ALBERT

Description: Available online 29 December 2012 Publication year: 2012 Source: FEBS Open Bio A water-soluble selenoxide (DHS ox ) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHS ox would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHS ox was extensively studied at pH 4–10 and 25°C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHS ox (375 mV at 25°C) compared to L-cystine supported the high ability of DHS ox for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the p K a value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTT red ) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains. Graphical abstract Highlights ▸ DHS ox was applied as a SS-forming reagent for five polythiol peptides and DTT red . ▸ The SS formation velocities depend on the kind of substrate and solvent conditions. ▸ The SS-formation rate constants are proportional to the number of free SH groups. ▸ Folded structures and SH p K a modify the stochastic nature and absolute values of the rate constants. ▸ DHS ox is a useful probe of chemical reactivity and intermediate structures in oxidative protein folding.

Description: Available online 19 December 2012 Publication year: 2012 Source: FEBS Open Bio The green fluorescent protein (GFP) is the most commonly used reporter protein for monitoring gene expression and protein localization in a variety of living and fixed cells, including not only prokaryotes, but also eukaryotes, e.g., yeasts, mammals, plants and fish. In general, it is thought that GFP is nontoxic to cells, although there are some reports on the side effect of GFP. However, details of the molecular mechanism concerning the side effect of GFP remain unclear. Here we show that Ku80, but not XRCC4, plays an important role in the mechanism of the resistance to cytotoxicity induced by enhanced GFP (EGFP). EGFP inhibited both cell proliferation and colony formation, and induced cell death in Ku80-deficient hamster cells, i.e., xrs-6 cells. In addition, Ku80 attenuated EGFP-induced cytotoxicity in the xrs-6 cells. No EGFP-induced cytotoxicity was observed in the NHEJ core protein XRCC4-deficient hamster cells, i.e., XR-1 cells. Furthermore, EGFP markedly enhanced X-ray-induced cytotoxicity in the xrs-6 cells. These results suggest that Ku80 plays a key role in the novel NHEJ-independent defense mechanism against EGFP-induced cytotoxicity. Caution should be taken in considering of the potential influence by the stress response mechanism, namely, the Ku80-dependent elimination mechanism of EGFP-induced cytotoxicity, being activated, even when using EGFP-expressing cells in which Ku80 functions normally.

Description: Publication year: 2012 Source: FEBS Open Bio Yusuke Ito, Suguru Shigemori, Takashi Sato, Tomoyuki Shimazu, Konomi Hatano, Hajime Otani, Haruki Kitazawa, Takeshi Shimosato We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′-TTAGGG-3′, which has a six-base loop head structure, and a 3′-oligo (dG) 3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)-immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)-6, IL-12p35, IL-12p40, and interferon (IFN) γ mRNA expression, but also IL-4 and IL-13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen-immunized mice, suggesting usefulness in the treatment of diseases induced by over-active immune activation.

Description: Publication year: 2012 Source: FEBS Open Bio Katsuhiro Tanaka, Maiko Soeda, Yoichiro Hashimoto, Shigeo Takenaka, Masayuki Komori Pex14p is a peroxisomal membrane protein that is involved in both peroxisome biogenesis and selective peroxisome degradation. Previously, we showed that Hansenula polymorpha Pex14p was phosphorylated in vivo . In this study, we identified its phosphorylation site by mass spectrometry. Recombinant His-tagged Pex14p (H6-Pex14p) was overexpressed and purified from the yeast. The protein band corresponding to H6-Pex14p was in-gel digested with trypsin and subjected to LC/MS. As a result of LC/MS, Thr 248 and Ser 258 were identified as the phosphorylated sites. To confirm the phosphorylation sites and explore its functions, we made Ala mutants of the candidate amino acids. In the western blot analysis with anti-Pex14p, S258A mutant gave doublet bands while wild type (WT) and T248A mutants gave triplet bands. Moreover, the double mutant (T248A/S258A) gave a single band. WT and all mutant Pex14p labeled with [ 32 P] orthophosphate were immunoprecipitated and analyzed by autoradiography. The phosphorylation of Pex14p was suppressed in S258A mutant, but enhanced in T248A mutant compared to WT. Moreover, the phosphorylated Pex14p was not detected in the T248A/S258A double mutant. All mutants were able to grow on methanol and their matrix proteins (alcohol oxidase and amine oxidase) were mostly localized in peroxisomes. Furthermore all mutants showed selective degradation of peroxisome like WT during the glucose-induced macropexophagy.

Description: Publication year: 2012 Source: FEBS Open Bio Miki Hieda, Michiko Koizumi, Chiduru Higashi, Taro Tachibana, Tomohiko Taguchi, Shigeki Higashiyama Heparin-binding epidermal growth factor (EGF) – like growth factor (HB-EGF) is synthesized in the ER, transported along the exocytic pathway, and expressed on the plasma membrane as a type I transmembrane protein. Upon extracellular stimulation, HB-EGF, either proHB-EGF or the shed form HB-EGF-CTF, undergoes endocytosis and is then transported retrogradely to the ER. In this study, we showed the essential contribution of the short cytoplasmic tail of HB-EGF (HB-EGF-cyto) to the bidirectional intracellular trafficking between the ER and plasma membrane and revealed several critical amino acids residues that are responsible for internalization from the plasma membrane and ER targeting. We suggest that these anterograde and retrograde sorting signals within HB-EGF-cyto are strictly regulated by protein modification and conformation. Highlights ▸ The cytoplasmic tail of HB-EGF contributes to bidirectional intracellular trafficking. ▸ The carboxyl-terminal five amino acids are indispensable for internalization. ▸ Amino acids S207 and K201 play essential roles in retrograde transport to the ER. ▸ Sorting signals in HB-EGF are regulated by protein modification and conformation. ▸ The extracellular domain of proHB-EGF is not required for intracellular trafficking.

Description: Publication year: 2012 Source: FEBS Open Bio Niege S. Mendes, Glauce L. Trevisan, Aline H.Silva Cruz, Rodrigo S. Santos, Nalu T.A. Peres, Nilce M. Martinez-Rossi, Antonio Rossi In fungi, ambient pH sensing involves the activation of the Pal/PacC signalling pathway. In the dermatophyte Trichophyton rubrum , pH-dependent secretion of keratinases, which are major virulence determinants, is affected by disruption of the pacC gene. Here, the transcription profiling of the genes coding for N- and O-linked mannosyltransferases, enzymes involved in protein glycosylation, was evaluated in T. rubrum in response to disruption of the pacC gene and growth in keratin, glucose, and glucose plus glycine. We show that transcription of these mannosyltransferase genes is affected by nutrients at acidic pH and by PacC.

Description: Publication year: 2012 Source: FEBS Open Bio Daisuke Sugimori, Kota Kano, Yusaku Matsumoto A novel metal ion-independent phospholipase A 1 of Streptpmyces albidoflavus isolated from Japanese soil has been purified and characterized. The enzyme consists of a 33-residue N-terminal signal secretion sequence and a 269-residue mature protein with a deduced molecular weight of 27,199. Efficient and extracellular production of the recombinant enzyme was successfully achieved using S. lividans cells and an expression vector. A large amount (25 mg protein, 14.7 kU) of recombinant enzyme with high specific activity (588 U/mg protein) was purified by simple purification steps. The maximum activity was found at pH 7.2 and 50°C. At pH 7.2, the enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine; however, the substrate specificity was dependent on the reaction pH. The enzyme hydrolyzed lysophosphatidylcholine and not triglyceride and the p -nitrophenyl ester of fatty acids. At the reaction equilibrium, the molar ratio of released free fatty acids ( sn -1: sn -2) was 65:35. The hydrolysis of phosphatidic acid at 50°C and pH 7.2 gave apparent V max and k cat values of 1389 μmol min −1 mg protein −1 and 630 s −1 , respectively. The apparent K m and k cat / K m values were 2.38 mM and 265 mM −1 s −1 , respectively. Mutagenesis analysis showed that Ser11 is essential for the catalytic function of the enzyme and the active site may include residues Ser216 and His218. Highlights ▸ A novel metal ion-independent phospholipase A 1 has been purified and characterized. ▸ We report the positional specific hydrolysis of glycerophospholipids. ▸ The enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine. ▸ The apparent K m , k cat , and K cat / K m values were 2.38 mM, 630 s −1 and 265 mM −1 s −1 . ▸ We demonstrate that the active site should be composed of Ser11, S216, and H218.

Description: Publication year: 2012 Source: FEBS Open Bio James A.L. Brown, John K. Eykelenboom, Noel F. Lowndes Under normal conditions histone H2AX is constitutively phosphorylated on tyrosine (Y) 142 by Williams-Beuren syndrome transcription factor kinase (WSTF). Following DNA double strand breaks (DSB), Y142 is de-phosphorylated and serine (S) 139 is phosphorylated. Here we explored DSB-dependent cross talk between H2AX residues S139 and Y142. H2axY142A mutation resulted in increased sensitivity to ionizing radiation (IR), compared to H2axS139A . Interestingly, co-mutation of S139A and Y142A rescued IR sensitivity. The DSB response proteins 53Bp1 and Rad51 were recruited to IR-induced foci (IRIF) in H2axS139A , H2axY142A and H2axS139A / Y142A cells. Our results suggest that H2axY142A IR sensitivity is dependent upon the C-terminal residue, S139.

Description: Publication year: 2012 Source: FEBS Open Bio Ambuj Kumar, Vidya Rajendran, Rao Sethumadhavan, R. Purohit Human STIL (SCL/TAL1 interrupting locus) protein maintains centriole stability and spindle pole localization. It helps in recruitment of CENPJ (Centromere protein J)/CPAP (centrosomal P4.1-associated protein) and other centrosomal proteins. Mutations in STIL protein are reported in several disorders, especially in deregulation of cell cycle cascades. In this work, we examined the non-synonymous single nucleotide polymorphisms (nsSNPs) reported in STIL protein for their disease association. Different SNP prediction tools were used to predict disease-associated nsSNPs. Our evaluation technique predicted rs147744459 (R242C) as a highly deleterious disease-associated nsSNP and its interaction behavior with CENPJ protein. Molecular modelling, docking and molecular dynamics simulation were conducted to examine the structural consequences of the predicted disease-associated mutation. By molecular dynamic simulation we observed structural consequences of R242C mutation which affects interaction of STIL and CENPJ functional domains. The result obtained in this study will provide a biophysical insight into future investigations of pathological nsSNPs using a computational platform.

Description: Publication year: 2012 Source: FEBS Open Bio Meng-Chun Chi, Yi-Yu Chen, Huei-Fen Lo, Long-Liu Lin The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase ( Bl GGT) was explored by site-directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal-affinity chromatography. SDS–PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only able to process itself, but also had a catalytic activity comparable to that of Bl GGT. As compared with the wild-type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of Bl GGT, E398A, E398D, E398K and E398Q followed the two-state unfolding process with a transition point ( T m ) of 47.7–69.4°C. Tryptophan fluorescence spectra of the mutant enzymes were different from the wild-type protein in terms of fluorescence intensity. Native Bl GGT started to unfold beyond ∼ 1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl] 0.5, N-U , at 3.07 M equivalent to free energy change ( Δ G N − U H 2 O ) of 14.53 kcal/mol for the N → U process, whereas the denaturation midpoints for the mutant enzymes were 1.31–2.99 M equivalent to Δ G N − U H 2 O of 3.29–12.05 kcal/mol. Taken together, these results strongly suggest that the explored glutamate residue is indeed important for the autocatalytic processing of Bl GGT. Highlights ▸ Bioinformatics was used to identify a key residue required for autocatalytic processing. ▸ Substitution of Glu398 with Ala, Asp or Lys blocked maturation of the enzyme. ▸ Wild-type and mutant enzymes exhibited different sensitivities towards thermal and GdnHCl-induced denaturation.

