secreted proteins such as cytokines, chemokines and proteases, and this condition is now known as senescence-associated secretory phenotype (SASP). These secreted factors are involved in myriad of physiological functions including tissue repair and clearance of damaged cells. Alternatively, these factors may promote detrimental effects, such as chronic inflammation or cancer progression, should the SASP persist. Recent scientific advances have indicated that innate immune responses, particularly involving the cGAS–STING pathway, trigger SASP induction. Therefore, developing a strategy to regulate SASP may provide scientific insights for the management of age-associated diseases and the implementation of healthy ageing in the future.

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Circ_0000218 plays a carcinogenic role in colorectal cancer progression by regulating miR-139-3p/RAB1A axis (2019)

Pei, Fu-Lai ; Cao, Ming-Zheng ; Li, Yue-Feng

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 55-65. Published 2019 Oct 09. doi: 10.1093/jb/mvz078.

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Publication Date: 2019-10-09

Description: Accumulating researches have confirmed that circRNA abnormal expression plays a prominent role in the progression of colorectal cancer (CRC). The role of circ_0000218 in CRC and its potential mechanism are not clear. In this study, real-time polymerase chain reaction (RT-PCR) was employed to measure the circ_0000218, miR-139-3p and RAB1A mRNA expression in CRC tissues and cells. Immunohistochemistry and western blot were conducted to determine the RAB1A expression in CRC tissues and cells, respectively. Colony formation assay and BrdU method were employed to monitor the effect of circ_0000218 on cell proliferation. Transwell assay was adopted to detect cell migration and invasion. Dual luciferase reporter assay and RNA immunoprecipitation assay were adopted to confirm the targeting relationship between circ_0000218 and miR-139-3p, miR-139-3p and RAB1A. We demonstrated that circ_0000218 was notably upregulated in CRC tissues and cell lines, and its high expression level was markedly linked to the increase of T staging and local lymph node metastasis. Circ_0000218 overexpression enhanced the proliferation and metastasis of CRC cells while knocking down circ_0000218 caused the opposite effects. We also observed that miR-139-3p was negatively regulated by circ_0000218, while RAB1A was positively regulated by it. Collectively, this study suggested that circ_0000218 upregulated RAB1A and promoted CRC proliferation and metastasis via sponging miR-139-3p.

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A novel angiotensin-I converting enzyme inhibitory peptide derived from the glutelin of vinegar soaked black soybean and its antihypertensive effect in spontaneously hypertensive rats (2019)

Zhang, Yueyuan ; Zhang, Yanling ; Chen, Peiyao ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(3): 223-230. Published 2019 Apr 20. doi: 10.1093/jb/mvz029.

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Publication Date: 2019-04-20

Description: Vinegar soaked black soybean is a traditional Chinese food widely used for the treatment of hypertension. While its pharmacodynamic substance was not fully unveiled. It contained abundant glutelin, thus the purpose of this study was to obtain potent antihypertensive peptides from vinegar soaked black soybean. Black soybean was soaked with vinegar and then glutelin was first catalyzed by alcalase. Ultrafiltration, ion exchange chromatography and reversed-phase high performance liquid chromatography were sequentially applied to separate and purify the angiotensin-I converting enzyme (ACE) inhibitory peptides from glutelin hydrolysates. As a result, the fraction L1-4 with the highest ACE inhibitory activity (83.41%) at the final concentration of 0.01 mg/ml was obtained and five peptides were then identified. These peptides were further optimized by virtual screening combining with in silico proteolysis. Finally, a novel tetrapeptide Phe-Gly-Ser-Phe (FGSF) was obtained. FGSF exhibited high in vitro ACE inhibitory activity (IC50 = 117.11 μM) and in vivo hypotensive effect which maximally reduced systolic blood pressure of 21.95 mmHg at 20 mg/kg body weight in spontaneously hypertensive rats. Our study demonstrated that FGSF derived from vinegar soaked black soybean might be used as a promising ingredient for pharmaceuticals against hypertension and its related diseases.

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MicroRNA-449b-5p suppresses cell proliferation, migration and invasion by targeting TPD52 in nasopharyngeal carcinoma (2019)

Yin, Wei ; Shi, Lei ; Mao, Yanjiao

Oxford University Press

In: Journal of Biochemistry. 2019; 166(5): 433-440. Published 2019 Jul 27. doi: 10.1093/jb/mvz057.

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Publication Date: 2019-07-27

Description: Nasopharyngeal carcinoma (NPC) is an important type of head and neck malignant cancer with geographical distribution. MicroRNA-449b-5p (miR-449b-5p) is related to the development of various cancers, while its function in NPC remains unknown. The present study aimed to investigate the role and target gene of miR-449b-5p in NPC. Expressions of miR-449b-5p in NPC cell lines and clinical tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was determined by MTT and colony formation assays. Migration and invasion abilities after different treatment were evaluated by wound healing and Transwell assays, respectively. Dual-luciferase reporter assay was performed to explore the relationship between miR-449b-5p and tumour protein D52 (TPD52). TPD52 expression was determined by qRT-PCR and western blot assay. miR-449b-5p was significantly downregulated in NPC cell lines and clinical tissues than the matched control. Overexpression of miR-449b-5p inhibited proliferation, migration and invasion of NPC cells. Dual-luciferase reporter assay indicated that miR-449b-5p directly targeted TPD52. Furthermore, shRNA-mediated downregulation of TPD52 rectified the promotion of cell migration and invasion by miR-449b-5p inhibition. In conclusion, the present study suggests that miR-449b-5p, as a novel tumour-suppressive miRNA against NPC, inhibits proliferation, migration and invasion of NPC cells via inhibiting TPD52 expression.

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ZBTB7A promotes migration, invasion and metastasis of human breast cancer cells through NF-κB-induced epithelial–mesenchymal transition in vitro and in vivo (2019)

Mao, Anyun ; Chen, Maojian ; Qin, Qinghong ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 485-493. Published 2019 Aug 06. doi: 10.1093/jb/mvz062.

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Publication Date: 2019-08-06

Description: It has been generally confirmed that zinc finger and BTB domain containing 7A (ZBTB7A) plays an important role in the occurrence and progression of malignant tumours, but the promotion or inhibition effect is related to tumour type. The mechanism between ZBTB7A and breast cancer is not well understood, so further research is needed. In this study, we first investigated the expression of ZBTB7A in tissue samples of clinical breast cancer patients, MDA-MB-231, MCF-7 and MCF-10A cells. Second, we overexpressed the ZBTB7A in MCF-7 cells and silenced the ZBTB7A in MDA-MB-231 cells using lentivirus transfection technology, respectively, and verified the effect of ZBTB7A on migration and invasion of breast cancer cell lines through in vitro cell function experiments, such as wound-healing assay, migration and invasion assay, quantitative real time reverse transcriptase (qRT-PCR) and western blot. Then, the correlation between the above influences, epithelial–mesenchymal transition (EMT) and NF-κB was analysed. Finally, in vivo tumour transplantation model in nude mice was established to verified the effect of ZBTB7A on metastasis of breast cancer MDA-MB-231 cells. In conclusion, ZBTB7A is highly expressed in cancer tissue, breast cancer cell line MDA-MB-231 and MCF-7. Meanwhile, the high expression of ZBTB7A may promote cell migration, invasion and tumour metastasis, which may be related to EMT events by regulating NF-κB.

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Dysregulation of miR484-TUSC5 axis takes part in the progression of hepatocellular carcinoma (2019)

Wang, Shanzong ; Wang, Weijuan ; Han, Xiaoguang ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(3): 271-279. Published 2019 May 07. doi: 10.1093/jb/mvz034.

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Publication Date: 2019-05-07

Description: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. miR-484 is previously reported to be a crucial modulator during the process from precancerous lesion to cancer. Tumour suppressor candidate 5 (TUSC5) is a potential tumour suppressor, but its expression and function in HCC are obscure. In this study, we aimed to explore the roles of miR-484 and TUSC5 in HCC, and clarify the relationship between them. We demonstrated that miR-484 was significantly up-regulated in HCC, while TUSC5 was down-regulated. TUSC5 was validated as the target gene of miR-484 and both of them were associated with the prognosis of HCC patients. miR-484 mimics markedly promoted the malignant phenotypes while TUSC5 plasmid had the opposite effect. In conclusion, miR-484/TUSC5 is potential diagnostic biomarkers and therapy targets for HCC.

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Redox regulation of tyrosine kinase signalling: more than meets the eye (2019)

Dustin, Christopher M ; Heppner, David E ; Lin, Miao-Chong J ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(2): 151-163. Published 2019 Oct 10. doi: 10.1093/jb/mvz085.

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Publication Date: 2019-10-10

Description: Protein kinases are essential mediators of cellular signal transduction and are often dysregulated in disease. Among these, protein tyrosine kinases (PTKs) have received specific interest due to their common roles in various diseases including cancer, and emerging observations indicating that PTK signalling pathways are susceptible to regulation by reactive oxygen species (ROS), which are also frequently implicated in disease pathology. While it is well recognized that ROS can impact on tyrosine kinase signalling by inhibiting tyrosine phosphatases, more recent studies highlight additional modes of redox-based regulation of tyrosine kinase signalling by direct redox modification of non-catalytic cysteines within tyrosine kinases or other protein components of this signalling pathway. In this review, we will present recent advancements with respect to redox-based mechanisms in regulating PTK signalling, with a specific focus on recent studies demonstrating direct redox regulation of Src-family kinases and epidermal growth factor receptor kinases. Importantly, redox-based modulation of tyrosine kinases may be relevant for many other kinases and has implications for current approaches to develop pharmacological inhibitors for these proteins.

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Epigenetic interplays between DNA demethylation and histone methylation for protecting oncogenesis (2019)

Furuya, Kanji ; Ikura, Masae ; Ikura, Tsuyoshi

Oxford University Press

In: Journal of Biochemistry. 2019; 165(4): 297-299. Published 2019 Jan 15. doi: 10.1093/jb/mvy124.

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Publication Date: 2019-01-15

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Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine (2019)

Klenk, Jan M ; Fischer, Max-Philipp ; Dubiel, Paulina ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 51-66. Published 2019 Feb 13. doi: 10.1093/jb/mvz010.

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Publication Date: 2019-02-13

Description: Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

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Mass spectrometry-based methods for analysing the mitochondrial interactome in mammalian cells (2019)

Koshiba, Takumi ; Kosako, Hidetaka

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 225-231. Published 2019 Oct 24. doi: 10.1093/jb/mvz090.

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Publication Date: 2019-10-24

Description: Protein–protein interactions are essential biologic processes that occur at inter- and intracellular levels. To gain insight into the various complex cellular functions of these interactions, it is necessary to assess them under physiologic conditions. Recent advances in various proteomic technologies allow to investigate protein–protein interaction networks in living cells. The combination of proximity-dependent labelling and chemical cross-linking will greatly enhance our understanding of multi-protein complexes that are difficult to prepare, such as organelle-bound membrane proteins. In this review, we describe our current understanding of mass spectrometry-based proteomics mapping methods for elucidating organelle-bound membrane protein complexes in living cells, with a focus on protein–protein interactions in mitochondrial subcellular compartments.

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Hepatitis B virus x protein accelerated the proliferation of hepatocellular carcinoma cell through lncRNA SNHG20/PTEN pathway (2019)

Tu, Wenhui ; Yang, Yonghong ; Song, Yulong ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 165(5): 423-431. Published 2019 Jan 25. doi: 10.1093/jb/mvy120.

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Publication Date: 2019-01-25

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CRY2 suppresses trophoblast migration and invasion in recurrent spontaneous abortion (2019)

Wu, Lianzhi ; Cheng, Biheng ; Liu, Qian ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 79-87. Published 2019 Oct 12. doi: 10.1093/jb/mvz076.

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Publication Date: 2019-10-12

Description: Disruption of circadian rhythms is associated with aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA). This study aims to explore the functional role and the mechanisms of cryptochrome 2 (CRY2), a fundamental component of the circadian clock, in regulating trophoblast migration and invasion. Human extravillous trophoblast cell line HTR-8/SVneo was used as a cell model. Cell migration and invasion were examined using wound healing assay and Transwell assay, respectively. The mRNA and protein levels were determined using quantitative real-time polymerase chain reaction and western blot, respectively. Luciferase reporter assay and chromatin immunoprecipitation assay were performed to explore the interaction between c-Myc to the brain and muscle ARNT-like protein 1 (BMAL1) promoter. CRY2 was highly expressed in human villous specimens of RSA. Furthermore, CRY2 overexpression impaired migration and invasion in HTR-8/SVneo cells, whereas CRY2 knockdown yielded the opposite results. Mechanistically, c-Myc bound to the BMAL1 promoter and induced BMAL1 transcription, both of which further activated matrix metalloproteinase 2/9 (MMP2/9) and facilitated migration and invasion in HTR-8/SVneo cells. CRY2 inhibited c-Myc-BMAL1 pathway and impaired migration and invasion of HTR-8/SVneo cells. Collectively, these findings demonstrate that CRY2 suppresses trophoblast migration and invasion via inhibiting c-Myc-BMAL1-MMP2/9 pathway.

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Mucin-type glycosylation as a regulatory factor of amyloid precursor protein processing (2019)

Nakamura, Naosuke ; Kurosaka, Akira

Oxford University Press

In: Journal of Biochemistry. 2019; 165(3): 205-208. Published 2019 Jan 23. doi: 10.1093/jb/mvy121.

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Publication Date: 2019-01-23

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GPR31 and GPR151 are activated under acidic conditions (2019)

Mashiko, Misaki ; Kurosawa, Aya ; Tani, Yuki ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 317-322. Published 2019 May 23. doi: 10.1093/jb/mvz042.

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Publication Date: 2019-05-23

Description: Recent studies have revealed that not only proton-sensing channels, but also one family of G protein-coupled receptors (GPCRs) comprising OGR1, GPR4, G2A and TDAG8 are responsible for the sensing of extracellular protons, or pH. Here, we report that two other GPCRs, GPR31 and GPR151, were also activated in acidic condition. Elevated pH of assay mixtures resulted in a remarkable increase in [35S]GTPγS binding by GPR31–Giα and GPR151–Giα fusion proteins in a narrow range between pH 6 and 5. Our reporter gene assays with CHO cells expressing recombinant GPR31 or GPR151 also showed that activation was maximal at pH ∼5.8. Although these results from in vitro and cellular assays revealed slightly different pH sensitivities, all of our results indicated that GPR31 and GPR151 sensed extracellular protons equally well as other proton-sensing GPCRs.

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Crystal structure of bacterial cyclopropane-fatty-acyl-phospholipid synthase with phospholipid (2019)

Ma, Yulong ; Pan, Chunli ; Wang, Qihai

Oxford University Press

In: Journal of Biochemistry. 2019; 166(2): 139-147. Published 2019 Mar 04. doi: 10.1093/jb/mvz018.

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Publication Date: 2019-03-04

Description: The lipids containing cyclopropane-fatty-acid (CFA) protect bacteria from adverse conditions such as acidity, freeze-drying desiccation and exposure to pollutants. CFA is synthesized when cyclopropane-fatty-acyl-phospholipid synthase (CFA synthase, CFAS) transfers a methylene group from S-adenosylmethionine (SAM) across the cis double bonds of unsaturated fatty acyl chains. Here, we reported a 2.7-Å crystal structure of CFAS from Lactobacillus acidophilus. The enzyme is composed of N- and C-terminal domain, which belong to the sterol carrier protein and methyltransferase superfamily, respectively. A phospholipid in the substrate binding site and a bicarbonate ion (BCI) acting as a general base in the active site were discovered. To elucidate the mechanism, a docking experiment using CFAS from L. acidophilus and SAM was carried out. The analysis of this structure demonstrated that three groups, the carbons from the substrate, the BCI and the methyl of S(CHn)3 group, were close enough to form a cyclopropane ring with the help of amino acids in the active site. Therefore, the structure supports the hypothesis that CFAS from L. acidophilus catalyzes methyl transfer via a carbocation mechanism. These findings provide a structural basis to more deeply understand enzymatic cyclopropanation.

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Not just a cargo receptor for large cargoes; an emerging role of TANGO1 as an organizer of ER exit sites (2019)

Saito, Kota ; Maeda, Miharu

Oxford University Press

In: Journal of Biochemistry. 2019; 166(2): 115-119. Published 2019 May 07. doi: 10.1093/jb/mvz036.

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Publication Date: 2019-05-07

Description: Proteins synthesized within the endoplasmic reticulum (ER) are exported from ER exit sites via coat protein complex II (COPII)-coated vesicles. Although the mechanisms of COPII-vesicle formation at the ER exit sites are highly conserved among species, vertebrate cells secrete a wide range of materials, including collagens and chylomicrons, which form bulky structures within the ER that are too large to fit into conventional carriers. Transport ANd Golgi Organization 1 (TANGO1) was initially identified as a cargo receptor for collagens but has been recently rediscovered as an organizer of ER exit sites. We would like to review recent advances in the mechanism of large cargo secretion and organization of ER exit sites through the function of TANGO1.

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Regulation of R-loops and genome instability in Fanconi anemia (2019)

Okamoto, Yusuke ; Hejna, James ; Takata, Minoru

Oxford University Press

In: Journal of Biochemistry. 2019; 165(6): 465-470. Published 2019 Mar 01. doi: 10.1093/jb/mvz019.

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Publication Date: 2019-03-01

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Identification of a chemical modulator of EZH2-mediated silencing by cell-based high-throughput screening assay (2019)

Murashima, Akihiro ; Shinjo, Keiko ; Katsushima, Keisuke ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 41-50. Published 2019 Jan 25. doi: 10.1093/jb/mvz007.

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Publication Date: 2019-01-25

Description: Dysregulation of enhancer of zeste homologue 2 (EZH2), a methyltransferase component of polycomb repressive complex 2, is found in many types of cancers especially those that are highly progressive and aggressive. Specific catalytic inhibitors of EZH2 have high anti-tumour activity, particularly in lymphomas with EZH2 activating mutations. However, the clinical benefits of EZH2 catalytic inhibitors in tumours overexpressing EZH2 are still limited. Here, we identified NPD13668, a novel modulator of EZH2-mediated gene silencing, from 329,049 small chemical compounds using a cell-based high-throughput screening assay. NPD13668 reactivated the expression of silenced H3K27me3 target genes together with depletion of the H3K27me3 modification. In addition, NPD13668 repressed the cell growth of prostate cancer cell lines (PC3 and LNCaP) and ovarian cancer cell lines (SKOV3 and NIH-OVCAR3). NPD13668 partially inhibited the methyltransferase activity of EZH2 in vitro. Genome-wide expression analysis revealed that after NPD13668 treatment, about half of the upregulated genes overlapped with genes upregulated after treatment with GSK126, well-known EZH2 catalytic inhibitor, indicating that NPD13668 is a potential modulator of EZH2 methyltransferase activity. Our data demonstrated that targeting the pharmacological inhibition of EZH2 activity by NPD13668 might be a novel cancer treatment.

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miR-146a promoted breast cancer proliferation and invasion by regulating NM23-H1 (2019)

Chen, Jun ; Jiang, Qiang ; Jiang, Xue-Qin ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 41-48. Published 2019 Oct 09. doi: 10.1093/jb/mvz079.