Description: Publication year: 2012 Source: FEBS Open Bio Masaharu Hazawa, Takeshi Yasuda, Katsuko Noshiro, Ai Saotome-Nakamura, Tomoko Fukuzaki, Yuichi Michikawa, Takaya Gotoh, Katsushi Tajima Vitronectin (VN) is a multi-functional protein involved in extracellular matrix (ECM)-cell binding through integrin receptors on the cell surface, which is an important environmental process for maintaining biological homeostasis. We investigated how VN affects the survival of endothelial cells after radiation damage. VN attenuated radiation-induced expression of p21, an inhibitor of cell cycle progression, and selectively inhibited Erk- and p38 MAPK-dependent p21 induction after radiation exposure through regulation of the activity of GSK-3β. VN also reduced the cleavage of caspase-3, thereby inhibiting radiation-induced apoptotic cell death. These results suggest that VN has important roles in cell survival after radiation damage.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Antimalarial chloroquine is also used for the treatment of immune-mediated diseases. The interference of chloroquine with interferon-γ-induced tryptophan breakdown and neopterin production has been investigated in human peripheral blood mononuclear cells (PBMC) in vitro . Micromolar concentrations (2–50 μM) of chloroquine dose-dependently suppressed mitogen-induced tryptophan breakdown in PBMC but not in the myelomonocytic THP-1-Blue cell line, after 48 h of treatment. In stimulated PBMC, neopterin production was super-induced by 10 μM chloroquine, while it was significantly suppressed at a concentration of 50 μM. These anti-inflammatory effects may relate to the therapeutic benefit of chloroquine in inflammatory conditions and may widen the spectrum of its clinical applications. Highlights ▸ 2–50 μM chloroquine suppresses mitogen-induced tryptophan breakdown in human PBMCs. ▸ The same effect was not seen in the myelomonocytic THP-1-Blue cell line. ▸ The anti-inflammatory property of chloroquine targets T cells more than monocytes. ▸ This anti-inflammatory effect may explain the drug's therapeutic benefit for malaria. ▸ Chloroquine treatment could be of benefit for other chronic inflammatory conditions.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Tespa1 has been recently reported to be a critical molecule in T-cell development, however, the precise molecular mechanisms of Tespa1 remain elusive. Here, we demonstrate that Tespa1 shows amino-acid sequence homology to KRAS -induced actin-interacting protein (KRAP), an inositol 1,4,5-trisphosphate receptor (IP 3 R) binding protein, and that Tespa1 physically associates with IP 3 R in T and B lymphocytes. Two-consecutive phenylalanine residues (Phe185/Phe186) in Tespa1, which are conserved between Tespa1 and KRAP, are indispensable for the association between Tespa1 and IP 3 R. These findings suggest that Tespa1 plays critical roles in the immune system through the regulation of the IP 3 R. Highlights ▸ We identified Tespa1 as a novel IP 3 R-associated protein in lymphoid tissues. ▸ Tespa1 interacts with multiple subtypes of IP 3 R in T and B lymphocytes. ▸ Amino-terminal region of IP 3 R is sufficient for the association with Tespa1. ▸ Two consecutive phenylalanines in Tespa1 are critical for the interaction with IP 3 R. ▸ Tespa1 is structurally and functionally related to KRAP.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Human STIL (SCL/TAL1 interrupting locus) protein maintains centriole stability and spindle pole localisation. It helps in recruitment of CENPJ (Centromere protein J)/CPAP (centrosomal P4.1-associated protein) and other centrosomal proteins. Mutations in STIL protein are reported in several disorders, especially in deregulation of cell cycle cascades. In this work, we examined the non-synonymous single nucleotide polymorphisms (nsSNPs) reported in STIL protein for their disease association. Different SNP prediction tools were used to predict disease-associated nsSNPs. Our evaluation technique predicted rs147744459 (R242C) as a highly deleterious disease-associated nsSNP and its interaction behaviour with CENPJ protein. Molecular modelling, docking and molecular dynamics simulation were conducted to examine the structural consequences of the predicted disease-associated mutation. By molecular dynamic simulation we observed structural consequences of R242C mutation which affects interaction of STIL and CENPJ functional domains. The result obtained in this study will provide a biophysical insight into future investigations of pathological nsSNPs using a computational platform.

Description: Publication year: 2012 Source: FEBS Open Bio Tadanobu Takahashi, Chairul A. Nidom, Mai thi QuynhLe, Takashi Suzuki, Yoshihiro Kawaoka Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans. Highlights ▸ All neuraminidases (NAs) of avian influenza A viruses (IAVs) are stable at low pH. ▸ Human 1918, 1957, and 1968 pandemic IAVs also have NAs that are stable at low pH. ▸ Most human epidemic IAVs have NAs that are unstable at low pH. ▸ The NAs of most H5N1 IAVs isolated since 2003 are unstable at low pH. ▸ Instability of H5N1 IAV NAs at low pH might explain frequent human infections.

Description: Publication year: 2012 Source: FEBS Open Bio Hiroshi Matsuzaki, Takahiro Fujimoto, Takeharu Ota, Masahiro Ogawa, Toshiyuki Tsunoda, Keiko Doi, Masato Hamabashiri, Masatoshi Tanaka, Senji Shirasawa Tespa1 has been recently reported to be a critical molecule in T-cell development, however, the precise molecular mechanisms of Tespa1 remain elusive. Here, we demonstrate that Tespa1 shows amino-acid sequence homology to KRAS -induced actin-interacting protein (KRAP), an inositol 1,4,5-trisphosphate receptor (IP 3 R) binding protein, and that Tespa1 physically associates with IP 3 R in T and B lymphocytes. Two-consecutive phenylalanine residues (Phe185/Phe186) in Tespa1, which are conserved between Tespa1 and KRAP, are indispensable for the association between Tespa1 and IP 3 R. These findings suggest that Tespa1 plays critical roles in the immune system through the regulation of the IP 3 R. Highlights ▸ We identified Tespa1 as a novel IP 3 R-associated protein in lymphoid tissues. ▸ Tespa1 interacts with multiple subtypes of IP 3 R in T and B lymphocytes. ▸ Amino-terminal region of IP 3 R is sufficient for the association with Tespa1. ▸ Two consecutive phenylalanines in Tespa1 are critical for the interaction with IP 3 R. ▸ Tespa1 is structurally and functionally related to KRAP.

Description: Publication year: 2012 Source: FEBS Open Bio Satoru Mizuno, Hiromichi Sakai, Masafumi Saito, Sayaka Kado, Fumio Sakane Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL-2 murine T-lymphocytes by interleukin-2 (IL-2) induced a significant increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1-, 40:5-, 40:6-diacyl-PA, suggesting that DGK IL-2-dependently and selectively generated these diacyl-PA species.

Description: Publication year: 2012 Source: FEBS Open Bio Nada El-Ekiaby, Nabila Hamdi, Mohamed Negm, Rasha Ahmed, Abdel Rahman Zekri, Gamal Esmat, Ahmed Ihab Abdelaziz MicroRNAs regulate the expression of many genes and subsequently control various cellular processes, such as the immune response to viral infections mediated by type I interferon (IFN). In this study, the expression pattern of two interferon-related microRNAs, miR-146a and miR-155, was examined in healthy and HCV-genotype-4-infected peripheral blood mononuclear cells (PBMCs) using qRT-PCR. In contrast to other viral infections, the expression pattern was similar in both healthly and infected PBMCs. This could be attributed to attenuation of IFN pathway by HCV, which was assessed by investigating the expression of MxA, an interferon-stimulated gene, that showed lower expression in HCV-infected PBMCs. To determine the site of interference of HCV in the IFN pathway, expression of both microRNAs was examined following stimulation of PBMCs with IFN-α2a, an activator of the JAK/STAT pathway as well as with imiquimod, a toll-like receptor-7 (TLR-7) agonist that promotes interferon release. IFN stimulation induced the expression of mir-146a and mir-155 in HCV-infected and healthy PBMCs. Stimulation with imiquimod led to a down-regulation of both microRNAs in infected PBMCs, while it increased their expression in healthy PBMCs, indicating that HCV might interfere with miR-146a and miR-155 expression at sites upstream of interferon release, specifically in the TLR-7 pathway. The pattern of expression of both miR-146a and miR-155 was very similar with a strong positive correlation, but showed no correlation to the patients’ clinical, or histopathological parameters or response to treatment. In conclusion, HCV infection might repress the induction of miR-146a and miR-155 by interfering with TLR-7 signaling.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Yutaro Kobayashi, Toshihiko Fukuda, Mitsuru Tanaka, Toshiro Matsui Trp-His is the only vasoactive di-peptide known to regulate intracellular Ca 2+ ([Ca 2+ ] i ) and prevent the onset of atherosclerosis in mice. In this study, we showed that Trp-His reduced the [Ca 2+ ] i elevation in phospholipase C-activated vascular smooth muscle cells (VSMCs), while a mixture of the corresponding constituent amino acids did not show significant reduction. Furthermore, Trp-His suppressed calmodulin-dependent kinase II (CaMK II) activity in angiotensin II-stimulated VSMCs, resulting in the inhibition of phosphorylation of voltage-dependent L-type Ca 2+ channels (VDCC). Therefore, Trp-His potentially regulates the VDCC phosphorylation cascade through Ca 2+ -CaM/CaMK II. Highlights ► Trp-His is the only vasoactive di-peptide known to prevent the onset of atherosclerosis in mice. ► The bioactivity of Trp-His was examined in angiotensin II-stimulated vascular smooth muscle cells. ► Trp-His inhibited CaMK II activity and the phosphorylation of voltage-dependent L-type Ca 2+ channels. ► Trp-His potentially regulates the VDCC phosphorylation cascade through Ca 2+ -CaM/CaMK II.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Sadanori Miyoshi, Shota Yamazaki, Asato Uchiumi, Yohtaro Katagata Hsp90 is essential for maintaining the activity of numerous signaling factors, and plays a key role in cellular signal transduction networks. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an ansamycin antibiotic that binds to Hsp90 and inhibits its function. HaCaT human keratinocytes were used to investigate the cellular and molecular functions of Hsp90 in keratinocyte differentiation. Inhibition of Hsp90 by 17-AAG leads to downregulation of the differentiation markers cytokeratin 1 and cytokeratin 10 at the protein and mRNA levels. Highlights ► 17-Allylamino-17-demethoxygeldanamycin (17-AAG) binds to Hsp90 and inhibits its function. ► Inhibition of Hsp90 downregulates differentiation marker cytokeratin 1 and cytokeratin 10. ► Phospho-p38 was upregulated by 17-AAG in a concentration-dependent manner. ► Involucrin expression was increased by upregulation of p38 MAPK phosphorylation. ► Inhibition of Hsp90 represses cell cycle arrest.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Zeng Wang, Wei Hu, Jia-Li Zhang, Xiu-Hua Wu, Hui-Jun Zhou Dihydroartemisinin (DHA), an active metabolite of artemisinin derivatives, is the most remarkable anti-malarial drug and has little toxicity to humans. Recent studies have shown that DHA effectively inhibits the growth of cancer cells. In the present study, we intended to elucidate the mechanisms underlying the inhibition of growth of iron-loaded human myeloid leukemia K562 cells by DHA. Mitochondria are important regulators of both autophagy and apoptosis, and one of the triggers for mitochondrial dysfunction is the generation of reactive oxygen species (ROS). We found that the DHA-induced autophagy of leukemia K562 cells, whose intracellular organelles are primarily mitochondria, was ROS dependent. The autophagy of these cells was followed by LC3-II protein expression and caspase-3 activation. In addition, we demonstrated that inhibition of the proliferation of leukemia K562 cells by DHA is also dependent upon iron. This inhibition includes the down-regulation of TfR expression and the induction of K562 cell growth arrest in the G 2 /M phase. Highlights ► DHA induced K562 cells autophagy followed by LC3-II protein expression. ► The DHA-induced autophagy might be ROS dependent. ► Inhibition of K562 cell proliferation by DHA was also dependent upon iron decrease.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Chad E.N. Reiter, Abdu I. Alayash We examined carbon monoxide (CO) delivery by carbon monoxide-releasing molecule 2 (CORM-2) or hemoglobin (Hb) on cellular oxygen sensing and mitochondrial respiration in bovine aortic endothelial cells (BAECs). CORM-2 reduced hypoxia-inducible factor-1α (HIF-1α) and endothelin-1 (ET-1) expression in normoxic and hypoxic cells, but while Hb alone significantly reduced HIF-1α stabilization in hypoxic cells, CO delivered by Hb (Hb-CO) had no effect on HIF-1α stabilization. CO dose-dependently increased basal oxygen consumption and reduced overall mitochondrial respiratory capacity. Hb-CO increased basal oxygen consumption but did not alter respiratory capacity. Together, CO reduced ET-1, and, at low doses, had no effect on endothelial mitochondria oxygen consumption. CO ligation to Hb may be developed further as non-vasoactive oxygen therapeutic without compromising mitochondrial function. Highlights ► CO delivered by CORM-2 reduces HIF-1α stabilization and expression of ET-1. ► CORM-2 dose-dependently disrupts mitochondrial function. ► CO ligated to Hb does not adversely affect mitochondrial function. ► CO ligated to Hb delivers less oxygen to target tissues than unligated Hb. ► This work provides new insight for CO therapeutics and effects on mitochondria.

Description: Publication year: 2012 Source: FEBS Open Bio Charlotte L. Hooper, Philip R. Dash, Samuel Y. Boateng Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats ( p 〈 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes ( P 〈 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions ( P 〈 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load. Highlights ► Lipoma preferred partner (LPP) is highly expressed in the heart, especially in cardiac fibroblasts. ► LPP increases in pressure overload hypertrophy but not in myocardial infarction. ► LPP may be used as a novel marker for heart failure due to hemodynamic overload. ► Inhibition of nitric oxide upregulates LPP, but not other fibroblast proteins. ► LPP may play an important role in cardiac homeostasis and heart disease.