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Publication Date: 2019-10-09

Description: The study aimed to investigate the regulatory effect of miR-146a in proliferation, invasion and migration of breast cancer and its possible mechanism via NM23-H1. The expression levels of miR-146a in breast cancer with different pathological classification were significantly increased, while the expression levels of NM23-H1 were significantly decreased, which were closely correlated. Double luciferase reporter gene was used to verify the target regulatory relationship between miR-146 and NM23-H1 on a human breast cancer cell line. miR-146a was closely related to the proliferation and metastasis of breast cancer. miR-146a also promoted the growth of breast cancer in vivo via targeting NM23-H1. In conclusion, miR-146 can promote the proliferation and invasion of breast cancer by targeting NM23-H1.

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Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter (2019)

Iwai, Yuma ; Kamatani, Setsuko ; Moriyama, Sawako ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 165(6): 479-486. Published 2019 Jan 14. doi: 10.1093/jb/mvz002.

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Publication Date: 2019-01-14

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Nonmuscle myosin IIA and IIB differently suppress microtubule growth to stabilize cell morphology (2019)

Sato, Yuta ; Kamijo, Keiju ; Tsutsumi, Motosuke ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 25-39. Published 2019 Oct 10. doi: 10.1093/jb/mvz082.

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Publication Date: 2019-10-10

Description: Precise regulation of cytoskeletal dynamics is important in many fundamental cellular processes such as cell shape determination. Actin and microtubule (MT) cytoskeletons mutually regulate their stability and dynamics. Nonmuscle myosin II (NMII) is a candidate protein that mediates the actin–MT crosstalk. NMII regulates the stability and dynamics of actin filaments to control cell morphology. Additionally, previous reports suggest that NMII-dependent cellular contractility regulates MT dynamics, and MTs also control cell morphology; however, the detailed mechanism whereby NMII regulates MT dynamics and the relationship among actin dynamics, MT dynamics and cell morphology remain unclear. The present study explores the roles of two well-characterized NMII isoforms, NMIIA and NMIIB, on the regulation of MT growth dynamics and cell morphology. We performed RNAi and drug experiments and demonstrated the NMII isoform-specific mechanisms—NMIIA-dependent cellular contractility upregulates the expression of some mammalian diaphanous-related formin (mDia) proteins that suppress MT dynamics; NMIIB-dependent inhibition of actin depolymerization suppresses MT growth independently of cellular contractility. The depletion of either NMIIA or NMIIB resulted in the increase in cellular morphological dynamicity, which was alleviated by the perturbation of MT dynamics. Thus, the NMII-dependent control of cell morphology significantly relies on MT dynamics.

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Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding (2019)

Nagata, Koji ; Okada, Akitoshi ; Ohtsuka, Jun ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 1-14. Published 2019 Oct 30. doi: 10.1093/jb/mvz087.

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Publication Date: 2019-10-30

Description: Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

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MSCs inhibits the angiogenesis of HUVECs through the miR-211/Prox1 pathway (2019)

Pan, Jian ; Wang, Xianglong ; Li, Dequan ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 107-113. Published 2019 May 29. doi: 10.1093/jb/mvz038.

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Publication Date: 2019-05-29

Description: The aim of this study was to investigate the effect of mesenchymal stem cells (MSCs) on the angiogenesis of human umbilical vein endothelial cells (HUVECs). MSCs were subconjunctival injected into rat corneal alkali burn models. Their impacts on the degree of corneal neovascularization (CNV) and corneal opacity were evaluated at 3, 6, 9 and 12 days after injection. An in vitro experiment of MSCs affecting HUVECs angiogenesis was performed and evaluated using the tube formation assay. The results showed that both CNV and corneal opacity were decreased in rats after MSCs injection. In HUVECs, angiogenesis of cells was inhibited by miR-211 overexpression. miR-211 negatively regulated Prox1 expression. Knockdown of miR-211 blocked the decrease of Prox1 expression induced by MSCs and the inhibitory effect of MSCs on the angiogenesis of HUVECs. The critical role of miR-211 in MSCs inhibition of corneal angiogenesis was confirmed in rat experiments. We concluded that MSCs inhibited the angiogenesis of HUVEC through miR-211 mediating the down-regulation of Prox1.

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Post-transcriptional regulation of inflammation by RNA-binding proteins via cis-elements of mRNAs (2019)

Uchida, Yutaro ; Chiba, Tomoki ; Kurimoto, Ryota ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(5): 375-382. Published 2019 Sep 12. doi: 10.1093/jb/mvz067.

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Publication Date: 2019-09-12

Description: In human genome, there are approximately 1,500 RNA-binding proteins (RBPs). They can regulate mRNA stability or translational efficiency via ribosomes and these processes are known as ‘post-transcriptional regulation’. Accumulating evidences indicate that post-transcriptional regulation is the determinant of the accurate levels of cytokines mRNAs. While transcriptional regulation of cytokines mRNAs has been well studied and found to be important for the rapid induction of mRNA and regulation of the acute phase of inflammation, post-transcriptional regulation by RBPs is essential for resolving inflammation in the later phase, and their dysfunction may lead to severe autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. For post-transcriptional regulation, RBPs recognize and directly bind to cis-regulatory elements in 3′ untranslated region of mRNAs such as AU-rich or constitutive decay elements and play various roles. In this review, we summarize the recent findings regarding the role of RBPs in the regulation of inflammation.

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Domains two and three of Escherichia coli ribosomal S1 protein confers 30S subunits a high affinity for downstream A/U-rich mRNAs (2019)

Cifuentes-Goches, Juan C ; Hernández-Ancheyta, Lizbeth ; Guarneros, Gabriel ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Jan 21. doi: 10.1093/jb/mvz006. [early online release]

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Publication Date: 2019-01-21

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A DNA-binding domain in the C-terminal region of Cdt2 enhances the DNA synthesis-coupled CRL4Cdt2 ubiquitin ligase activity for Cdt1 (2019)

Mazian, Muadz Ahmad ; Suenaga, Naohiro ; Ishii, Takashi ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 165(6): 505-516. Published 2019 Jan 12. doi: 10.1093/jb/mvz001.

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Publication Date: 2019-01-12

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Harnessing biomolecular condensates in living cells (2019)

Nakamura, Hideki ; DeRose, Robert ; Inoue, Takanari

Oxford University Press

In: Journal of Biochemistry. 2019; 166(1): 13-27. Published 2019 Apr 24. doi: 10.1093/jb/mvz028.

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Publication Date: 2019-04-24

Description: As part of the ‘Central Dogma’ of molecular biology, the function of proteins and nucleic acids within a cell is determined by their primary sequence. Recent work, however, has shown that within living cells the role of many proteins and RNA molecules can be influenced by the physical state in which the molecule is found. Within living cells, both protein and RNA molecules are observed to condense into non-membrane-bound yet distinct structures such as liquid droplets, hydrogels and insoluble aggregates. These unique intracellular organizations, collectively termed biomolecular condensates, have been found to be vital in both normal and pathological conditions. Here, we review the latest studies that have developed molecular tools attempting to recreate artificial biomolecular condensates in living cells. We will describe their design principles, implementation and unique characteristics, along with limitations. We will also introduce how these tools can be used to probe and perturb normal and pathological cell functions, which will then be complemented with discussions of remaining areas for technological advance under this exciting theme.

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Cellular and molecular mechanisms of sterile inflammation in ischaemic stroke (2019)

Nakamura, Koutarou ; Shichita, Takashi

Oxford University Press

In: Journal of Biochemistry. 2019; 165(6): 459-464. Published 2019 Feb 22. doi: 10.1093/jb/mvz017.

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Publication Date: 2019-02-22

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Matrin3 promotes homologous recombinational repair by regulation of RAD51 (2019)

Shi, Lin ; Sun, Jiying ; Kinomura, Aiko ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 343-351. Published 2019 May 23. doi: 10.1093/jb/mvz041.

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Publication Date: 2019-05-23

Description: Matrin3 is a highly conserved inner nuclear matrix protein involved in multiple stages of RNA metabolism. Although Matrin3 may also play a role in DNA repair, its precise roles have remained unclear. In this study, we showed that the depletion of Matrin3 led to decreased homologous recombination (HR) efficiency and increased radiation sensitivity of cells. Matrin3-depleted cells showed impaired DNA damage-dependent focus formation of RAD51, a key protein in HR. These findings suggest that Matrin3 promotes HR by regulating RAD51.

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lncRNA CCAT1/miR-490-3p/MAPK1/c-Myc positive feedback loop drives progression of acute myeloid leukaemia (2019)

Wang, Chenghong ; Chen, Fangping ; Fan, Zili ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(4): 379-388. Published 2019 Dec 02. doi: 10.1093/jb/mvz107.

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Publication Date: 2019-12-02

Description: Acute myeloid leukaemia (AML) is a frequently diagnosed malignancy in adults. Long non-coding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) has been well known to play vital roles in multiple malignancies including AML. Unfortunately, the detailed mechanism of CCAT1 in AML progression remains obscure. In this study, we demonstrated that CCAT1 was up-regulated in AML samples while its target, miR-490-3p, was relatively down-regulated. CCAT1 markedly increased viability and metastasis of AML cells, while miR-490-3p had opposite effects. CCAT1 could specifically bind to miR-490-3p and reduce its expression and activity, and MAPK1 was a target gene of miR-490-3p. Overexpressed CCAT1 could induce MAPK1 expression and c-Myc reciprocally increased CCAT1 expression. Our data implied that miR-490-3p could be a novel therapeutic target for AML, and highlights the crucial role of CCAT1/miR-490-3p/MAPK1/c-Myc positive feedback loop in AML progression.

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A AAA ATPase Cdc48 with a cofactor Ubx2 facilitates ubiquitylation of a mitochondrial fusion-promoting factor Fzo1 for proteasomal degradation (2019)

Nahar, Sabiqun ; Chowdhury, Abhijit ; Ogura, Teru ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 279-286. Published 2019 Dec 05. doi: 10.1093/jb/mvz104.

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Publication Date: 2019-12-05

Description: Dynamic functionality of mitochondria is maintained by continual fusion and fission events. A mitochondrial outer membrane protein Fzo1 plays a pivotal role upon mitochondrial fusion by homo-oligomerization to tether fusing mitochondria. Fzo1 is tightly regulated by ubiquitylations and the ubiquitin-responsible AAA protein Cdc48. Here, we show that a Cdc48 cofactor Ubx2 facilitates Fzo1 turnover. The Cdc48-Ubx2 complex has been shown to facilitate degradation of ubiquitylated proteins stacked at the protein translocation complex in the mitochondrial outer membrane by releasing them from the translocase. By contrast, in the degradation process of Fzo1, the Cdc48-Ubx2 complex appears to facilitate the degradation-signalling ubiquitylation of the substrate itself. In addition, the Cdc48-Ubx2 complex interacts with Ubp2, a deubiquitylase reversing the degradation-signalling ubiquitylation of Fzo1. These results suggest that the Cdc48-Ubx2 complex regulates Fzo1 turnover by modulating ubiquitylation status of the substrate.

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Mitochondrial division, fusion and degradation (2019)

Murata, Daisuke ; Arai, Kenta ; Iijima, Miho ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 233-241. Published 2019 Dec 04. doi: 10.1093/jb/mvz106.

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Publication Date: 2019-12-04

Description: The mitochondrion is an essential organelle for a wide range of cellular processes, including energy production, metabolism, signal transduction and cell death. To execute these functions, mitochondria regulate their size, number, morphology and distribution in cells via mitochondrial division and fusion. In addition, mitochondrial division and fusion control the autophagic degradation of dysfunctional mitochondria to maintain a healthy population. Defects in these dynamic membrane processes are linked to many human diseases that include metabolic syndrome, myopathy and neurodegenerative disorders. In the last several years, our fundamental understanding of mitochondrial fusion, division and degradation has been significantly advanced by high resolution structural analyses, protein-lipid biochemistry, super resolution microscopy and in vivo analyses using animal models. Here, we summarize and discuss this exciting recent progress in the mechanism and function of mitochondrial division and fusion.

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Dynamic regulation of subcellular mitochondrial position for localized metabolite levels (2019)

Alshaabi, Haya ; Heininger, Meara ; Cunniff, Brian

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Jul 29. doi: 10.1093/jb/mvz058. [early online release]

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Publication Date: 2019-07-29

Description: Mitochondria are not passive bystanders aimlessly floating throughout our cell’s cytoplasm. Instead, mitochondria actively move, anchor, divide, fuse, self-destruct and transfer between cells in a coordinated fashion, all to ensure proper structure and position supporting cell function. The existence of the mitochondria in our cells has long been appreciated, but their dynamic nature and interaction with other subcellular compartments has only recently been fully realized with the advancement of high-resolution live-cell microscopy and improved fractionization techniques. The how and why that dictates positioning of mitochondria to specific subcellular sites is an ever-expanding research area. Furthermore, the advent of new and improved functional probes, sensitive to changes in subcellular metabolite levels has increased our understanding of local mitochondrial populations. In this review, we will address the evidence for intentional mitochondrial positioning in supporting subcellular mitochondrial metabolite levels, including calcium, adenosine triphosphate and reactive oxygen species and the role mitochondrial metabolites play in dictating cell outcomes.

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Phosphorylation of TMEM55B by Erk/MAPK regulates lysosomal positioning (2019)

Takemasu, Shinya ; Nigorikawa, Kiyomi ; Yamada, Miho ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(2): 175-185. Published 2019 Apr 09. doi: 10.1093/jb/mvz026.

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Publication Date: 2019-04-09

Description: TMEM55B is first identified as phosphatidylinositol-4,5-P24-phosphatases (PtdIns-4,5-P24-phosphatases) that catalyse dephosphorylation of PtdIns-4,5-P2 to PtdIns-5-P. We demonstrate for the first time that TMEM55B is phosphorylated by Erk/MAPK and that this mechanism participates in regulation of lysosomal clustering. Exposure of RAW264.7 macrophages to various stimuli induces phosphorylation of TMEM55B on Ser76 and Ser169, sites corresponding to consensus sequences (PX(S/T)P) for phosphorylation by MAPK. Of these stimuli, Toll-like receptor ligands most strongly induce TMEM55B phosphorylation, and this is blocked by the MEK1/2 inhibitor U0126. However, phosphorylation does not impact intrinsic phosphatase activity of TMEM55B. TMEM55B has recently been implicated in starvation induced lysosomal translocation. Amino acid starvation induces perinuclear lamp1 clustering in RAW264.7 macrophages, which was attenuated by shRNA-mediated knock-down or CRISPR/Cas9-mediated knock-out of TMEM55B. Cells exposed to U0126 also exhibit attenuated lamp1 clustering. Overexpression of TMEM55B but not TMEM55A notably enhances lamp1 clustering, with TMEM55B mutants (lacking phosphorylation sites or mimicking the phosphorylated state) exhibiting lower and higher efficacies (respectively) than wild-type TMEM55B. Collectively, results suggest that phosphorylation of TMEM55B by Erk/MAPK impacts lysosomal dynamics.

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Regulation of collagen type XVII expression by miR203a-3p in oral squamous cell carcinoma cells (2019)

Yasukochi, Atsushi ; Kawakubo-Yasukochi, Tomoyo ; Morioka, Masahiko ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(2): 163-173. Published 2019 Mar 27. doi: 10.1093/jb/mvz024.

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Publication Date: 2019-03-27

Description: Collagen type XVII (COL17) is expressed in various tissues and its aberrant expression is associated with tumour progression. In this study, we investigated the regulation of COL17 expression in oral squamous cell carcinoma (OSCC) using the cell lines NA, SAS, Ca9-22, and Sa3. COL17 was induced upon p53 activation by cisplatin in SAS; however, this effect was more limited in NA and hardly in Ca9-22 and Sa3, with mutated p53. Moreover, COL17 was found to be regulated by miR203a-3p in all cell lines. Our data suggest that COL17 expression in OSCC cell lines is regulated by p53 and miR203a-3p.

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miR-146a and miR-196a-2 genes polymorphisms and its circulating levels in lung cancer patients (2019)

Mohamed, Randa H ; Pasha, Heba F ; Gad, Doaa M ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 323-329. Published 2019 May 24. doi: 10.1093/jb/mvz044.

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Publication Date: 2019-05-24

Description: Recently, MicroRNAs polymorphisms and their serum expression have been linked to increase risk of various cancers. The aim of this study was to elucidate the association between single nucleotide polymorphisms of miR-146a and miR-196a-2 and their serum expression and lung cancer risk. One hundred and twenty lung cancer patients and 120 health controls were included in this study. Genotyping and expression for miR-146a and miR-196a-2 were performed using polymerase chain reaction (PCR)-restriction fragment length polymorphism and quantitative real-time PCR. Individuals carrying miR-146a CG and CC genotypes had significantly increased risk for lung cancer than those carrying miR-146a GG genotype. MiR-146a expression significantly decreased in miR-146a CG and CC genotypes carriers as compared with GG genotype carriers. MiR-196a-2 CT and TT genotypes were significantly associated with increased lung cancer while the highest expression of MiR-196a-2 was detected in miR-196a-2 CC genotype carriers. Serum miR-146a was significantly decreased in lung cancer patients while serum miR-196a-2 expression was significantly increased in lung cancer patients. In conclusion, miR-146a and miR-196a-2 genes polymorphisms and their circulating levels were associated with lung cancer risk in Egyptians and may be helpful in early detection of lung cancer.

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Signal amplification in flow cytometry for cell surface antigen analysis (2019)

Mori, Takeshi ; Katayama, Yoshiki

Oxford University Press

In: Journal of Biochemistry. 2019; 166(3): 205-212. Published 2019 Jun 28. doi: 10.1093/jb/mvz052.

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Publication Date: 2019-06-28

Description: Signal enhancing systems have been introduced to enable detection of cell surface antigens by flow cytometry. Cell surface antigens are important targets that describe the function and lineage of cells. Although flow cytometry is an effective tool for analysing cell surface antigens, this technique has poor sensitivity, which prohibits the detection of many important antigens on cell membranes. Thus, signal amplification is essential for developing practical tools for evaluating cell surface antigens by flow cytometry. Using a bright fluorophore or fluorescent polymer incorporated into antibodies is a straightforward strategy to improve flow cytometry sensitivity but may affect the functional characteristics of the labelled antibody. In contrast, enzymatic signal amplification is a more practical and efficient strategy to improve sensitivity that should not affect antibody activity. Although enzymatic signal amplification still has a number of drawbacks, this approach is a promising strategy to analyse cell surface antigens.

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Cross-talk between the three furA orthologs in Mycobacterium smegmatis and the contribution to isoniazid resistance (2019)

Gao, Chun-Hui ; Wei, Wen-Ping ; Tao, Hui-Ling ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(3): 237-243. Published 2019 Apr 16. doi: 10.1093/jb/mvz030.

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Publication Date: 2019-04-16

Description: The ferric uptake regulator A (FurA) plays an essential role in responding to oxidative stress in mycobacteria. The genome of Mycobacterium smegmatis harbours three FurA orthologs; however, the potential cross-talk and contribution to drug resistance of different furA operon remain underdetermined. In this study, we characterized the cross-regulation and effect in drug resistance of these orthologs from M. smegmatis. Cross-binding of FurA protein to furA promoter was observed. The binding of FurA1 to furA3p and FurA2 to furA1p or furA3p is even more pronounced than their self-binding. The three FurA proteins are all functional at repressing the expression of the peroxidase enzyme katG1/katG2 in vivo. When overexpressing any of the furA orthologs in M. smegmatis, the bacteria become more resistant to isoniazid (INH). This pattern is consistent with that in Mycobacterium bovis. However, the knockdown of furA does not affect the INH sensitivity. This is the first report of cross-talk and contribution to drug resistance of all three furA orthologs in M. smegmatis.