Description: Publication year: 2012 Source: FEBS Open Bio Mariana Freitas, Inês Baldeiras, Teresa Proença, Vera Alves, Anabela Mota-Pinto, Ana Sarmento-Ribeiro Oxidative stress has been associated with prostate cancer development and progression due to an increase of reactive oxygen species (ROS). However, the mechanisms whereby ROS and the antioxidant system participate in cancer progression remain unclear. In order to clarify the influence of oxidative stress in prostate cancer progression, we performed this study in two human prostate cancer cell lines, PC3 and HPV10 (from metastasis and from cancer in situ, respectively) and RWPE1 cells derived from normal prostate epithelium. Cells were treated with hydrogen peroxide (H 2 O 2 ) and PC3 cells were also treated with diethyl maleate (DEM). The effect on cell growth, viability, mitochondria membrane potential and oxidative stress was analysed. Oxidative stress was evaluated based on ROS production, oxidative lesion of lipids (MDA) and on determination of antioxidants, including enzyme activity of glutathione peroxidase (Gl-Px), glutathione reductase (Gl-Red) and on the quantification of glutathione (GSH), glutathione-s-transferase (GST) and total antioxidant status (TAS). PC3 shows higher ROS production but also the highest GSH levels and Gl-Red activity, possibly contributing to oxidative stress resistance. This is also associated with higher mitochondrial membrane potential, TAS and lower lipid peroxidation. On the other hand, we identified Gl-Red activity reduction as a new strategy in overcoming oxidative stress resistance, by inducing H 2 O 2 cytotoxicity. Therefore these results suggest Gl-Red activity reduction as a new potential therapeutic approach, in prostate cancer. Highlights ► Oxidative stress was evaluated on a cell line model of prostate cancer progression. ► Metastatic cell lines show the highest ROS, total antioxidant status and resistance to H 2 O 2 . ► Metastatic cell lines show the highest levels of GSH levels and Gl-Red activity. ► Decrease in GSH levels and Gl-Red activity induced a decrease in H 2 O 2 resistance. ► Gl-Red activity reduction may be a new therapeutic approach in prostate cancer.

Description: Publication year: 2011 Source: FEBS Open Bio, Volume 1 Małgorzata Pietrowska-Borek, Katarzyna Nuc, Małgorzata Zielezińska, Andrzej Guranowski It is known that cells under stress accumulate various dinucleoside polyphosphates, compounds suggested to function as alarmones. In plants, the phenylpropanoid pathways yield metabolites protecting these organisms against various types of stress. Observations reported in this communication link these two phenomena and provide an example of a metabolic “addressee” for an “alarm” signaled by diadenosine triphosphate (Ap 3 A) or diadenosine tetraphosphate (Ap 4 A). In response to added Ap 3 A or Ap 4 A, seedlings of Arabidopsis thaliana incubated in full nutrition medium increased both the expression of the genes for and the specific activity of phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase, enzymes that control the beginning of the phenylpropanoid pathway. Neither adenine mononucleotides (AMP, ADP or ATP) nor adenosine evoked such effects. Reactions catalyzed in vitro by these enzymes were not affected by Ap 3 A or Ap 4 A.

Description: Publication year: 2012 Source: FEBS Open Bio Michael Gamble, Georg Künze, Andrea Brancale, Keith S. Wilson, D.Dafydd Jone The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100 fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca 2+ , are proposed to be important for ISP activity through structural changes. The presence of 〉0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca 2+ at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions.

Description: Publication year: 2012 Source: FEBS Open Bio Siân Lax, Ewan A. Ross, Andrea White, Jennifer L. Marshall, William E. Jenkinson, Clare M. Isacke, David L. Huso, Adam F. Cunningham, Graham Anderson, Christopher D. Buckley The role of mesenschymal stromal cells (MSCs) in regulating immune responses in the thymus is currently unclear. Here we report the existence and role of a MSC population in the thymus that expresses the pericyte and MSC marker CD248 (endosialin). We show using a CD248 deficient mouse model, that CD248 expression on these cells is required for full post-natal thymus development and regeneration post- Salmonella infection. In CD248 −/− mice the thymus is hypocellular and regeneration is poorer, with significant loss of all thymocyte populations. This identifies the requirement of CD248 to maintain optimal thymic cellularity post-partum and infection. Highlights ▸ The mesenchymal stromal cell marker CD248 is expressed in the thymus on pericytes. ▸ Genetic deletion of CD248 prevents full post-natal development of the thymus. ▸ Genetic deletion of CD248 prevents infection-dependent regeneration following thymic atrophy. ▸ CD248 regulates blood endothelial vessel formation in the thymus.

Description: Publication year: 2012 Source: FEBS Open Bio Daigo Tsubokawa, Yukinobu Goso, Rei Kawashima, Hiroyoshi Ota, Takeshi Nakamura, Kazuo Nakamura, Noriko Sato, Makoto Kurihara, Taeko Dohi, Yuki I. Kawamura, Takafumi Ichikawa, Kazuhiko Ishihara Rat small intestinal goblet cell mucins reacting with monoclonal antibody HCM31 increase significantly during regeneration from experimental mucosal damage and at the period of expulsion of parasitic nematode, Nippostrongylus brasiliensis ( N.b ). The reduction in reactivity of HCM31 with mucin upon neuraminidase treatment, suggested that HCM31 recognizes sialylated oligosaccharide on mucin. HCM31-reactive sialomucins are therefore considered to play an important role in the physiological and pathological changes in the gastrointestinal mucosa. To determine the epitope for HCM31, oligosaccharide-alditols reacted with HCM31 were obtained from the small intestinal mucins of N.b -infected rats and purified by ion-exchange chromatography followed by normal-phase HPLC. Two HCM31-reactive oligosaccharide-alditols were obtained. Analyses using tandem mass spectrometry and NMR spectroscopy showed that these oligosaccharides were core 4 mucin-type oligosaccharides having a common tetrasaccharide sequence, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ- (Sd a blood group antigen). These structures were not found in the small intestinal mucin oligosaccharides from uninfected rats. This epitope specificity of HCM31 was also confirmed using previously established anti-GM2 and anti-Sd a antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin-type oligosaccharides with the Sd a tetrasaccharide sequence. Immunohistochemical examination of human gastrointestinal tracts showed that HCM31 site-specifically stained the goblet cells in normal sigmoid colon and normal rectum, but the goblet cells stained with HCM31 were reduced in the corresponding cancer tissues. HCM31 seems to be useful for diagnosis of colonic cancer and for examining the function of secretory-type mucin with Sd a antigen. Graphical abstract Graphical abstract Highlights ▸ We analyzed the epitope for the monoclonal antibody HCM31. ▸ HCM31 recognizes core 4 oligosaccharides with the Sd a antigen obtained from small intestinal mucins of N. brasiliensis -infected rats. ▸ We propose that the Sd a tetrasaccharide sequence, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-, is the epitope for HCM31. ▸ Goblet cells stained with HCM31 decrease with malignant change in human colon tissues. ▸ HCM31 might be useful for the diagnosis of colonic cancer and for examining the function of mucin with Sd a tetrasaccharide.

Description: Publication year: 2012 Source: FEBS Open Bio Sedighe Karimi, Jana Wetzel, Johannes Wöstemeyer, Anke Burmester Transformation of fungi by complementation of auxotrophs is generally much more reliable than usage of antibiotic resistance markers. In order to establish such a system for the model zygomycete Absidia glauca , a stable methionine auxotrophic mutant was isolated after X-ray mutagenesis of the minus mating type and characterized at the molecular level. The mutant is disrupted in the coding region of the Met2–1 gene, encoding homoserine O-acetyltransferase. The corresponding wild type gene was cloned, sequenced and inserted into appropriate vector plasmids. Transformants are prototrophs and show restored methionine-independent growth, based on complementation by the autonomously replicating plasmids.

Description: Publication year: 2012 Source: FEBS Open Bio Shoji Ohuchi, Yusuke Mori, Yoshikazu Nakamura Aptamers are promising gene components that can be used for the construction of synthetic gene circuits. In this study, we isolated an RNA aptamer that specifically inhibits transcription of T7 RNA polymerase (RNAP). The 38-nucleotide aptamer, which was a shortened variant of an initial SELEX isolate, showed moderate inhibitory activity. By stepwise doped-SELEX, we isolated evolved variants with strong inhibitory activity. A 29-nucleotide variant of a doped-SELEX isolate showed 50% inhibitory concentration at 11 nM under typical in vitro transcription conditions. Pull-down experiments revealed that the aptamer inhibited the association of T7 RNAP with T7 promoter DNA. Highlights ▸ An inhibitory RNA aptamer against T7 RNA polymerase was isolated. ▸ Evolved aptamers with strong inhibitory activity were isolated by doped-SELEX. ▸ Aptamer sequence/structure requirements were elucidated based on phylogeny. ▸ A 29-nucleotide variant of an evolved aptamer has an IC 50 of 11 nM. ▸ The aptamer inhibits the association of T7 RNA polymerase with T7 promoter DNA.

Description: Publication year: 2012 Source: FEBS Open Bio Zhanxiang Wang, Debbie C. Thurmond Human type 2 diabetes is associated with β-cell apoptosis, and human islets from diabetic donors are ∼80% deficient in PAK1 protein. Toward addressing linkage of PAK1 to β-cell survival, PAK1-siRNA targeted MIN6 β-cells were found to exhibit increased caspase-3 cleavage, cytosolic cytochrome-C and the pro-apoptotic protein Bad. PAK1 +/− heterozygous mouse islets recapitulated the upregulation of Bad protein expression, as did hyperglycemic treatment of human or mouse islets; Bad levels were exacerbated most in PAK1+/- islets subjected to hyperglycemic stress. These data implicate PAK1 in β-cell survival via quenching of Bad protein expression, and suggest PAK1 as potential molecular target to preserve β-cell mass. Highlights ▸ Bad expression in islets was increased by chronic exposure to hyperglycemic conditions. ▸ Depletion of PAK1 in the absence of glucose induced Bad expression. ▸ Bad expression increased most in PAK1 +/− heterozygous islets subjected to hyperglycemia. ▸ PAK1 is required to protect against β-cell apoptosis under hyperglycemic conditions.

Description: Publication year: 2012 Source: FEBS Open Bio Johann M. Gostner, Sebastian Schröcksnadel, Kathrin Becker, Marcel Jenny, Harald Schennach, Florian Überall, Dietmar Fuchs Antimalarial chloroquine is also used for the treatment of immune-mediated diseases. The interference of chloroquine with interferon-γ-induced tryptophan breakdown and neopterin production has been investigated in human peripheral blood mononuclear cells (PBMC) in vitro . Micromolar concentrations (2–50 μM) of chloroquine dose-dependently suppressed mitogen-induced tryptophan breakdown in PBMC but not in the myelomonocytic THP-1 -Blue cell line, after 48 h of treatment. In stimulated PBMC, neopterin production was super-induced by 10 μM chloroquine, while it was significantly suppressed at a concentration of 50 μM. These anti-inflammatory effects may relate to the therapeutic benefit of chloroquine in inflammatory conditions and may widen the spectrum of its clinical applications. Highlights ▸ 2–50 μM chloroquine suppresses mitogen-induced tryptophan breakdown in human PBMCs. ▸ The same effect was not seen in the myelomonocytic THP-1 Blue cell line. ▸ The anti-inflammatory property of chloroquine targets T cells more than monocytes. ▸ This anti-inflammatory effect may explain the drug's therapeutic benefit for malaria. ▸ Chloroquine treatment could be of benefit for other chronic inflammatory conditions.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Nicolas Sprynski, Christine Felix, David O’Callaghan, Annette C. Vergunst Complementation for virulence of a non-polar virB5 mutant in Brucella suis 1330 was not possible using a pBBR-based plasmid but was with low copy vector pGL10. Presence of the pBBR-based replicon in wildtype B. suis had a dominant negative effect, leading to complete attenuation in J774 macrophages. This was due to pleiotropic effects on VirB protein expression due to multiple copies of the virB promoter region and over expression of VirB5. Functional complementation of mutants in individual components of multiprotein complexes such as bacterial secretion systems, are often problematic; this study highlights the importance of using a low copy vector. Highlights ► A Brucella suis virB5 mutant is attenuated in J774 macrophages. ► VirB5 over expression and/or multiple virB promoter copies attenuate WT B. suis. ► VirB5 over expression and/or multiple virB promoter copies affect other VirB proteins. ► Expressing virB5 from a low copy number plasmid fully complements a virB5 mutant.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Kenta Arai, Fumio Kumakura, Michio Iwaoka Redox-coupled folding pathways of bovine pancreatic ribonuclease A (RNase A) with four intramolecular disulfide (SS) bonds comprise three phases: (I) SS formation to generate partially oxidized intermediate ensembles with no rigid folded structure; (II) SS rearrangement from the three SS intermediate ensemble (3S) to the des intermediates having three native SS linkages; (III) final oxidation of the last native SS linkage to generate native RNase A. We previously demonstrated that DHS ox , a water-soluble selenoxide reagent for rapid and quantitative SS formation, allows clear separation of the three folding phases. In this study, the main conformational folding phase (phase II) has been extensively analyzed at pH 8.0 under a wide range of temperatures (5–45 °C), and thermodynamic and kinetic parameters for the four des intermediates were determined. The free-energy differences (Δ G ) as a function of temperature suggested that the each SS linkage has different thermodynamic and kinetic roles in stability of the native structure. On the other hand, comparison of the rate constants and the activation energies for 3S → des with those reported for the conformational folding of SS-intact RNase A suggested that unfolded des species (desU) having three native SS linkages but not yet being folded are involved in very small amounts (〈1%) in the 3S intermediate ensemble and the desU species would gain the native-like structures via X-Pro isomerization like SS-intact RNase A. It was revealed that DHS ox is useful for kinetic and thermodynamic analysis of the conformational folding process on the oxidative folding pathways of SS-reduced proteins. Graphical abstract Graphical abstract Highlights ► Three phases of the oxidative folding of RNase A were temporally separated using DHS ox . ► Thermodynamic and kinetic parameters of the four des intermediates were obtained using DHS ox . ► Each SS linkage in the native RNase A has different thermodynamic and kinetic roles. ► Unfolded des species (desU) gain native-like structures via X-Pro isomerization. ► All four des species were isolated and allowed to fold to N under aerobic conditions.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Patrick J. Halvey, Daniel C. Liebler, Robbert J.C. Slebos Agents to induce readthrough of premature termination codons (PTCs) are useful research tools and potential therapeutics. Reporters used to detect PTC readthrough are gene-specific and thus are not suited to for general assessment of readthrough activity or in cases where PTC-inactivated genes are unknown. Here we describe a GFP-based reporter construct pMHG-W57 ∗ which is capable of detecting dose-dependent drug-induced PTC readthrough both by fluorescence microscopy and flow cytometry. pMHG-W57 ∗ may be used as a general indicator of PTC readthrough in living cells and obviates the need for gene-specific recoding sequences in reporter constructs. Highlights ► Current reporter systems for translational readthrough are gene-specific and cannot be used in living cells. ► We developed a readthrough reporter based on green fluorescent protein. ► The reporter is gene-independent and can be used to sort living cells based on fluorescence.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Nan Zhang, Martin Buck The widely distributed bacterial σ 54 -dependent transcription regulates pathogenicity and numerous adaptive responses in diverse bacteria. Formation of the σ 54 -dependent open promoter complex is a multi-step process driven by AAA + ATPases. Non-hydrolysable nucleotide analogues are particularly suitable for studying such complexity by capturing various intermediate states along the energy coupling pathway. Here we report a novel ATP analogue, ADP–MgF 3 − , which traps an AAA + ATPase with its target σ 54 . The MgF 3 − -dependent complex is highly homogeneous and functional assays suggest it may represent an early transcription intermediate state valuable for structural studies. Highlights ► Novel ATP analogue ADP–MgF 3 − traps an AAA + ATPase with its target σ 54 . ► MgF 3 − -dependent complex forms a highly homogenous population. ► MgF 3 − -dependent complex represents an early transcription intermediate state.

Description: Publication year: 2012 Source: FEBS Open Bio Hanna-Kirsti S. Leiros, Anita-Elin Fedøy, Ingar Leiros, Ida Helene Steen Isocitrate dehydrogenase (IDH) catalyses the oxidative NAD(P) + -dependent decarboxylation of isocitrate into α-ketoglutarate and CO 2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH ( Ct IDH), a native open apo Ct IDH to 2.35 Å and a quaternary complex of Ct IDH with NADP + , isocitrate and Mg 2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH ( Dp IDH) was also resolved to 1.93 Å. Ct IDH and Dp IDH showed similar global thermal stabilities with melting temperatures of 67.9°C and 66.9°C, respectively. Ct IDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. Ct IDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to Dp IDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in Dp IDH, were absent in Ct IDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251’ and Arg255 ( Ct IDH). These interactions lock the large domain to the small domain and direct NADP + into the correct orientation, which together are important for NADP + selectivity. Graphical abstract Graphical abstract Highlights ► Focus is on a thermophilic (CtIDH) and cold active (DpIDH) isocitrate dehydrogenase. ► Biochemical characterization and three new IDH crystal structures are presented. ► CtIDH has many ionic interactions and hydrogen bonds, which explain the T m of 68°C. ► Prokaryotic IDH subfamily II seems to have a unique locking mechanism. ► The large domain is locked to the small domain and NADP + is also directed correctly.

Description: Publication year: 2011 Source: FEBS Open Bio, Volume 1 Tarlan Mamedov, Vidadi Yusibov Green algae have a great potential as biofactories for the production of proteins. Chlamydomonas reinhardtii , a representative of eukaryotic microalgae, has been extensively used as a model organism to study light-induced gene expression, chloroplast biogenesis, photosynthesis, light perception, cell–cell recognition, and cell cycle control. However, little is known about the glycosylation machinery and N-linked glycan structures of green algae. In this study, we performed mass spectrometry analysis of N-linked oligosaccharides released from total extracts of Chlamydomonas reinhardtii and demonstrated that C. reinhardtii algae possess glycoproteins with mammalian-like sialylated N-linked oligosaccharides. These findings suggest that C. reinhardtii may be an attractive system for expression of target proteins.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Annica Vlad-Fiegen, Anette Langerak, Sonja Eberth, Oliver Müller The Wnt pathway regulates cell proliferation, mobility and differentiation. Among the many Wnt target genes is CCND1 which codes for cyclin D1. Cyclin D1, in complex with cdk4 and cdk6, regulates G1/S phase transition during cell cycle. Independently of CDK, cyclin D1 also regulates the migration of macrophages. Here we analyzed the effects of cyclin D1 on the migration of cancer cell lines using the transwell migration and scratch assays. We also tested the effect of cyclin D1 and β-catenin on E-cadherin-mediated cell–cell contacts. Our results show that the Wnt pathway promotes cellular migration via its target gene cyclin D1. Moreover we show that cyclin D1 influences the actin cytoskeleton and destabilizes adherens junctions. Highlights ► We analyzed the effects of cyclin D1 on the migration of cancer cell lines. ► The Wnt pathway promotes cellular migration via its target gene cyclin D1. ► Cyclin D1 rearranges the actin cytoskeleton and destabilizes adherens junctions.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Hongchang Shen, Yan Fang, Wei Dong, Xueru Mu, Qi Liu, Jiajun Du The insulin like growth factor receptor subtype 1(IGF-1R) plays an important role in cancers transformation and progression. The aim is to investigate the effects of sunitinib on IGF-1R cell signaling transduction, especially on receptor phosphorylation and ubiquitination. In HEK293 cells, IGF-1R signaling pathways are activated in response to IGF-1, which induces obvious phosphorylations of receptor tyrosine and Akt, ERK. However, the phosphorylations of receptor tyrosine, Akt and ERK were significant inhibited by sunitinib. We found that both IGF-1 and sunitinib obviously down regulated the IGF-1R expression. For analysis the ubiquitination, HEK293 cells were simulated with 100 ng/ml IGF-1 or 10 nM sunitinib for 10 min after serum starvation for 24 h. Both IGF-1 and sunitinib could obviously induce the IGF-1R ubiquitination at 10 min compared with control (only serum free, no stimulation), indicating IGF-1 and sunitinib down-regulate the IGF-1R by increasing the receptor degradation through ubiquitination dependent proteasome pathway. We also found that MDM2 combined to IGF-1R in response to sunitinib stimulation. To confirm it, HEK293 cells were transfected with human HA-MDM2 (+MDM2) or siRNA to MDM2 (−MDM2). Following 24 h serum starvation, cells were stimulated with 10 nM sunitinib for 10 min. In over-expressed MDM2 cells, IGF-1R was more ubiquitinated than that in mock-transfected cells (control), and no ubiquitination in −MDM2 cells. These results mean that sunitinib mediates ubiquitination of IGF-1R dependent on MDM2. In summary, sunitinib could block signaling transduction and mediate degradation of IGF-1R.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Leonardo Acuña, Gianluca Picariello, Fernando Sesma, Roberto D. Morero, Augusto Bellomio Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries. Graphical abstract Graphical abstract Highlights ► A chimerical gene encoding a hybrid bacteriocin–microcin was generated by PCR. ► Ent35–MccV is a hybrid bacteriocin containing enterocin CRL35 and microcin V. ► Ent35–MccV is active against Gram-positive and Gram-negative bacteria. ► Enterohemorrhagic Escherichia coli and Listeria monocytogenes are sensitive to Ent35–MccV.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mike J.F. Broderick, Andrey Bobkov, Steve J. Winder Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F-actin have revealed a number of different conformations with calponin homology domains in ‘open’ and ‘closed’ positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F-actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F-actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino-terminal utrophin actin binding domain is in an open conformation in solution and when bound to F-actin. Highlights ► Utrophin actin binding domain crystallises as an open dimer but is monomeric in solution. ► We introduced cysteines in the CH domains to lock the monomer closed. ► Utrophin bound to F-actin with reduced affinity in the closed conformation. ► Differential scanning calorimetry confirmed the open conformation of utrophin on actin.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Vinay Kumar, Gagan D. Gupta Crystals of native Drosophila melanogaster translin diffracted to 7 Å resolution. Reductive methylation of the protein improved crystal quality. The native and methylated proteins showed similar profiles in size-exclusion chromatography analyses but the methylated protein displayed reduced DNA-binding activity. Crystals of the methylated protein diffracted to 4.2 Å resolution at BM14 of the ESRF synchrotron. Crystals with 49% solvent content belonged to monoclinic space group P 2 1 with eight protomers in the asymmetric unit. Only 2% of low-resolution structures with similar low percentage solvent content were found in the PDB. The crystal structure, solved by molecular replacement method, refined to R work ( R free ) of 0.24 (0.29) with excellent stereochemistry. The crystal structure clearly shows that drosophila protein exists as an octamer, and not as a decamer as expected from gel-filtration elution profiles. The similar octameric quaternary fold in translin orthologs and in translin–TRAX complexes suggests an up-down dimer as the basic structural subunit of translin-like proteins. The drosophila oligomer displays asymmetric assembly and increased radius of gyration that accounts for the observed differences between the elution profiles of human and drosophila proteins on gel-filtration columns. This study demonstrates clearly that low-resolution X-ray structure can be useful in understanding complex biological oligomers. Highlights ► Methylation yielded diffraction-quality crystals of Drosophila translin. ► The crystal structure revealed asymmetric octameric assembly of Drosophila translin. ► Up-down dimer is the basic structural subunit of translin-like proteins. ► The radius of gyration of the Drosophila oligomer is larger than that of human translin. ► Low-resolution X-ray structures facilitate understanding of biological complexes.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mamoru Hagihara, Ayaka Takei, Takeshi Ishii, Fumio Hayashi, Kenji Kubota, Kaori Wakamatsu, Nobukazu Nameki Choline- O -sulfate (2-(trimethylammonio)ethyl sulfate, COS) is a naturally occurring osmolyte that is synthesized by plants, lichens, algae, fungi, and several bacterial species. We examined the inhibitory effects of COS on amyloid formation of the human islet amyloid polypeptide (hIAPP or amylin) using a thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy and transmission electron microscopy. The results showed that COS suppresses a conformational change of hIAPP from a random coil to a β-sheet structure, resulting in the inhibition of amyloid formation. Comparisons with various structural analogs including carnitine, acetylcholine and non-detergent sulfobetaines (NDSBs) using the ThT fluorescence assay showed that COS is the most effective inhibitor of hIAPP amyloid formation, suggesting that the sulfate group, which is unique to COS, significantly contributes to the inhibition. Highlight ► Choline- O -sulfate (COS), an osmolyte, inhibits amyloid formation of hIAPP. ► COS is the most effective inhibitor of amyloid formation among structural analogs. ► The sulfate group appears to be involved in the inhibition.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mark J. Holness, Gulrez Zariwala, Celia G. Walker, Mary C. Sugden We studied adipocytes from 8-week-old control rat offspring (CON) or rat offspring subjected to maternal low (8%) protein (MLP) feeding during pregnancy/lactation, a procedure predisposing to obesity. Acute exposure to isoproterenol or adenosine enhanced PDK4 and PPARγ mRNA gene expression in CON and MLP adipocytes. Enhanced adipocyte Pdk4 expression correlated with increased PPARγ expression. Higher levels of PDK4 and PPARγ were observed in MLP adipocytes. SCD1 is a PPARγ target. Isoproterenol enhanced adipocyte PDK4 and SCD1 gene expression in parallel. This could reflect augmented PPARγ expression together with enhanced lipolytic stimulation to supply endogenous PPARγ ligands, allowing enhanced adipocyte PDK4 and SCD1 expression via PPARγ activation. In contrast, the effect of adenosine to increase PDK4 expression is independent of stimulation of lipolysis and, as SCD1 expression was unaffected by adenosine, unlikely to reflect PPARγ activation. Increased adipocyte expression of both PDK4 and SCD1 in the MLP model could participate as components of a “thrifty” phenotype, favouring the development of obesity. Highlights ► An early-life dietary intervention increasing the risk of obesity enhances adipocyte Pdk4 expression. ► Adenosine increases adipocyte PDK4 expression. ► An early-life dietary intervention augments PPARγ expression and enhances lipolytic stimulation. ► Enhanced lipolysis could supply PPARγ ligands enhancing adipocyte PDK4 via PPARγ activation.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Carol A. Chrestensen, Jonathan L. McMurry, John C. Salerno Endothelial nitric oxide synthase (eNOS) contains a motif similar to recognition sequences in known MAPK binding partners. In optical biosensing experiments, eNOS bound p38 and ERK with ∼100 nM affinity and complex kinetics. Binding is diffusion-limited ( k on ∼ .15 × 10 6 M −1 s −1 ). Neuronal NOS also bound p38 but exhibited much slower and weaker binding. p38-eNOS binding was inhibited by calmodulin. Evidence for a ternary complex was found when eNOS bound p38 was exposed to CaM, increasing the apparent dissociation rate. These observations strongly suggest a direct role for MAPK in regulation of NOS with implications for signaling pathways including angiogenesis and control of vascular tone. Highlights ► MAP kinases bind to a pentabasic sequence in the eNOS autoinhibitory insertion. ► MAP kinases bind poorly or not at all to nNOS. ► MAP kinases interact weakly with CaM binding, increasing CaM release. ► The MAPK binding site is adjacent to the phosphorylation sites of other kinases.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Mariana Tamazato Longhi, Nathalie Cella Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation. Highlights ► Three endogenous maspin tyrosine phosphoforms were identified in non-transformed mammary epithelial cells. ► Tyrosine phosphatase inhibition results in rapid increase in maspin levels. ► Tyrosine phosphatase inhibition results in maspin accumulation in the cytoplasm.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Hiromitsu Araki, Christoph Knapp, Peter Tsai, Cristin Print Most “omics” experiments require comprehensive interpretation of the biological meaning of gene lists. To address this requirement, a number of gene set analysis (GSA) tools have been developed. Although the biological value of GSA is strictly limited by the breadth of the gene sets used, very few methods exist for simultaneously analysing multiple publically available gene set databases. Therefore, we constructed GeneSetDB ( http://genesetdb.auckland.ac.nz/haeremai.html ), a comprehensive meta-database, which integrates 26 public databases containing diverse biological information with a particular focus on human disease and pharmacology. GeneSetDB enables users to search for gene sets containing a gene identifier or keyword, generate their own gene sets, or statistically test for enrichment of an uploaded gene list across all gene sets, and visualise gene set enrichment and overlap using a clustered heat map. Highlights ► Gene Set Analysis (GSA) can reveal biological meaning from “omics” experiments. ► It is currently difficult to apply GSA to the range of available gene set databases. ► We constructed GeneSetDB, a comprehensive meta-database for gene set analysis. ► GeneSetDB integrates 26 databases containing different types of information. ► GeneSetDB lets biologists visualise the gene sets represented in their “omics” results.