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Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate (2019)

Hamada, Hiroshi ; Goto, Yuta ; Arakawa, Jun ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(4): 363-373. Published 2019 Jun 14. doi: 10.1093/jb/mvz047.

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Publication Date: 2019-06-14

Description: The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 5′-upstream of the E2F family genes. The motifs are especially rich in the 5′-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5′-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

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Propofol alleviates oxidative stress via upregulating lncRNA-TUG1/Brg1 pathway in hypoxia/reoxygenation hepatic cells (2019)

Ming, Nuo ; Na, Ha Sen Ta ; He, Jin-Ling ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(5): 415-421. Published 2019 Jul 12. doi: 10.1093/jb/mvz054.

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Publication Date: 2019-07-12

Description: Reducing oxidative stress is an effective method to prevent hepatic ischaemia/reperfusion injury (HIRI). This study focuses on the role of propofol on the oxidative stress of hepatic cells and the involved lncRNA-TUG1/Brahma-related gene 1 (Brg1) pathway in HIRI mice. The mouse HIRI model was established and was intraperitoneally injected with propofol postconditioning. Hepatic injury indexes were used to evaluate HIRI. The oxidative stress was indicated by increasing 8-isoprostane concentration. Mouse hepatic cell line AML12 was treated with hypoxia and subsequent reoxygenation (H/R). The targeted regulation of lncRNA-TUG1 on Brg1 was proved by RNA pull-down, RIP (RNA-binding protein immunoprecipitation) and the expression level of Brg1 responds to silencing or overexpression of lncRNA-TUG1. Propofol alleviates HIRI and induces the upregulation of lncRNA-TUG1 in the mouse HIRI model. Propofol increases cell viability and lncRNA-TUG1 expression level in H/R-treated hepatic cells. In H/R plus propofol-treated hepatic cells, lncRNA-TUG1 silencing reduces cell viability and increased oxidative stress. LncRNA-TUG1 interacts with Brg1 protein and keeps its level via inhibiting its degradation. Brg1 overexpression reverses lncRNA-TUG1 induced the reduction of cell viability and the increase in oxidative stress. LncRNA-TUG1 silencing abrogates the protective role of propofol against HIRI in the mouse HIRI model. LncRNA-TUG1 has a targeted regulation of Brg1, and thereby affects the oxidative stress induced by HIRI. This pathway mediates the protective effect of propofol against HIRI of hepatic cell.

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From germ cells to neonates: the beginning of life and the KEAP1–NRF2 system (2019)

Matsumaru, Daisuke ; Motohashi, Hozumi

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Sep 13. doi: 10.1093/jb/mvz070. [early online release]

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Publication Date: 2019-09-13

Description: The Kelch-like ECH-associated protein 1(KEAP1)–NF-E2-related factor 2 (NRF2) system is one of the most studied environmental stress response systems. In the presence of oxidative and electrophilic insults, the thiols of cysteine residues in KEAP1 are modified, and subsequently stabilized NRF2 activates its target genes that are involved in detoxification and cytoprotection. A myriad of recent studies has revealed the broad range of contributions of the KEAP1–NRF2 system to physiological and pathological processes. However, its functions during gametic and embryonic development are still open for investigation. Although oxidative stress is harmful for embryos, Nrf2−/− mice do not show any apparent morphological abnormalities during development, probably because of the compensatory antioxidant functions of NF-E2-related factor 1 (NRF1). It can also be considered that the antioxidant system is essential for protecting germ cells during reproduction. The maturation processes of germ cells in both sexes are affected by Nrf2 mutation. Hence, in this review, we focus on the stress response system related to reproduction and embryonic development through the functions of the KEAP1–NRF2 system.

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Plasmalogen mediates integration of adherens junction (2019)

Takahashi, Takanori ; Honsho, Masanori ; Abe, Yuichi ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(5): 423-432. Published 2019 Jun 24. doi: 10.1093/jb/mvz049.

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Publication Date: 2019-06-24

Description: Ether glycerolipids, plasmalogens are found in various mammalian cells and tissues. However, physiological role of plasmalogens in epithelial cells remains unknown. We herein show that synthesis of ethanolamine-containing plasmalogens, plasmenylethanolamine (PlsEtn), is deficient in MCF7 cells, an epithelial cell line, with severely reduced expression of alkyl-dihydroxyacetonephosphate synthase (ADAPS), the second enzyme in the PlsEtn biosynthesis. Moreover, expression of ADAPS or supplementation of PlsEtn containing C18-alkenyl residue delays the migration of MCF7 cells as compared to that mock-treated MCF7 and C16-alkenyl-PlsEtn-supplemented MCF7 cells. Localization of E-cadherin to cell–cell junctions is highly augmented in cells containing C18-alkenyl-PlsEtn. Together, these results suggest that PlsEtn containing C18-alkenyl residue plays a distinct role in the integrity of E-cadherin-mediated adherens junction.

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Multifunctional transcriptional coactivator PC4 is a global co-regulator of p53-dependent stress response and gene regulation (2019)

Mondal, Priya ; Saleem, Suraiya ; Sikder, Sweta ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(5): 403-413. Published 2019 Jun 24. doi: 10.1093/jb/mvz050.

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Publication Date: 2019-06-24

Description: Human positive coactivator 4 (PC4), a multifunctional chromatin-associated protein, is known to directly interact with p53 and modulate expressions of a few p53-dependent genes. However, the role of PC4 in p53's myriad of other regulatory functions is not known. The p53–PC4 interaction was selectively perturbed by a small peptide which led to abrogation of genotoxic stress-induced up-regulation of many p53-dependent genes and reduction of apoptosis in A549 cells. Over-expression of a PC4 point mutant, incapable of binding p53, recapitulated many of the effects of the peptide. Global gene expression profiling in A549 cells, upon peptide treatment, revealed PC4's involvement in the regulation of many p53-dependent pathways, including the Hippo pathway. Introduction of the peptide in neuronal cells significantly reduced its amyloid-β-induced death. Thus, PC4 emerges as a global co-regulator of p53 and a therapeutic target against pathogeneses where the p53-dependent cell death process plays a crucial role.

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Cooperative action of APJ and α1A-adrenergic receptor in vascular smooth muscle cells induces vasoconstriction (2019)

Nagano, Katsumasa ; Kwon, Chulwon ; Ishida, Junji ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(5): 383-392. Published 2019 Sep 03. doi: 10.1093/jb/mvz071.

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Publication Date: 2019-09-03

Description: The apelin receptor (APJ), a receptor for apelin and elabela/apela, induces vasodilation and vasoconstriction in blood vessels. However, the prolonged effects of increased APJ-mediated signalling, involving vasoconstriction, in smooth muscle cells have not been fully characterized. Here, we investigated the vasoactive effects of APJ gain of function under the control of the smooth muscle actin (SMA) gene promoter in mice. Transgenic overexpression of APJ (SMA-APJ) conferred sensitivity to blood pressure and vascular contraction induced by apelin administration in vivo. Interestingly, ex vivo experiments showed that apelin markedly increased the vasoconstriction of isolated aorta induced by noradrenaline (NA), an agonist for α- and β-adrenergic receptors, or phenylephrine, a specific agonist for α1-adrenergic receptor (α1-AR). In addition, intracellular calcium influx was augmented by apelin with NA in HEK293T cells expressing APJ and α1A-AR. To examine the cooperative action of APJ and α1A-AR in the regulation of vasoconstriction, we developed α1A-AR deficient mice using a genome-editing technique, and then established SMA-APJ/α1A-AR-KO mice. In the latter mouse line, aortic vasoconstriction induced by a specific agonist for α1A-AR, A-61603, were significantly less than in SMA-APJ mice. These results suggest that the APJ-enhanced response requires α1A-AR to contract vessels coordinately.

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Mitochondrial dynamics and interorganellar communication in the development and dysmorphism of mammalian oocytes (2019)

Udagawa, Osamu ; Ishihara, Naotada

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 257-266. Published 2019 Dec 09. doi: 10.1093/jb/mvz093.

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Publication Date: 2019-12-09

Description: Mitochondria play many critical roles in cells, not only by supplying energy, but also by supplying metabolites, buffering Ca2+ levels and regulating apoptosis. During oocyte maturation and subsequent embryo development, mitochondria change their morphology by membrane fusion and fission, and coordinately undergo multiple cellular events with the endoplasmic reticulum (ER) closely apposed. Mitochondrial fusion and fission, known as mitochondrial dynamics, are regulated by family members of dynamin GTPases. Oocytes in animal models with these regulators artificially altered exhibit morphological abnormalities in nearby mitochondria and at the ER interface that are reminiscent of major cytoplasmic dysmorphisms in human assisted reproductive technology, in which a portion of mature oocytes retrieved from patients contain cytoplasmic dysmorphisms associated with mitochondria and ER abnormal morphologies. Understanding organelle morpho-homeostasis in oocytes obtained from animal models will contribute to the development of novel methods for determining oocyte health and for how to deal with dysmorphic oocytes.

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Aurora kinase A-mediated phosphorylation of mPOU at a specific site drives skeletal muscle differentiation (2019)

Karthigeyan, Dhanasekan ; Bose, Arnab ; Boopathi, Ramachandran ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(2): 195-201. Published 2019 Oct 30. doi: 10.1093/jb/mvz088.

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Publication Date: 2019-10-30

Description: Aurora kinases are Ser/Thr-directed protein kinases which play pivotal roles in mitosis. Recent evidences highlight the importance of these kinases in multiple biological events including skeletal muscle differentiation. Our earlier study identified the transcription factor POU6F1 (or mPOU) as a novel Aurora kinase (Aurk) A substrate. Here, we report that Aurora kinase A phosphorylates mPOU at Ser197 and inhibit its DNA-binding ability. Delving into mPOU physiology, we find that the phospho-mimic (S197D) mPOU mutant exhibits enhancement, while the wild type or the phospho-deficient mutant shows retardation in C2C12 myoblast differentiation. Interestingly, POU6F1 depletion phenocopies S197D-mPOU overexpression in the differentiation context. Collectively, our results signify mPOU as a negative regulator of skeletal muscle differentiation and strengthen the importance of AurkA in skeletal myogenesis.

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Chemical toolbox for ‘live’ biochemistry to understand enzymatic functions in living systems (2019)

Komatsu, Toru ; Urano, Yasuteru

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Sep 25. doi: 10.1093/jb/mvz074. [early online release]

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Publication Date: 2019-09-25

Description: In this review, we present an overview of the recent advances in chemical toolboxes that are used to provide insights into ‘live’ protein functions in living systems. Protein functions are mediated by various factors inside of cells, such as protein−protein interactions, posttranslational modifications, and they are also subject to environmental factors such as pH, redox states and crowding conditions. Obtaining a true understanding of protein functions in living systems is therefore a considerably difficult task. Recent advances in research tools have allowed us to consider ‘live’ biochemistry as a valid approach to precisely understand how proteins function in a live cell context.

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Pathological consequences of the unfolded protein response and downstream protein disulphide isomerases in pulmonary viral infection and disease (2019)

Chamberlain, Nicolas ; Anathy, Vikas

Oxford University Press

In: Journal of Biochemistry. 2019; 167(2): 173-184. Published 2019 Dec 02. doi: 10.1093/jb/mvz101.

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Publication Date: 2019-12-02

Description: Protein folding within the endoplasmic reticulum (ER) exists in a delicate balance; perturbations of this balance can overload the folding capacity of the ER and disruptions of ER homoeostasis is implicated in numerous diseases. The unfolded protein response (UPR), a complex adaptive stress response, attempts to restore normal proteostasis, in part, through the up-regulation of various foldases and chaperone proteins including redox-active protein disulphide isomerases (PDIs). There are currently over 20 members of the PDI family each consisting of varying numbers of thioredoxin-like domains which, generally, assist in oxidative folding and disulphide bond rearrangement of peptides. While there is a large amount of redundancy in client proteins of the various PDIs, the size of the family would indicate more nuanced roles for the individual PDIs. However, the role of individual PDIs in disease pathogenesis remains uncertain. The following review briefly discusses recent findings of ER stress, the UPR and the role of individual PDIs in various respiratory disease states.

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Mitochondrial nucleoid morphology and respiratory function are altered in Drp1-deficient HeLa cells (2019)

Ota, Azusa ; Ishihara, Takaya ; Ishihara, Naotada

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 287-294. Published 2019 Dec 24. doi: 10.1093/jb/mvz112.

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Publication Date: 2019-12-24

Description: Mitochondria are dynamic organelles that frequently divide and fuse with each other. The dynamin-related GTPase protein Drp1 has a key role in mitochondrial fission. To analyse the physiological roles of Drp1 in cultured human cells, we analysed Drp1-deficient HeLa cells established by genome editing using CRISPR/Cas9. Under fluorescent microscopy, not only mitochondria were elongated but their DNA (mtDNA) nucleoids were extremely enlarged in bulb-like mitochondrial structures (‘mito-bulbs’) in the Drp1-deficient HeLa cells. We further found that respiratory activity, as measured by oxygen consumption rates, was severely repressed in Drp1-deficient HeLa cells and that this was reversible by the co-repression of mitochondrial fusion factors. Although mtDNA copy number was not affected, several respiratory subunits were repressed in Drp1-deficient HeLa cells. These results suggest that mitochondrial fission is required for the maintenance of active respiratory activity and the morphology of mtDNA nucleoids in human cells.

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Developments of human adrenomedullin-IgG1 Fc fusion proteins (2019)

Nagata, Sayaka ; Yamasaki, Motoo ; Kawano, Aya ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(2): 157-162. Published 2019 Mar 20. doi: 10.1093/jb/mvz023.

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Publication Date: 2019-03-20

Description: Human adrenomedullin (hAM) is a hypotensive peptide hormone that exerts powerful anti-inflammatory effects. However, treatment required continuous administration of hAM, as the half-life of native hAM is quite short in blood. To resolve this problem, we designed two kinds of human IgG1 Fc fusion proteins containing either full-length hAM (IgG1-AM) or hAM residues 6–52 [IgG1-AM (6–52)]. A DNA construct was constructed by connecting DNA sequences encoding hAM and the IgG1 Fc region with a DNA sequence encoding a (GGGGS)3 linker. The molecular weights of IgG1-AM and IgG1-AM (6–52) were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. By protein sequencing, the N-terminal sequence of both recombinant AM-Fc fusions showed the expected human IgG1 sequence. Sufficient concentrations of both AM-Fc fusions were observed in blood 2 days after a single subcutaneous administration. IgG1-AM and IgG1-AM (6–52) stimulated cAMP production in human embryonic kidney-293 cells stably expressing the AM1 receptor. The activity of IgG1-AM (6–52) was higher than that of IgG1-AM. Treatment with IgG1-AM (6–52) inhibited blood pressure increase in spontaneously hypertensive rats. In addition, IgG1-AM (6–52) reduced total inflammation scores in the dextran sulfate sodium colitis model. Therefore, AM-IgG1 Fc fusions represent potential novel therapeutic agents.

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Screening of subtype-specific activators and inhibitors for diacylglycerol kinase (2019)

Hayashi, Daiki ; Tsumagari, Ryosuke ; Liu, Ke ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 165(6): 517-522. Published 2019 Feb 04. doi: 10.1093/jb/mvz008.

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Publication Date: 2019-02-04

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Age- and gender-dependent D-amino acid oxidase activity in mouse brain and peripheral tissues: implication for aging and neurodegeneration (2019)

Kim, Soo Hyeon ; Shishido, Yuji ; Sogabe, Hirofumi ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(2): 187-196. Published 2019 Apr 02. doi: 10.1093/jb/mvz025.

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Publication Date: 2019-04-02

Description: D-amino acid oxidase (DAO) is a flavoenzyme, catalysing oxidative deamination of D-amino acids to produce corresponding α-keto acids, ammonia and hydrogen peroxide. In our search for DAO activity among various tissues, we developed a sensitive assay based on hydrogen peroxide production involving enzyme-coupled colorimetric assay with peroxidase. We first optimized buffer components to extract DAO protein from mouse tissues. Here we show that DAO activity was detected in kidney, cerebellum, medulla oblongata, midbrain and spinal cord, but not in liver. In addition, we observed that DAO activity and expression were decreased in thoracic and lumbar regions of spinal cord in aged mice when compared with young mice, indicating that decreased DAO is involved in motoneuron degeneration during senescence. We also found gender difference in DAO activity in the kidney, suggesting that DAO activity is influenced by sexual dimorphism. We newly detected DAO activity in the epididymis, although undetected in testis. Furthermore, DAO activity was significantly higher in the caput region than corpus and cauda regions of epididymis, indicating that D-amino acids present in the testis are eliminated in epididymis. Taken together, age- and gender-dependent DAO activity in each organ may underlie the human pathophysiology regulated by D-amino acid metabolism.

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Toll-like receptor 7 regulates osteoclastogenesis in rheumatoid arthritis (2019)

Kim, Kyoung-Woon ; Kim, Bo-Mi ; Won, Ji-Yeon ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(3): 259-270. Published 2019 May 13. doi: 10.1093/jb/mvz033.

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Publication Date: 2019-05-13

Description: This study aimed to determine the regulatory role of toll-like receptor 7 (TLR7) in receptor activator of nuclear factor kappa-B ligand (RANKL) production and osteoclast differentiation in rheumatoid arthritis (RA). In confocal microscopy, the co-expression of TLR7, CD55 and RANKL was determined in RA synovial fibroblasts. After RA synovial fibroblasts were treated with imiquimod, the RANKL gene expression and protein production were determined by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Osteoclastogenesis from peripheral blood CD14+ monocytes which were cultured with imiquimod was assessed by determining the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. The signal pathways mediating the TLR7-induced RANKL expression and osteoclastogenesis were analysed after inhibition of intracellular signal molecules and their phosphorylation. Imiquimod stimulated the expression of TLR7 and RANKL and production of RANKL in RA synovial fibroblasts, increasing the phosphorylation of TRAF6, IRF7, mitogen-activated protein kinases (MAPK), c-Jun and NFATc1. When CD14+ monocytes were cultured with imiquimod or co-cultured with imiquimod-pre-treated RA synovial fibroblasts, they were differentiated into TRAP+ multinucleated osteoclasts in the absence of RANKL. TLR7 activation-induced osteoclastogenesis in RA through direct induction of osteoclast differentiation from its precursors and up-regulation of RANKL production in RA synovial fibroblasts. Thus, the blockage of TLR7 pathway could be a promising therapeutic strategy for preventing bone destruction in RA.

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Multiple functions of the ER-resident VAP and its extracellular role in neural development and disease (2019)

Kamemura, Kosuke ; Chihara, Takahiro

Oxford University Press

In: Journal of Biochemistry. 2019; 165(5): 391-400. Published 2019 Feb 06. doi: 10.1093/jb/mvz011.

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Publication Date: 2019-02-06

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Deciphering the anti-Parkinson’s activity of sulphated polysaccharides from Chlamydomonas reinhardtii on the α-Synuclein mutants A30P, A53T, E46K, E57K and E35K (2019)

Panigrahi, Gitanjali P ; Rane, Ankita R ; Vavilala, Sirisha L ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 463-474. Published 2019 Aug 06. doi: 10.1093/jb/mvz064.