Description: Publication year: 2012 Source: FEBS Open Bio Ryo Furukawa, Yuma Yamada, Yuichi Matsushima, Yu-ichi Goto, Hideyoshi Harashima Mitochondrial transcription factor A (TFAM) packages mitochondrial DNA (mtDNA) and plays a critical role in mtDNA maintenance, replication and transcription. We report herein on the effects of DNA packaged with TFAM on the transcription process. Our initial findings indicated that the transcription of the TFAM/DNA complex was activated, when the complex was formed at an optimal ratio. We also found that TFAM has a significant advantage over protamine, a nuclear DNA packaging protein, from the viewpoint of transcription efficiency. This result indicates that TFAM can be useful packaging protein for exogenous DNA to achieve mitochondrial transgene expression. Graphical abstract Graphical abstract Highlights ► The base-pair interval at which mitochondrial transcription factor A (TFAM) binds to DNA is critical for transcription. ► The TFAM/DNA complex was transcriptionally active only when the packaging of DNA was optimal. ► The inhibition of the transcription of a TFAM/DNA complex at high ratios appears to result from the overpackaging of DNA by TFAM. ► Our findings indicated that the manner in which DNA is packaged with TFAM has an impact on transcription activation.

Description: Publication year: 2012 Source: FEBS Open Bio Yossi Laviv, Helen Toledano, Shalom Michowiz, Olga Dratviman-Storobinsky, Yuval Turm, Suzana Fichman-Horn, Ella Kagnovski, Nitza Goldenberg-Cohen Fifty-two samples of pediatric low-grade glioma (48 primary, 4 recurrent) were analyzed for BRAF copy number variation (digital PCR analysis, CopyCaller) and point mutations of BRAF V600E, and exon 5 Q209 in GNAQ , and GNA11 , using the MALDI-TOF mass spectrometer with validation by direct sequencing. An increased BRAF copy number was found in 18/47 primary samples tested; 15 of them (83.3%) were pilocytic astrocytomas. A BRAF mutation was found in 3/48 primary tumors, all with a normal BRAF copy number and no GNAQ mutation. One sample had a GNAQ209 mutation (Q209P626) with a normal BRAF gene ; none of the tumors had a GNA11Q209 mutation. Recurrent or progressive tumors, analyzed in four patients, had the same molecular genotype as their primary. Increased BRAF copy number and activating BRAF mutations may be involved in the development of low-grade glioma via overactivation of the Ras/Raf pathway. This is the first report of a mutation in GNAQ209 in pediatric low-grade glioma. Understanding the molecular mechanisms underlying glioma initiation and growth may assist in the development of targeted therapies. Highlights ► We investigated the occurrence of BRAF , GNAQ , and GNA11 mutations in pediatric low-grade glioma. ► We found an increase in the copy number of BRAF. ► BRAF and GNAQ mutations are mutually exclusive. ► Understanding the molecular basis of low-grade glioma may increase treatment options.

Description: Publication year: 2011 Source: FEBS Open Bio, Volume 1 Mariana Giró, Romina D. Ceccoli, Hugo O. Poli, Néstor Carrillo, Anabella F. Lodeyro Oxidative stress in plants causes ferredoxin down-regulation and NADP + shortage, over-reduction of the photosynthetic electron transport chain, electron leakage to oxygen and generation of reactive oxygen species (ROS). Expression of cyanobacterial flavodoxin in tobacco chloroplasts compensates for ferredoxin decline and restores electron delivery to productive routes, resulting in enhanced stress tolerance. We have designed an in vivo system to optimize flavodoxin reduction and NADP + regeneration under stress using a version of cyanobacterial ferredoxin–NADP + reductase without the thylakoid-binding domain. Co-expression of the two soluble flavoproteins in the chloroplast stroma resulted in lines displaying maximal tolerance to redox-cycling oxidants, lower damage and decreased ROS accumulation. The results underscore the importance of chloroplast redox homeostasis in plants exposed to adverse conditions, and provide a tool to improve crop tolerance toward environmental hardships. Graphical abstract Graphical abstract Highlights ► Expression of cyanobacterial flavodoxin in tobacco plants confers increased tolerance to oxidative stress. ► Expression of ferredoxin–NADP + reductase in tobacco does not provide oxidative stress tolerance. ► The presence of ferredoxin–NADP + reductase significantly increases oxidative stress tolerance conferred by flavodoxin in transgenic plants. ► Stress tolerance in plants depends on chloroplast redox homeostasis.

Description: Publication year: 2011 Source: FEBS Open Bio, Volume 1 Sabrina Siamer, Oriane Patrit, Mathilde Fagard, Naïma Belgareh-Touzé, Marie-Anne Barny Erwinia amylovora is responsible for fire blight, a necrotic disease of apples and pears. E. amylovora relies on a type III secretion system (T3SS) to induce disease on host plants. DspA/E belongs to the AvrE family of type III effector. Effectors of the AvrE family are injected via the T3SS in plant cell and are important to promote bacterial growth following infection and to suppress plant defense responses. Their mode of action in the plant cells is unknown. Here we study the physiological effects induced by dsp A/E expression in the yeast Saccharomyces cerevisiae . Expression of dspA/E in the yeast inhibits cell growth. This growth inhibition is associated with perturbations of the actin cytoskeleton and endocytosis.

Description: Publication year: 2012 Source: FEBS Open Bio, Volume 2 Yoshihiro Kawamura, Shigeru Takasaki, Masashi Mizokami The recommended treatment for patients with chronic hepatitis C, pegylated interferon α (PEG-IFN-α) plus rebavirin (RBV), does not provide a sustained virologic response in all patients, especially those with hepatitis C virus (HCV) genotype 1. It is therefore important to predict whether or not a new patient with HCV genotype 1 will be cured by the recommended treatment. We propose a prediction method for a new patient using a decision tree learning model based on SNPs evaluated in a genome-wide association study. By the decision tree learning for 142 Japanese patients with HCV genotype 1 (78 with null virologic response and 64 with virologic response), we can predict with high probability (93%) whether or not a new patient with HCV will be helped by the recommended treatment. Highlights ► We modeled drug responses using decision tree learning based on SNPs in a genome-wide association study. ► We can predict the drug responses of a new patient with HCV genotype 1. ► Responsiveness to pegylated interferon α (PEG-IFN-α) plus rebavirin (RBV) treatment was predicted. ► We can predict with 93% probability whether a new patient with HCV genotype 1 will be helped by drug treatment.

Description: Publication year: 2012 Source: FEBS Open Bio Karolina Stefanowicz, Nausicaä Lannoo, Paul Proost, Els J.M. Van Damme The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N - and O -glycans containing N -acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly- N- acetyllactosamine ([Galβ1-4GlcNAc] n ) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides. Highlights ► Recombinant expression of a new type of Arabidopsis F-box protein. ► Arabidopsis F-box-Nictaba protein is a functional lectin. ► Arabidopsis F-box-Nictaba recognizes N-acetyllactosamine and Lewis structures.

Description: Publication year: 2012 Source: FEBS Open Bio Elias Tannous, Koji Yokoyama, Dong-Ju You, Yuichi Koga, Shigenori Kanaya RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNH1) consists of an N-terminal domain with unknown function and a C-terminal RNase H domain. It is characterized by the high content of acidic residues on the protein surface. The far- and near-UV CD spectra of Halo-RNH1 suggested that Halo-RNH1 assumes a partially folded structure in the absence of salt and divalent metal ions. It requires either salt or divalent metal ions for folding. However, thermal denaturation of Halo-RNH1 analyzed in the presence of salt and/or divalent metal ions by CD spectroscopy suggested that salt and divalent metal ions independently stabilize the protein and thereby facilitate folding. Divalent metal ions stabilize the protein probably by binding mainly to the active site and suppressing negative charge repulsions at this site. Salt stabilizes the protein probably by increasing hydrophobic interactions at the protein core and decreasing negative charge repulsions on the protein surface. Halo-RNH1 exhibited activity in the presence of divalent metal ions regardless of the presence or absence of 3 M NaCl. However, higher concentrations of divalent metal ions are required for activity in the absence of salt to facilitate folding. Thus, divalent metal ions play a dual role in catalysis and folding of Halo-RNH1. Construction of the Halo-RNH1 derivatives lacking an N- or C-terminal domain, followed by biochemical characterizations, indicated that an N-terminal domain is dispensable for stability, activity, folding, and substrate binding of Halo-RNH1. Highlights ▸ Halophilic RNase H1 is partially folded in the absence of salt. ▸ Salt induces folding by decreasing negative charge repulsions on the protein surface. ▸ Divalent metal ions induce folding by binding to the active site. ▸ Divalent metal ions play a dual role in catalysis and folding of the enzyme.

Description: Publication year: 2012 Source: FEBS Open Bio Sushil Kumar Tomar, Prashant Kumar, Soneya Majumdar, Varun Bhaskar, Prasun Dutta, Balaji Prakash EngA is an essential protein involved in ribosome biogenesis. It is a unique GTPase, possessing two consecutive G-domains. Using sequence and phylogenetic analysis, we found two intriguing variants among EngA homologues – one with a shorter linker joining the G-domains and another with a longer linker, which additionally possesses an extended C-terminus. Interestingly, while the former variant is mainly restricted to Gram-positive bacteria, the latter is found in Gram-negative bacteria. Chimeric proteins with interchanged linkers and extensions were generated to gauge the importance of these elements. Ribosome interaction experiments employing the chimeric proteins suggest that a precise combination of the linker and C-terminal extension are important features regulating EngA ribosome interactions in a variant-specific manner. Highlights ▸ Homologues of the GTPase EngA exist as two distinct variants. ▸ EngA from Gram-positive bacteria have smaller linkers connecting the G-domains. ▸ A longer linker and an extended C-terminus are found in Gram-negative homologues. ▸ These species-specific features appear to regulate the EngA–ribosome association.

Description: Publication year: 2012 Source: FEBS Open Bio Abu Rafay, Soneya Majumdar, Balaji Prakash GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a ‘K-loop’ present in switch-I, were suggested as attributes of GTPases employing a K + -mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K + -activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases – Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) – and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K + -mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K + -mechanism in GTPases and hence warrant extensive structural investigations. Highlights ▸ An emerging alternative mechanism of GTP hydrolysis is mediated by K + ions. ▸ Features characteristic of the K + -mediated mechanism were suggested. ▸ As TEES family GTPases possess these, they were suggested to employ this mechanism. ▸ Not all GTPases that possess these features utilize the K + -mediated mechanism. ▸ Unambiguously identifying determinants of K + mechanism requires extensive studies.

Description: Publication year: 2012 Source: FEBS Open Bio Mohammed Al Bratty, Venkateswara R. Chintapalli, Julian A.T. Dow, Tong Zhang, David G. Watson Yellow ( y ) encodes a protein which is closely similar to major royal jelly proteins produced by bees. However, the function of y remains largely unknown. Metabolomic profiling was carried out on third instar Oregon R (OR) and yellow ( y ) Drosophila melanogaster larvae. Phenylalanine, tyrosine and DOPA were all elevated in y as might be expected since the mutation blocks melanin biosynthesis. The most consistent effects were related to lysine metabolism, with the lysine metabolite saccharopine being much higher in y . In addition, lysine acetate was elevated, and the levels of methyl lysines were lower, in y than in OR. Highlights ▸ The yellow (y) protein is widespread in nature but its function is unclear. ▸ we used mass spectrometric based metabolomic profiling to probe the function of Y. ▸ The absence of Y does not disrupt the formation of melanin precursors. ▸ Y was found to influence lysine metabolism and this might affect histone metabolism. ▸ Y may affect melanin formation indirectly, via its regulation of chitin biosynthesis.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Gloeobacter violaceus PCC 7421 is considered, by molecular phylogenetic analyses, to be an early-branching cyanobacterium within the cyanobacterial clade. G. violaceus is the only known oxygenic photosynthetic organism that lacks thylakoid membranes. There is only one report on the development of a transformation system for G. violaceus [H. Guo, X. Xu, Prog. Nat. Sci. 14 (2004) 31–35] and further studies using the system have not been reported. In the present study, we succeeded in introducing an expression vector (pKUT1121) derived from a broad-host-range plasmid, RSF1010, into G. violaceus by conjugation. The frequency of transformation of our system is significantly higher than that described in the previous report. In addition, luciferase heterologously expressed in G. violaceus functioned as a reporter. The established system will promote the molecular genetic studies on G. violaceus .