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Publication Date: 2019-08-06

Description: Parkinsonism-linked mutations in alanine and glutamic acid residues of the pre-synaptic protein α-Synuclein (α-Syn) affect specific tertiary interactions essential for stability of the native state and make it prone to more aggregation. Many of the currently available drugs used for the treatment of Parkinson’s disease (PD) are not very effective and are associated with multiple side effects. Recently, marine algae have been reported to have sulphated polysaccharides which offers multiple pharmaceutical properties. With this background, we have isolated sulphated polysaccharides from Chlamydomonas reinhardtii (Cr-SPs) and investigated their effects on inhibition of fibrillation/aggregation of α-Syn mutants through a combination of spectroscopic and microscopic techniques. The kinetics of α-Syn fibrillation establishes that Cr-SPs are very effective in inhibiting fibrillation of α-Syn mutants. The morphological changes associated with the fibrillation/aggregation process have been monitored by transmission electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel image suggests that Cr-SPs increase the amount of soluble protein after completion of the fibrillation/aggregation process. The circular dichroism results showed that Cr-SPs efficiently delay the conversion of native protein into β-sheet-rich structures. Thus, the current work has considerable therapeutic implications towards deciphering the potential of Cr-SPs to act against PD and other protein aggregation-related disorders.

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Purification, biochemical and molecular study of lipase producing from a newly thermoalkaliphilic Aeribacillus pallidus for oily wastewater treatment (2019)

Ktata, Ameni ; Krayem, Najeh ; Aloulou, Ahmed ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 89-99. Published 2019 Oct 10. doi: 10.1093/jb/mvz083.

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Publication Date: 2019-10-10

Description: Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.

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MiRNA-96-5p contributed to the proliferation of gastric cancer cells by targeting FOXO3 (2019)

He, Xionghui ; Zou, Kejian

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 101-108. Published 2019 Oct 09. doi: 10.1093/jb/mvz080.

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Publication Date: 2019-10-09

Description: Various microRNAs (miRNAs, miRs) and the forkhead box O (FOXO) family proteins have been shown to influence gastric cancer progression and development. Here, we aimed to identify the gastric cancer related miRNAs and their relationship with the FOXO family. MiRNA profiles were generated by miRNA microarray screening from pre-operative plasma samples. Quantitative reverse transcription PCR and western bolt were used to determine the expression levels of miR-96 and FOXO family. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay and colony formation assay were used to test the cell viability. The miR-96-5p and FOXO3 interaction were confirmed by luciferase reporter assay. Our results demonstrated the excessive expression of miR-96-5p in gastric cancer cell lines and plasma samples from gastric cancer patients. In addition, the protein levels of FOXO3 were decreased in tissue samples from gastric cancer patients. Moreover, miR-96-5p accelerated the gastric cancer cell proliferation by directly targeting FOXO3. Therefore, we conclude that iR-96-5p might promote the progression of gastric cancer by directly targeting FOXO3 mRNA and downregulating the expression of FOXO3 protein, which provides new insights for the molecular mechanism of gastric cancer.

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Anti-melanogenic activity of salacinol by inhibition of tyrosinase oligosaccharide processing (2019)

Homma, Toshiyuki ; Kageyama, Shigeki ; Nishikawa, Atsushi ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(5): 503-511. Published 2019 Dec 27. doi: 10.1093/jb/mvz115.

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Publication Date: 2019-12-27

Description: Hyperpigmentation that manifests through melasma and solar lentigo (age spots), although mostly harmless for health, bothers many people. Controlling the rate-limiting activity of tyrosinase is most effective for suppressing excessive melanin formation and accordingly recent research has focused on the maturation of tyrosinase. Salacia, a medicinal plant, has been used to treat diabetes in India and Sri Lanka. Salacia extract reportedly contains components that inhibit the activity of α-glucosidase. Salacinol, the active ingredient in Salacia extract, has unique thiosugar sulphonium sulphate inner salt structure. Here, we observed that the salacinol component of Salacia extract possesses anti-melanogenic activity in comparison to various existing whitening agents. Although the anti-melanogenic mechanism of salacinol is presumably medicated by inhibition of tyrosinase activity, which is often found in existing whitening agents, salacinol did not inhibit tyrosinase activity in vitro. Analysis of the intracellular state of tyrosinase showed a decrease in the mature tyrosinase form due to inhibition of N-linked oligosaccharide processing. Salacinol inhibited the processing glucosidase I/II, which are involved in the initial stage of N-linked glycosylation. Owing to high activity, low cytotoxicity and high hydrophilicity, salacinol is a promising candidate compound in whitening agents aimed for external application on skin.

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A monoclonal antibody recognizing a new epitope on CD81 inhibits T-cell migration without inducing cytokine production (2019)

Hasezaki, Takuya ; Yoshima, Tadahiko ; Mattsson, Mikael ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(4): 399-409. Published 2019 Dec 03. doi: 10.1093/jb/mvz103.

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Publication Date: 2019-12-03

Description: CD81 is involved in leukocyte migration and cytokine induction. Previous work found that anti-CD81 monoclonal antibodies (mAbs) showed therapeutic potential for several immune diseases via inhibiting leukocyte migration. Although the suppression of cell migration is a promising approach for treating immune diseases, some anti-CD81 mAbs can induce cytokine production, which may exacerbate disease. To obtain new anti-human CD81 mAbs that inhibited migration in the absence of cytokine production enhancement activity, we screened a human single chain variable fragment by phage library. One of the new anti-CD81 mAbs isolated, DSP-8250, had equivalent inhibitory cell migration activity with the established anti-CD81 mAb 5A6, but it lacked cytokine induction activity. These mAbs recognized different epitopes on CD81. mAb 5A6, which had inhibitory activity on T-cell migration and increased cytokine production, bound to three residues, Ser179, Asn180 and Phe186 of CD81. In contrast, DSP-8250, which had inhibitory activity on T-cell migration but no cytokine enhancement activity, bound to four residues, His151, Ala164, Ser168 and Asn172 of CD81 as a unique epitope. These results indicate that the set of His151, Ala164, Ser168 and Asn172 forms a novel epitope that might make the application of anti-CD81 mAb therapeutically useful.

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Thanking All Peer Reviewers (2019)

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 449-451. Published 2019 Nov 28. doi: 10.1093/jb/mvz094.

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Publication Date: 2019-11-28

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miR-206 regulates non-small-cell lung cancer cell aerobic glycolysis by targeting hexokinase 2 (2019)

Jia, Ke-Gang ; Feng, Gang ; Tong, Yu-Suo ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(4): 365-370. Published 2019 Nov 19. doi: 10.1093/jb/mvz099.

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Publication Date: 2019-11-19

Description: Aerobic glycolysis was closely associated with the malignant transformation and prognosis of tumours. miR-206 was found to be downregulated in several cancers. However, whether miR-206 functions in non-small-cell lung cancers (NSCLCs) via the process of aerobic glycolysis remains poorly characterized. Quantitative real-time PCR was performed to detect miR-206 level in NSCLC cells and tissues. The effect of miR-206 on hexokinase 2 (HK2) expression was examined through miR-206 overexpression or miR-206 knockdown. CCK-8 assay and colony formation assay were carried out to explore the role of miR-206 on cell proliferation and colony formation, respectively. The relationship between miR-206 and HK2 was measured by dual-luciferase reporter assay. Glucose consumption, lactate production assay and ATP generation were performed in NSCLC cells following miR-206 and HK2 overexpression. We found that miR-206 was downregulated in NSCLC tissues and cells. miR-206 overexpression downregulated the expression of HK2 via targeting HK2 3′UTR in NSCLC cells. In addition, miR-206 decreased the cell viability and colony formation in NSCLC cells. Furthermore, miR-206 reduced glucose uptake, lactate production and ATP generation in NSCLC cells via HK2 repression. In conclusion, these findings suggested that miR-206 regulated NSCLC cell aerobic glycolysis by targeting HK2.

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Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor (2019)

Nishikino, Tatsuro ; Iwatsuki, Hiroto ; Mino, Taira ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(4): 389-398. Published 2019 Nov 18. doi: 10.1093/jb/mvz102.

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Publication Date: 2019-11-18

Description: The bacterial flagellar motor is a rotary nanomachine driven by ion flow. The flagellar stator complex, which is composed of two proteins, PomA and PomB, performs energy transduction in marine Vibrio. PomA is a four transmembrane (TM) protein and the cytoplasmic region between TM2 and TM3 (loop2–3) interacts with the rotor protein FliG to generate torque. The periplasmic regions between TM1 and TM2 (loop1–2) and TM3 and TM4 (loop3–4) are candidates to be at the entrance to the transmembrane ion channel of the stator. In this study, we purified the stator complex with cysteine replacements in the periplasmic loops and assessed the reactivity of the protein with biotin maleimide (BM). BM easily modified Cys residues in loop3–4 but hardly labelled Cys residues in loop1–2. We could not purify the plug deletion stator (ΔL stator) composed of PomBΔ41–120 and WT-PomA but could do the ΔL stator with PomA-D31C of loop1–2 or with PomB-D24N of TM. When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop1–2 region regulate this activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB.

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Histidine 131 in presenilin 1 is the pH-sensitive residue that causes the increase in Aβ42 level in acidic pH (2019)

Cai, Tetsuo ; Hatano, Aki ; Kanatsu, Kunihiko ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(5): 463-471. Published 2019 Dec 09. doi: 10.1093/jb/mvz110.

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Publication Date: 2019-12-09

Description: Alzheimer disease (AD) is the most common neurodegenerative disease worldwide. The pathological hallmark of AD is the presence of senile plaques in the brain, which are accumulations of amyloid-β peptide (Aβ) ending at the 42nd residue (i.e. Aβ42), which is produced through multistep cleavage by γ-secretase. Thus, methods to regulate γ-secretase activity to attenuate the production of Aβ42 are in urgent demand towards the development of treatments for AD. We and others have demonstrated that γ-secretase activity is affected by its localization and ambient environment. In particular, an increase in Aβ42 production is correlated with the intracellular transport of γ-secretase and endosomal maturation-dependent luminal acidification. In this study, we focused on the mechanism by which γ-secretase affects Aβ42 production together with alterations in pH. Histidine is known to function as a pH sensor in many proteins, to regulate their activities through the protonation state of the imidazole side chain. Among the histidines facing the luminal side of presenilin (PS) 1, which is the catalytic subunit of γ-secretase, point mutations at H131 had no effect on the Aβ42 production ratio in an acidic environment. We also observed an increase in Aβ42 ratio when histidine was introduced into N137 of PS2, which is the corresponding residue of H131 in PS1. These results indicated that H131 serves as the pH sensor in PS1, which contains γ-secretase, to regulate Aβ42 production depending on the luminal pH. Our findings provide new insights into therapeutic strategies for AD targeting endosomes or the intracellular transport of γ-secretase.