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231–294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Rat small intestinal goblet cell mucins reacting with monoclonal antibody HCM31 increase significantly during regeneration from experimental mucosal damage and at the period of expulsion of parasitic nematode, Nippostrongylus brasiliensis ( N.b ). The reduction in reactivity of HCM31 with mucin upon neuraminidase treatment, suggested that HCM31 recognizes sialylated oligosaccharide on mucin. HCM31-reactive sialomucins are therefore considered to play an important role in the physiological and pathological changes in the gastrointestinal mucosa. To determine the epitope for HCM31, oligosaccharide–alditols reacted with HCM31 were obtained from the small intestinal mucins of N.b -infected rats and purified by ion-exchange chromatography followed by normal-phase HPLC. Two HCM31-reactive oligosaccharide-alditols were obtained. Analyses using tandem mass spectrometry and NMR spectroscopy showed that these oligosaccharides were core 4 mucin-type oligosaccharides having a common tetrasaccharide sequence, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ- (Sd a blood group antigen). These structures were not found in the small intestinal mucin oligosaccharides from uninfected rats. This epitope specificity of HCM31 was also confirmed using previously established anti-GM2 and anti-Sd a antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin-type oligosaccharides with the Sd a tetrasaccharide sequence. Immunohistochemical examination of human gastrointestinal tracts showed that HCM31 site-specifically stained the goblet cells in normal sigmoid colon and normal rectum, but the goblet cells stained with HCM31 were reduced in the corresponding cancer tissues. HCM31 seems to be useful for diagnosis of colonic cancer and for examining the function of secretory-type mucin with Sd a antigen. Graphical abstract Highlights ▸ We analyzed the epitope for the monoclonal antibody HCM31. ▸ HCM31 recognizes core 4 oligosaccharides with the Sd a antigen obtained from small intestinal mucins of N. brasiliensis -infected rats. ▸ We propose that the Sd a tetrasaccharide sequence, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-, is the epitope for HCM31. ▸ Goblet cells stained with HCM31 decrease with malignant change in human colon tissues. ▸ HCM31 might be useful for the diagnosis of colonic cancer and for examining the function of mucin with Sd a tetrasaccharide.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 MicroRNAs regulate the expression of many genes and subsequently control various cellular processes, such as the immune response to viral infections mediated by type I interferon (IFN). In this study, the expression pattern of two interferon-related microRNAs, miR-146a and miR-155, was examined in healthy and HCV-genotype-4-infected peripheral blood mononuclear cells (PBMCs) using qRT-PCR. In contrast to other viral infections, the expression pattern was similar in both healthy and infected PBMCs. This could be attributed to attenuation of IFN pathway by HCV, which was assessed by investigating the expression of MxA, an interferon-stimulated gene, that showed lower expression in HCV-infected PBMCs. To determine the site of interference of HCV in the IFN pathway, expression of both microRNAs was examined following stimulation of PBMCs with IFN-α2a, an activator of the JAK/STAT pathway as well as with imiquimod, a toll-like receptor-7 (TLR-7) agonist that promotes interferon release. IFN stimulation induced the expression of miR-146a and miR-155 in HCV-infected and healthy PBMCs. Stimulation with imiquimod led to a down-regulation of both microRNAs in infected PBMCs, while it increased their expression in healthy PBMCs, indicating that HCV might interfere with miR-146a and miR-155 expression at sites upstream of interferon release, specifically in the TLR-7 pathway. The pattern of expression of both miR-146a and miR-155 was very similar with a strong positive correlation, but showed no correlation to the patients’ clinical or histopathological parameters or response to treatment. In conclusion, HCV infection might repress the induction of miR-146a and miR-155 by interfering with TLR-7 signaling.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Human type 2 diabetes is associated with β-cell apoptosis, and human islets from diabetic donors are ∼80% deficient in PAK1 protein. Toward addressing linkage of PAK1 to β-cell survival, PAK1–siRNA targeted MIN6 pancreatic β-cells were found to exhibit increased caspase-3 cleavage, cytosolic cytochrome-C and the pro-apoptotic protein Bad. PAK1 +/− heterozygous mouse islets recapitulated the upregulation of Bad protein expression, as did hyperglycemic treatment of human or mouse islets; Bad levels were exacerbated most in PAK1 +/− islets subjected to hyperglycemic stress. These data implicate PAK1 in β-cell survival via quenching of Bad protein expression, and suggest PAK1 as potential molecular target to preserve β-cell mass. Highlights ▸ Bad expression in islets was increased by chronic exposure to hyperglycemic conditions. ▸ Depletion of PAK1 in the absence of glucose induced Bad expression. ▸ Bad expression increased most in PAK1 +/− heterozygous islets subjected to hyperglycemia. ▸ PAK1 is required to protect against β-cell apoptosis under hyperglycemic conditions.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100-fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca 2+ , are proposed to be important for ISP activity through structural changes. The presence of 〉0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca 2+ at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans. Highlights ▸ All neuraminidases (NAs) of avian influenza A viruses (IAVs) are stable at low pH. ▸ Human 1918, 1957, and 1968 pandemic IAVs also have NAs that are stable at low pH. ▸ Most human epidemic IAVs have NAs that are unstable at low pH. ▸ The NAs of most H5N1 IAVs isolated since 2003 are unstable at low pH. ▸ Instability of H5N1 IAV NAs at low pH might explain frequent human infections.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Subtype-selective thyromimetics have potential as new pharmaceuticals for the prevention or treatment of heart disease, high LDL cholesterol and obesity, but there are only a few methods that can detect agonistic behavior of TR-active compounds. Among these are the rat pituitary GH 3 cell assay and transcriptional activation assays in engineered yeast and mammalian cells. We report the construction and validation of a newly designed TRα-1 bacterial biosensor, which indicates the presence of thyroid active compounds through their impacts on the growth of an engineered Escherichia coli strain in a simple defined medium. This biosensor couples the configuration of a hormone receptor ligand-binding domain to the activity of a thymidylate synthase reporter enzyme through an engineered allosteric fusion protein. The result is a hormone-dependent growth phenotype in the expressing E. coli cells. This sensor can be combined with our previously published TRβ-1 biosensor to detect potentially therapeutic subtype-selective compounds such as GC-1 and KB-141. To demonstrate this capability, we determined the half-maximal effective concentration (EC 50 ) for the compounds T 3 , Triac, GC-1 and KB-141 using our biosensors, and determined their relative potency in each biosensor strain. Our results are similar to those reported by mammalian cell reporter gene assays, confirming the utility of our assay in identifying TR subtype-selective therapeutics. This biosensor thus provides a high-throughput, receptor-specific, and economical method (less than US$ 0.10 per well at laboratory scale) for identifying important therapeutics against these targets. Graphical abstract Highlights ▸ Engineered E. coli cells react to thyromimetic compounds through growth phenotype. ▸ This provides a simple method to identify subtype-selective thyroid active compounds. ▸ Phenotype is linked to ligand binding through an engineered sensor protein. ▸ Subtype-selectivity and EC 50 values for T 3 , Triac, GC-1 and KB-141 were determined

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Tespa1 has been recently reported to be a critical molecule in T-cell development, however, the precise molecular mechanisms of Tespa1 remain elusive. Here, we demonstrate that Tespa1 shows amino-acid sequence homology to KRAS -induced actin-interacting protein (KRAP), an inositol 1,4,5-trisphosphate receptor (IP 3 R) binding protein, and that Tespa1 physically associates with IP 3 R in T and B lymphocytes. Two-consecutive phenylalanine residues (Phe185/Phe186) in Tespa1, which are conserved between Tespa1 and KRAP, are indispensable for the association between Tespa1 and IP 3 R. These findings suggest that Tespa1 plays critical roles in the immune system through the regulation of the IP 3 R. Highlights ▸ We identified Tespa1 as a novel IP 3 R-associated protein in lymphoid tissues. ▸ Tespa1 interacts with multiple subtypes of IP 3 R in T and B lymphocytes. ▸ Amino-terminal region of IP 3 R is sufficient for the association with Tespa1. ▸ Two consecutive phenylalanines in Tespa1 are critical for the interaction with IP 3 R. ▸ Tespa1 is structurally and functionally related to KRAP.

Description: Available online 7 December 2012 Publication year: 2012 Source: FEBS Open Bio l -Rhamnose isomerase ( l -RhI) catalyzes the reversible isomerization of l -rhamnose to l -rhamnulose. Previously determined X-ray structures of l -RhI showed a hydride-shift mechanism for the isomerization of substrates in a linear form, but the mechanism for opening of the sugar-ring is still unclear. To elucidate this mechanism, we determined X-ray structures of a mutant l -RhI in complex with l -rhamnopyranose and d -allopyranose. Results suggest that a catalytic water molecule, which acts as an acid/base catalyst in the isomerization reaction, is likely to be involved in pyranose-ring opening, and that a newly found substrate sub-binding site in the vicinity of the catalytic site may recognize different anomers of substrates.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The insulin like growth factor receptor subtype 1(IGF-1R) plays an important role in cancers transformation and progression. The aim is to investigate the effects of sunitinib on IGF-1R cell signaling transduction, especially on receptor phosphorylation and ubiquitination. In HEK293 cells, IGF-1R signaling pathways are activated in response to IGF-1, which induces obvious phosphorylations of receptor tyrosine and Akt, ERK. However, the phosphorylations of receptor tyrosine, Akt and ERK were significant inhibited by sunitinib. We found that both IGF-1 and sunitinib obviously down regulated the IGF-1R expression. For analysis the ubiquitination, HEK293 cells were simulated with 100 ng/ml IGF-1 or 10 nM sunitinib for 10 min after serum starvation for 24 h. Both IGF-1 and sunitinib could obviously induce the IGF-1R ubiquitination at 10 min compared with control (only serum free, no stimulation), indicating IGF-1 and sunitinib down-regulate the IGF-1R by increasing the receptor degradation through ubiquitination dependent proteasome pathway. We also found that MDM2 combined to IGF-1R in response to sunitinib stimulation. To confirm it, HEK293 cells were transfected with human HA-MDM2 (+MDM2) or siRNA to MDM2 (−MDM2). Following 24 h serum starvation, cells were stimulated with 10 nM sunitinib for 10 min. In over-expressed MDM2 cells, IGF-1R was more ubiquitinated than that in mock-transfected cells (control), and no ubiquitination in −MDM2 cells. These results mean that sunitinib mediates ubiquitination of IGF-1R dependent on MDM2. In summary, sunitinib could block signaling transduction and mediate degradation of IGF-1R.