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Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13 (2019)

Muramatsu, Hisashi ; Miyaoku, Haruna ; Kurita, Syuya ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 333-341. Published 2019 Nov 14. doi: 10.1093/jb/mvz098.

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Publication Date: 2019-11-14

Description: A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The Km and Vmax values of thiourocanate hydratase towards thiourocanic acid were 30 μM and 7.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

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Reaction of threonine synthase with the substrate analogue 2-amino-5-phosphonopentanoate: implications into the proton transfer at the active site (2019)

Machida, Yasuhiro ; Murakawa, Takeshi ; Sakai, Akiko ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(4): 357-364. Published 2019 Nov 13. doi: 10.1093/jb/mvz100.

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Publication Date: 2019-11-13

Description: Threonine synthase catalyses the conversion of O-phospho-l-homoserine and a water molecule to l-threonine and has the most complex catalytic mechanism among the pyridoxal 5′-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The Kd for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site.

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Biochemical and structural analyses of the nucleosome containing human histone H2A.J (2019)

Tanaka, Hiroki ; Sato, Shoko ; Koyama, Masako ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(4): 419-427. Published 2019 Dec 03. doi: 10.1093/jb/mvz109.

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Publication Date: 2019-12-03

Description: Histone H2A.J, a histone H2A variant conserved in mammals, may function in the expression of genes related to inflammation and cell proliferation. In the present study, we purified the human histone H2A.J variant and found that H2A.J is efficiently incorporated into the nucleosome in vitro. H2A.J formed the stable nucleosome, which accommodated the DNA ends. Mutations in the H2A.J-specific residues did not affect the nucleosome stability, although the mutation of the H2A.J Ala40 residue, which is conserved in some members of the canonical H2A class, reduced the nucleosome stability. Consistently, the crystal structure of the H2A.J nucleosome revealed that the H2A.J-specific residues, including the Ala40 residue, did not affect the nucleosome structure. These results provide basic information for understanding the function of the H2A.J nucleosome.

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Kinetic and solvent isotope effects in oxidation of halogen derivatives of tyramine catalyzed by monoamine oxidase A (2019)

Pająk, Małgorzata

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 49-54. Published 2019 Oct 24. doi: 10.1093/jb/mvz089.

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Publication Date: 2019-10-24

Description: The isotope effects approach was used to elucidate the mechanism of oxidative deamination of 3′-halotyramines, catalyzed by monoamine oxidase A (EC 1.4.3.4). The numerical values of kinetic isotope effect (KIE) and solvent isotope effect (SIE) were established using a non-competitive spectrophotometric technique. Based upon KIE and SIE values, some of the mechanistic details of investigated reaction were discussed.

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Improving the catalytic efficiency of thermostable Geobacillus stearothermophilus xylanase XT6 by single-amino acid substitution (2019)

Azouz, Rasha A M ; Hegazy, Usama M ; Said, Mahmoud M ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(2): 203-215. Published 2019 Oct 16. doi: 10.1093/jb/mvz086.

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Publication Date: 2019-10-16

Description: Directed evolution using error-prone polymerase chain reaction was employed in the current study to enhance the catalytic efficiency of a thermostable Geobacillus stearothermophilus xylanase XT6 parent. High-throughput screening identified two variants with enhanced activity. Sequencing analysis revealed the presence of a single-amino acid substitution (P209L or V161L) in each variant. The maximum activity of mutant V161L and P209L was at 85°C and 70°C, respectively. Both mutants exhibited maximum activity at pH 7. The thermal and alkaline tolerance of mutant V161L only were markedly improved. The two mutants were more resistant to ethanol inhibition than the parent. Substrate specificity of the two mutants was shifted from beechwood xylan to birchwood xylan. The potential of the two mutants to hydrolyze rice straw and sugarcane bagasse increased. Both turnover number (kcat) and catalytic efficiency (kcat/kM) increased 12.2- and 5.7-folds for variant P209L and 13- and 6.5-folds for variant V161L, respectively, towards birchwood xylan. Based on the previously published crystal structure of extracellular G. stearothermophilus xylanase XT6, V161L and P209L mutation locate on βα-loops. Conformational changes of the respective loops could potentiate the loop swinging, product release and consequently result in enhancement of the catalytic performance.

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Electronic ISSN: 1756-2651

Topics: Biology , Chemistry and Pharmacology

Published by Oxford University Press on behalf of Japanese Biochemical Society.

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Interleukin-6 confers radio-resistance by inducing Akt mediated glycolysis and reducing mitochondrial damage in cells (2019)

Kumari, Neeraj ; Das, Asmita ; Bhatt, Anant Narayan

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Oct 31. doi: 10.1093/jb/mvz091. [early online release]

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Publication Date: 2019-10-31

Description: Interleukin-6 (IL-6) induced glycolysis and therapeutic resistance is reported in some cell systems; however the mechanism of IL-6 induced glycolysis in radio-resistance is un-explored. Therefore to investigate, we treated Raw264.7 cells with IL-6 (1hr prior to irradiation) and examined the glycolytic flux. Increased expression of mRNA and protein levels of key glycolytic enzymes was observed after IL-6 treatment, which conferred glycolysis dependent resistance from radiation induced cell death. We further established that IL-6 induced glycolysis is activated by Akt signaling and knocking down Akt or inhibition of pan Akt phosphorylation significantly abrogated the IL-6 induced radio-resistance. Moreover, reduction of IL-6 induced pAkt level suppressed the expression of Hexokinase-2 and its translocation to the mitochondria, thereby inhibiting the glycolysis induced resistance to radiation. IL-6 induced glycolysis also minimized the radiation induced mitochondrial damage. These results suggest that IL-6 induced glycolysis observed in cells may be responsible for IL-6 mediated therapeutic radio-resistance in cancer cells, partly by activation of Akt signaling.

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Topics: Biology , Chemistry and Pharmacology

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iTRAQ-based quantitative proteomic analysis of two transgenic soybean lines and the corresponding non-genetically modified isogenic variety (2019)

Liu, Weixiao ; Zhang, Zhe ; Liu, Xuri ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(1): 67-78. Published 2019 Oct 09. doi: 10.1093/jb/mvz081.

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Publication Date: 2019-10-09

Description: To investigate the unintended effects of genetically modified (GM) crops, an isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic analysis was performed with seed cotyledons of two GM soybean lines, MON87705 and MON87701×MON89788, and the corresponding non-transgenic isogenic variety A3525. Thirty-five differentially abundant proteins (DAPs) were identified in MON87705/A3525, 27 of which were upregulated and 8 downregulated. Thirty-eight DAPs were identified from the MON87701×MON89788/A3525 sample, including 29 upregulated proteins and 9 downregulated proteins. Pathway analysis showed that most of these DAPs participate in protein processing in endoplasmic reticulum and in metabolic pathways. Protein–protein interaction analysis of these DAPs demonstrated that the main interacting proteins are associated with post-translational modification, protein turnover, chaperones and signal transduction mechanisms. Nevertheless, these DAPs were not identified as new unintended toxins or allergens and only showed changes in abundance. All these results suggest that the seed cotyledon proteomic profiles of the two GM soybean lines studied were not dramatically altered compared with that of their natural isogenic control.

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The JB Award (2019)

Oxford University Press

In: Journal of Biochemistry. 2019; 166(6): 453-453. Published 2019 Nov 28. doi: 10.1093/jb/mvz095.

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Publication Date: 2019-11-28

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Topics: Biology , Chemistry and Pharmacology

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Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity (2019)

Feng, An-Ning ; Huang, Chih-Wei ; Lin, Chi-Huei ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; 167(3): 315-322. Published 2019 Nov 13. doi: 10.1093/jb/mvz092.

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Publication Date: 2019-11-13

Description: 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

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Hypermetabolism of glutathione, glutamate and ornithine via redox imbalance in methylglyoxal-induced peritoneal injury rats (2019)

Hirahara, Ichiro ; Kusano, Eiji ; Jin, Denan ; [et al.]

Oxford University Press

In: Journal of Biochemistry. 2019; Published 2019 Oct 08. doi: 10.1093/jb/mvz077. [early online release]

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Publication Date: 2019-10-08

Description: Peritoneal dialysis (PD) is a blood purification treatment for patients with reduced renal function. However, the peritoneum is exposed to oxidative stress during PD and long-term PD results in peritoneal damage, leading to the termination of PD. Methylglyoxal (MGO) contained in commercial PD fluids is a source of strong oxidative stress. The aim of this study was to clarify the mechanism of MGO-induced peritoneal injury using metabolome analysis in rats. We prepared peritoneal fibrosis rats by intraperitoneal administration of PD fluids containing MGO for 21 days. As a result, MGO-induced excessive proliferation of mesenchymal cells with an accumulation of advanced glycation end-products (AGEs) at the surface of the thickened peritoneum in rats. The effluent levels of methionine sulfoxide, an oxidative stress marker and glutathione peroxidase activity were increased in the MGO-treated rats. The levels of glutathione, glutamate, aspartate, ornithine and AGEs were also increased in these rats. MGO upregulated the gene expression of transporters and enzymes related to the metabolism of glutathione, glutamate and ornithine in the peritoneum. These results suggest that MGO may induce peritoneal injury with mesenchymal cell proliferation via increased redox metabolism, directly or through the formation of AGEs during PD.

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