Description: Available online 12 November 2012 Publication year: 2012 Source: FEBS Open Bio We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′-TTAGGG-3′, which has a six-base loop head structure, and a 3′-oligo (dG) 3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)-immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)-6, IL-12p35, IL-12p40, and interferon (IFN) γ mRNA expression, but also IL-4 and IL-13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen-immunized mice, suggesting usefulness in the treatment of diseases induced by over-active immune activation.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Choline- O -sulfate (2-(trimethylammonio)ethyl sulfate, COS) is a naturally occurring osmolyte that is synthesized by plants, lichens, algae, fungi, and several bacterial species. We examined the inhibitory effects of COS on amyloid formation of the human islet amyloid polypeptide (hIAPP or amylin) using a thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy and transmission electron microscopy. The results showed that COS suppresses a conformational change of hIAPP from a random coil to a β-sheet structure, resulting in the inhibition of amyloid formation. Comparisons with various structural analogs including carnitine, acetylcholine and non-detergent sulfobetaines (NDSBs) using the ThT fluorescence assay showed that COS is the most effective inhibitor of hIAPP amyloid formation, suggesting that the sulfate group, which is unique to COS, significantly contributes to the inhibition. Highlights ► Choline- O -sulfate (COS), an osmolyte, inhibits amyloid formation of hIAPP. ► COS is the most effective inhibitor of amyloid formation among structural analogs. ► The sulfate group appears to be involved in the inhibition.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Some ligand–receptor couples involve spare receptors, which are apparent when a maximal response is achieved with only a small fraction of the receptor population occupied. This situation favours cross-reactions with low-affinity ligands, which may be detrimental for cell signaling. In the case of the adenosine A 2A receptors (A 2A R), which have an immunosuppressive effect on lymphocytes through cAMP production, the presence of spare A 2A R remains to be established. We examined the situation using patients over-expressing lymphocyte A 2A R and an agonist-like mAb to A 2A R. We found that maximal mAb binding and functional response varied among the patients whereas the dissociation constant and half-maximal effective concentration had similar mean values (0.19 and 0.18 μM, respectively). Lymphocyte A 2A R expression was correlated to plasma adenosine level and A 2A R occupation but not to A 2A R response. These results are consistent with a lack of a reserve of functional A 2A R on human lymphocytes as a general rule and suggest that the amount and functional state of the expressed A 2A R determine the maximal level of the lymphocyte response to adenosine.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Pex14p is a peroxisomal membrane protein that is involved in both peroxisome biogenesis and selective peroxisome degradation. Previously, we showed that Hansenula polymorpha Pex14p was phosphorylated in vivo . In this study, we identified its phosphorylation site by mass spectrometry. Recombinant His-tagged Pex14p (H6-Pex14p) was overexpressed and purified from the yeast. The protein band corresponding to H6-Pex14p was in-gel digested with trypsin and subjected to LC/MS. As a result of LC/MS, Thr 248 and Ser 258 were identified as the phosphorylated sites. To confirm the phosphorylation sites and explore its functions, we made Ala mutants of the candidate amino acids. In the western blot analysis with anti-Pex14p, S258A mutant gave doublet bands while wild type (WT) and T248A mutants gave triplet bands. Moreover, the double mutant (T248A/S258A) gave a single band. WT and all mutant Pex14p labeled with [ 32 P] orthophosphate were immunoprecipitated and analyzed by autoradiography. The phosphorylation of Pex14p was suppressed in S258A mutant, but enhanced in T248A mutant compared to WT. Moreover, the phosphorylated Pex14p was not detected in the T248A/S258A double mutant. All mutants were able to grow on methanol and their matrix proteins (alcohol oxidase and amine oxidase) were mostly localized in peroxisomes. Furthermore all mutants showed selective degradation of peroxisome like WT during the glucose-induced macropexophagy.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Accumulated evidence suggests that aberrant regulation of δ-catenin leads to pathological consequences such as mental retardation and cognitive dysfunction. This study revealed that 14-3-3ɛ/ζ stabilizes δ-catenin, with different binding regions involved in the interaction. Furthermore, the specific inhibition of the interaction of 14-3-3 with δ-catenin reduced levels of δ-catenin and significantly impaired the capacity of δ-catenin to induce dendritic branching in both NIH3T3 fibroblasts and primary hippocampal neurons. However, the S1094A δ-catenin mutant, which cannot interact with 14-3-3ζ, still retained the capability of inducing dendrogenesis. Taken together, these results elucidate the underlying events that regulate the stability of δ-catenin and δ-catenin-induced dendrogenesis.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F-actin have revealed a number of different conformations with calponin homology domains in ‘open’ and ‘closed’ positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F-actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F-actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino-terminal utrophin actin binding domain is in an open conformation in solution and when bound to F-actin. Highlights ► Utrophin actin binding domain crystallises as an open dimer but is monomeric in solution. ► We introduced cysteines in the CH domains to lock the monomer closed. ► Utrophin bound to F-actin with reduced affinity in the closed conformation. ► Differential scanning calorimetry confirmed the open conformation of utrophin on actin.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The Wnt pathway regulates cell proliferation, mobility and differentiation. Among the many Wnt target genes is CCND1 which codes for cyclin D1. Cyclin D1, in complex with cdk4 and cdk6, regulates G1/S phase transition during cell cycle. Independently of CDK, cyclin D1 also regulates the migration of macrophages. Here we analyzed the effects of cyclin D1 on the migration of cancer cell lines using the transwell migration and scratch assays. We also tested the effect of cyclin D1 and β-catenin on E-cadherin-mediated cell–cell contacts. Our results show that the Wnt pathway promotes cellular migration via its target gene cyclin D1. Moreover we show that cyclin D1 influences the actin cytoskeleton and destabilizes adherens junctions. Highlights ► We analyzed the effects of cyclin D1 on the migration of cancer cell lines. ► The Wnt pathway promotes cellular migration via its target gene cyclin D1. ► Cyclin D1 rearranges the actin cytoskeleton and destabilizes adherens junctions.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The widely distributed bacterial σ 54 -dependent transcription regulates pathogenicity and numerous adaptive responses in diverse bacteria. Formation of the σ 54 -dependent open promoter complex is a multi-step process driven by AAA + ATPases. Non-hydrolysable nucleotide analogues are particularly suitable for studying such complexity by capturing various intermediate states along the energy coupling pathway. Here we report a novel ATP analogue, ADP–MgF 3 − , which traps an AAA + ATPase with its target σ 54 . The MgF 3 − -dependent complex is highly homogeneous and functional assays suggest it may represent an early transcription intermediate state valuable for structural studies. Highlights ► Novel ATP analogue ADP–MgF 3 − traps an AAA + ATPase with its target σ 54 . ► MgF 3 − -dependent complex forms a highly homogenous population. ► MgF 3 − -dependent complex represents an early transcription intermediate state.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Redox-coupled folding pathways of bovine pancreatic ribonuclease A (RNase A) with four intramolecular disulfide (SS) bonds comprise three phases: (I) SS formation to generate partially oxidized intermediate ensembles with no rigid folded structure; (II) SS rearrangement from the three SS intermediate ensemble (3S) to the des intermediates having three native SS linkages; (III) final oxidation of the last native SS linkage to generate native RNase A. We previously demonstrated that DHS ox , a water-soluble selenoxide reagent for rapid and quantitative SS formation, allows clear separation of the three folding phases. In this study, the main conformational folding phase (phase II) has been extensively analyzed at pH 8.0 under a wide range of temperatures (5–45 °C), and thermodynamic and kinetic parameters for the four des intermediates were determined. The free-energy differences (Δ G ) as a function of temperature suggested that the each SS linkage has different thermodynamic and kinetic roles in stability of the native structure. On the other hand, comparison of the rate constants and the activation energies for 3S → des with those reported for the conformational folding of SS-intact RNase A suggested that unfolded des species (desU) having three native SS linkages but not yet being folded are involved in very small amounts (〈1%) in the 3S intermediate ensemble and the desU species would gain the native-like structures via X-Pro isomerization like SS-intact RNase A. It was revealed that DHS ox is useful for kinetic and thermodynamic analysis of the conformational folding process on the oxidative folding pathways of SS-reduced proteins. Graphical abstract Highlights ► Three phases of the oxidative folding of RNase A were temporally separated using DHS ox . ► Thermodynamic and kinetic parameters of the four des intermediates were obtained using DHS ox . ► Each SS linkage in the native RNase A has different thermodynamic and kinetic roles. ► Unfolded des species (desU) gain native-like structures via X-Pro isomerization. ► All four des species were isolated and allowed to fold to N under aerobic conditions.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Oxidative stress has been associated with prostate cancer development and progression due to an increase of reactive oxygen species (ROS). However, the mechanisms whereby ROS and the antioxidant system participate in cancer progression remain unclear. In order to clarify the influence of oxidative stress in prostate cancer progression, we performed this study in two human prostate cancer cell lines, PC3 and HPV10 (from metastasis and from localized cancer, respectively) and RWPE1 cells derived from normal prostate epithelium. Cells were treated with hydrogen peroxide (H 2 O 2 ) and PC3 cells were also treated with diethyl maleate (DEM). The effect on cell growth, viability, mitochondria membrane potential and oxidative stress was analysed. Oxidative stress was evaluated based on ROS production, oxidative lesion of lipids (MDA) and on determination of antioxidants, including enzyme activity of glutathione peroxidase (Gl-Px), glutathione reductase (Gl-Red) and on the quantification of glutathione (GSH), glutathione-s-transferase (GST) and total antioxidant status (TAS). PC3 shows higher ROS production but also the highest GSH levels and Gl-Red activity, possibly contributing to oxidative stress resistance. This is also associated with higher mitochondrial membrane potential, TAS and lower lipid peroxidation. On the other hand, we identified Gl-Red activity reduction as a new strategy in overcoming oxidative stress resistance, by inducing H 2 O 2 cytotoxicity. Therefore these results suggest Gl-Red activity reduction as a new potential therapeutic approach, in prostate cancer. Highlights ► Oxidative stress was evaluated on a cell line model of prostate cancer progression. ► Metastatic cell lines show the highest ROS, total antioxidant status and resistance to H 2 O 2 . ► Metastatic cell lines show the highest levels of GSH and Gl-Red activity. ► Decrease in GSH levels and Gl-Red activity induced a decrease in H 2 O 2 resistance. ► Gl-Red activity reduction may be a new therapeutic approach in prostate cancer.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 For successful mitochondrial transgene expression, an optimal packaging exogenous DNA is an important issue. We report herein on the effects of DNA packaged with mitochondrial transcription factor A (TFAM), which packages mitochondrial DNA (mtDNA), on the transcription process. Our initial findings indicated that the transcription of the TFAM/DNA complex was activated, when the complex was formed at an optimal ratio. We also found that TFAM has a significant advantage over protamine, a nuclear DNA packaging protein, from the viewpoint of transcription efficiency. This result indicates that TFAM can be useful packaging protein for exogenous DNA to achieve mitochondrial transgene expression. Graphical abstract Highlights ► The base-pair interval at TFAM binds to DNA is critical for transcription. ► TFAM/DNA complex was transcriptionally active only when DNA was optimally packaged. ► The transcription inhibition appears to result from the overpackaging of DNA by TFAM. ► The manner in which DNA is packaged with TFAM has an impact on transcription.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation. Highlights ► Three endogenous maspin tyrosine phosphoforms were identified in non-transformed mammary epithelial cells. ► Tyrosine phosphatase inhibition results in rapid increase in maspin levels. ► Tyrosine phosphatase inhibition results in maspin accumulation in the cytoplasm.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Yellow ( y ) encodes a protein which is closely similar to major royal jelly proteins produced by bees. However, the function of y remains largely unknown. Metabolomic profiling was carried out on third instar Oregon R (OR) and yellow ( y ) Drosophila melanogaster larvae. Phenylalanine, tyrosine and DOPA were all elevated in y as might be expected since the mutation blocks melanin biosynthesis. The most consistent effects were related to lysine metabolism, with the lysine metabolite saccharopine being much higher in y . In addition, lysine acetate was elevated, and the levels of methyl lysines were lower, in y than in OR. Highlights ▸ The yellow (y) protein is widespread in nature but its function is unclear. ▸ we used mass spectrometric based metabolomic profiling to probe the function of Y. ▸ The absence of Y does not disrupt the formation of melanin precursors. ▸ Y was found to influence lysine metabolism and this might affect histone metabolism. ▸ Y may affect melanin formation indirectly, via its regulation of chitin biosynthesis.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Dihydroartemisinin (DHA), an active metabolite of artemisinin derivatives, is the most remarkable anti-malarial drug and has little toxicity to humans. Recent studies have shown that DHA effectively inhibits the growth of cancer cells. In the present study, we intended to elucidate the mechanisms underlying the inhibition of growth of iron-loaded human myeloid leukemia K562 cells by DHA. Mitochondria are important regulators of both autophagy and apoptosis, and one of the triggers for mitochondrial dysfunction is the generation of reactive oxygen species (ROS). We found that the DHA-induced autophagy of leukemia K562 cells, whose intracellular organelles are primarily mitochondria, was ROS dependent. The autophagy of these cells was followed by LC3-II protein expression and caspase-3 activation. In addition, we demonstrated that inhibition of the proliferation of leukemia K562 cells by DHA is also dependent upon iron. This inhibition includes the down-regulation of TfR expression and the induction of K562 cell growth arrest in the G 2 /M phase. Highlights ► DHA induced K562 cells autophagy followed by LC3-II protein expression. ► The DHA-induced autophagy might be ROS dependent. ► Inhibition of K562 cell proliferation by DHA was also dependent upon iron decrease.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a ‘K-loop’ present in switch-I, were suggested as attributes of GTPases employing a K + -mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K + -activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases – Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) – and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K + -mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K + -mechanism in GTPases and hence warrant extensive structural investigations. Highlights ▸ An emerging alternative mechanism of GTP hydrolysis is mediated by K + ions. ▸ Features characteristic of the K + -mediated mechanism were suggested. ▸ As TEES family GTPases possess these, they were suggested to employ this mechanism. ▸ Not all GTPases that possess these features utilize the K + -mediated mechanism. ▸ Unambiguously identifying determinants of K + mechanism requires extensive studies.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Aptamers are promising gene components that can be used for the construction of synthetic gene circuits. In this study, we isolated an RNA aptamer that specifically inhibits transcription of T7 RNA polymerase (RNAP). The 38-nucleotide aptamer, which was a shortened variant of an initial SELEX isolate, showed moderate inhibitory activity. By stepwise doped-SELEX, we isolated evolved variants with strong inhibitory activity. A 29-nucleotide variant of a doped-SELEX isolate showed 50% inhibitory concentration at 11 nM under typical in vitro transcription conditions. Pull-down experiments revealed that the aptamer inhibited the association of T7 RNAP with T7 promoter DNA. Highlights ▸ An inhibitory RNA aptamer against T7 RNA polymerase was isolated. ▸ Evolved aptamers with strong inhibitory activity were isolated by doped-SELEX. ▸ Aptamer sequence/structure requirements were elucidated based on phylogeny. ▸ A 29-nucleotide variant of an evolved aptamer has an IC 50 of 11 nM. ▸ The aptamer inhibits the association of T7 RNA polymerase with T7 promoter DNA.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Human STIL (SCL/TAL1 interrupting locus) protein maintains centriole stability and spindle pole localisation. It helps in recruitment of CENPJ (Centromere protein J)/CPAP (centrosomal P4.1-associated protein) and other centrosomal proteins. Mutations in STIL protein are reported in several disorders, especially in deregulation of cell cycle cascades. In this work, we examined the non-synonymous single nucleotide polymorphisms (nsSNPs) reported in STIL protein for their disease association. Different SNP prediction tools were used to predict disease-associated nsSNPs. Our evaluation technique predicted rs147744459 (R242C) as a highly deleterious disease-associated nsSNP and its interaction behaviour with CENPJ protein. Molecular modelling, docking and molecular dynamics simulation were conducted to examine the structural consequences of the predicted disease-associated mutation. By molecular dynamic simulation we observed structural consequences of R242C mutation which affects interaction of STIL and CENPJ functional domains. The result obtained in this study will provide a biophysical insight into future investigations of pathological nsSNPs using a computational platform.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Antimalarial chloroquine is also used for the treatment of immune-mediated diseases. The interference of chloroquine with interferon-γ-induced tryptophan breakdown and neopterin production has been investigated in human peripheral blood mononuclear cells (PBMC) in vitro . Micromolar concentrations (2–50 μM) of chloroquine dose-dependently suppressed mitogen-induced tryptophan breakdown in PBMC but not in the myelomonocytic THP-1-Blue cell line, after 48 h of treatment. In stimulated PBMC, neopterin production was super-induced by 10 μM chloroquine, while it was significantly suppressed at a concentration of 50 μM. These anti-inflammatory effects may relate to the therapeutic benefit of chloroquine in inflammatory conditions and may widen the spectrum of its clinical applications. Highlights ▸ 2–50 μM chloroquine suppresses mitogen-induced tryptophan breakdown in human PBMCs. ▸ The same effect was not seen in the myelomonocytic THP-1-Blue cell line. ▸ The anti-inflammatory property of chloroquine targets T cells more than monocytes. ▸ This anti-inflammatory effect may explain the drug's therapeutic benefit for malaria. ▸ Chloroquine treatment could be of benefit for other chronic inflammatory conditions.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Postmenopausal women with chronic hepatitis C exhibited a poor response to interferon (IFN) therapy compared to premenopausal women. Osteoporosis is the typical complication that occurs in postmenopausal women. Recently, it was reported that an osteoporotic reagent, vitamin D3, exhibited anti-hepatitis C virus (HCV) activity. Therefore, we investigated whether or not another osteoporotic reagent, raloxifene, would exhibit anti-HCV activity in cell culture systems. Here, we demonstrated that raloxifene inhibited HCV RNA replication in genotype 1b and infection in genotype 2a. Raloxifene enhanced the anti-HCV activity of IFN-α. These results suggest a link between the molecular biology of osteoporosis and the HCV life cycle. Highlights ▸ The osteoporotic reagent, raloxifene, inhibits HCV infection and replication. ▸ Raloxifene enhanced the anti-HCV activity of interferon. ▸ Raloxifene is a candidate for add-on therapy in patients with chronic hepatitis C. ▸ The biology of osteoporosis is associated with the HCV life cycle.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Some ligand–receptor couples involve spare receptors, which are apparent when a maximal response is achieved with only a small fraction of the receptor population occupied. This situation favours cross-reactions with low-affinity ligands, which may be detrimental for cell signaling. In the case of the adenosine A 2A receptors (A 2A R), which have an immunosuppressive effect on lymphocytes through cAMP production, the presence of spare A 2A R remains to be established. We examined the situation using patients over-expressing lymphocyte A 2A R and an agonist-like mAb to A 2A R. We found that maximal mAb binding and functional response varied among the patients whereas the dissociation constant and half-maximal effective concentration had similar mean values (0.19 and 0.18 μM, respectively). Lymphocyte A 2A R expression was correlated to plasma adenosine level and A 2A R occupation but not to A 2A R response. These results are consistent with a lack of a reserve of functional A 2A R on human lymphocytes as a general rule and suggest that the amount and functional state of the expressed A 2A R determine the maximal level of the lymphocyte response to adenosine.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Pex14p is a peroxisomal membrane protein that is involved in both peroxisome biogenesis and selective peroxisome degradation. Previously, we showed that Hansenula polymorpha Pex14p was phosphorylated in vivo . In this study, we identified its phosphorylation site by mass spectrometry. Recombinant His-tagged Pex14p (H6-Pex14p) was overexpressed and purified from the yeast. The protein band corresponding to H6-Pex14p was in-gel digested with trypsin and subjected to LC/MS. As a result of LC/MS, Thr 248 and Ser 258 were identified as the phosphorylated sites. To confirm the phosphorylation sites and explore its functions, we made Ala mutants of the candidate amino acids. In the western blot analysis with anti-Pex14p, S258A mutant gave doublet bands while wild type (WT) and T248A mutants gave triplet bands. Moreover, the double mutant (T248A/S258A) gave a single band. WT and all mutant Pex14p labeled with [ 32 P] orthophosphate were immunoprecipitated and analyzed by autoradiography. The phosphorylation of Pex14p was suppressed in S258A mutant, but enhanced in T248A mutant compared to WT. Moreover, the phosphorylated Pex14p was not detected in the T248A/S258A double mutant. All mutants were able to grow on methanol and their matrix proteins (alcohol oxidase and amine oxidase) were mostly localized in peroxisomes. Furthermore all mutants showed selective degradation of peroxisome like WT during the glucose-induced macropexophagy.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Gloeobacter violaceus PCC 7421 is considered, by molecular phylogenetic analyses, to be an early-branching cyanobacterium within the cyanobacterial clade. G. violaceus is the only known oxygenic photosynthetic organism that lacks thylakoid membranes. There is only one report on the development of a transformation system for G. violaceus [H. Guo, X. Xu, Prog. Nat. Sci. 14 (2004) 31–35] and further studies using the system have not been reported. In the present study, we succeeded in introducing an expression vector (pKUT1121) derived from a broad-host-range plasmid, RSF1010, into G. violaceus by conjugation. The frequency of transformation of our system is significantly higher than that described in the previous report. In addition, luciferase heterologously expressed in G. violaceus functioned as a reporter. The established system will promote the molecular genetic studies on G. violaceus .

Description: Available online 27 November 2012 Publication year: 2012 Source: FEBS Open Bio Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231–294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The Wnt pathway regulates cell proliferation, mobility and differentiation. Among the many Wnt target genes is CCND1 which codes for cyclin D1. Cyclin D1, in complex with cdk4 and cdk6, regulates G1/S phase transition during cell cycle. Independently of CDK, cyclin D1 also regulates the migration of macrophages. Here we analyzed the effects of cyclin D1 on the migration of cancer cell lines using the transwell migration and scratch assays. We also tested the effect of cyclin D1 and β-catenin on E-cadherin-mediated cell–cell contacts. Our results show that the Wnt pathway promotes cellular migration via its target gene cyclin D1. Moreover we show that cyclin D1 influences the actin cytoskeleton and destabilizes adherens junctions. Highlights ► We analyzed the effects of cyclin D1 on the migration of cancer cell lines. ► The Wnt pathway promotes cellular migration via its target gene cyclin D1. ► Cyclin D1 rearranges the actin cytoskeleton and destabilizes adherens junctions.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries. Graphical abstract Highlights ► A chimerical gene encoding a hybrid bacteriocin–microcin was generated by PCR. ► Ent35–MccV is a hybrid bacteriocin containing enterocin CRL35 and microcin V. ► Ent35–MccV is active against Gram-positive and Gram-negative bacteria. ► Enterohemorrhagic Escherichia coli and Listeria monocytogenes are sensitive to Ent35–MccV.

Description: Available online 12 November 2012 Publication year: 2012 Source: FEBS Open Bio We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′-TTAGGG-3′, which has a six-base loop head structure, and a 3′-oligo (dG) 3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)-immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)-6, IL-12p35, IL-12p40, and interferon (IFN) γ mRNA expression, but also IL-4 and IL-13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen-immunized mice, suggesting usefulness in the treatment of diseases induced by over-active immune activation.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The widely distributed bacterial σ 54 -dependent transcription regulates pathogenicity and numerous adaptive responses in diverse bacteria. Formation of the σ 54 -dependent open promoter complex is a multi-step process driven by AAA + ATPases. Non-hydrolysable nucleotide analogues are particularly suitable for studying such complexity by capturing various intermediate states along the energy coupling pathway. Here we report a novel ATP analogue, ADP–MgF 3 − , which traps an AAA + ATPase with its target σ 54 . The MgF 3 − -dependent complex is highly homogeneous and functional assays suggest it may represent an early transcription intermediate state valuable for structural studies. Highlights ► Novel ATP analogue ADP–MgF 3 − traps an AAA + ATPase with its target σ 54 . ► MgF 3 − -dependent complex forms a highly homogenous population. ► MgF 3 − -dependent complex represents an early transcription intermediate state.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The insulin like growth factor receptor subtype 1(IGF-1R) plays an important role in cancers transformation and progression. The aim is to investigate the effects of sunitinib on IGF-1R cell signaling transduction, especially on receptor phosphorylation and ubiquitination. In HEK293 cells, IGF-1R signaling pathways are activated in response to IGF-1, which induces obvious phosphorylations of receptor tyrosine and Akt, ERK. However, the phosphorylations of receptor tyrosine, Akt and ERK were significant inhibited by sunitinib. We found that both IGF-1 and sunitinib obviously down regulated the IGF-1R expression. For analysis the ubiquitination, HEK293 cells were simulated with 100 ng/ml IGF-1 or 10 nM sunitinib for 10 min after serum starvation for 24 h. Both IGF-1 and sunitinib could obviously induce the IGF-1R ubiquitination at 10 min compared with control (only serum free, no stimulation), indicating IGF-1 and sunitinib down-regulate the IGF-1R by increasing the receptor degradation through ubiquitination dependent proteasome pathway. We also found that MDM2 combined to IGF-1R in response to sunitinib stimulation. To confirm it, HEK293 cells were transfected with human HA-MDM2 (+MDM2) or siRNA to MDM2 (−MDM2). Following 24 h serum starvation, cells were stimulated with 10 nM sunitinib for 10 min. In over-expressed MDM2 cells, IGF-1R was more ubiquitinated than that in mock-transfected cells (control), and no ubiquitination in −MDM2 cells. These results mean that sunitinib mediates ubiquitination of IGF-1R dependent on MDM2. In summary, sunitinib could block signaling transduction and mediate degradation of IGF-1R.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F-actin have revealed a number of different conformations with calponin homology domains in ‘open’ and ‘closed’ positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F-actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F-actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino-terminal utrophin actin binding domain is in an open conformation in solution and when bound to F-actin. Highlights ► Utrophin actin binding domain crystallises as an open dimer but is monomeric in solution. ► We introduced cysteines in the CH domains to lock the monomer closed. ► Utrophin bound to F-actin with reduced affinity in the closed conformation. ► Differential scanning calorimetry confirmed the open conformation of utrophin on actin.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Oxidative stress has been associated with prostate cancer development and progression due to an increase of reactive oxygen species (ROS). However, the mechanisms whereby ROS and the antioxidant system participate in cancer progression remain unclear. In order to clarify the influence of oxidative stress in prostate cancer progression, we performed this study in two human prostate cancer cell lines, PC3 and HPV10 (from metastasis and from localized cancer, respectively) and RWPE1 cells derived from normal prostate epithelium. Cells were treated with hydrogen peroxide (H 2 O 2 ) and PC3 cells were also treated with diethyl maleate (DEM). The effect on cell growth, viability, mitochondria membrane potential and oxidative stress was analysed. Oxidative stress was evaluated based on ROS production, oxidative lesion of lipids (MDA) and on determination of antioxidants, including enzyme activity of glutathione peroxidase (Gl-Px), glutathione reductase (Gl-Red) and on the quantification of glutathione (GSH), glutathione-s-transferase (GST) and total antioxidant status (TAS). PC3 shows higher ROS production but also the highest GSH levels and Gl-Red activity, possibly contributing to oxidative stress resistance. This is also associated with higher mitochondrial membrane potential, TAS and lower lipid peroxidation. On the other hand, we identified Gl-Red activity reduction as a new strategy in overcoming oxidative stress resistance, by inducing H 2 O 2 cytotoxicity. Therefore these results suggest Gl-Red activity reduction as a new potential therapeutic approach, in prostate cancer. Highlights ► Oxidative stress was evaluated on a cell line model of prostate cancer progression. ► Metastatic cell lines show the highest ROS, total antioxidant status and resistance to H 2 O 2 . ► Metastatic cell lines show the highest levels of GSH and Gl-Red activity. ► Decrease in GSH levels and Gl-Red activity induced a decrease in H 2 O 2 resistance. ► Gl-Red activity reduction may be a new therapeutic approach in prostate cancer.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats ( p 〈 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes ( p 〈 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions ( p 〈 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load. Highlights ► Lipoma preferred partner (LPP) is highly expressed in the heart, especially in cardiac fibroblasts. ► LPP increases in pressure overload hypertrophy but not in myocardial infarction. ► LPP may be used as a novel marker for heart failure due to hemodynamic overload. ► Inhibition of nitric oxide upregulates LPP, but not other fibroblast proteins. ► LPP may play an important role in cardiac homeostasis and heart disease.