ALBERT

Description: 〈p〉Publication date: Available online 8 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Journal of Molecular Biology〈/p〉 〈p〉Author(s): Adam J.H. Quek, Blake A. Mazzitelli, Guojie Wu, Eleanor W.W. Leung, Tom T. Caradoc-Davies, Gordon J. Lloyd, Devadharshini Jeevarajah, Paul J. Conroy, Martina Sanderson-Smith, Yue Yuan, Yetunde A. Ayinuola, Francis J. Castellino, James C. Whisstock, Ruby H.P. Law〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Plasminogen (Plg)-binding M protein (PAM) is a group A streptococcal cell surface receptor that is crucial for bacterial virulence. Previous studies revealed that, by binding to the kringle 2 (KR2) domain of host Plg, the pathogen attains a proteolytic microenvironment on the cell surface that facilitates its dissemination from the primary infection site. Each of the PAM molecules in their dimeric assembly consists of two Plg binding motifs (called the a1 and a2 repeats). To date, the molecular interactions between the a1 repeat and KR2 have been structurally characterized, whereas the role of the a2 repeat is less well defined. Here, we report the 1.7-Å x-ray crystal structure of KR2 in complex with a monomeric PAM peptide that contains both the a1 and a2 motifs. The structure reveals how the PAM peptide forms key interactions simultaneously with two KR2 via the high-affinity lysine isosteres within the a1a2 motifs. Further studies, through combined mutagenesis and functional characterization, show that a2 is a stronger KR2 binder than a1, suggesting that these two motifs may play discrete roles in mediating the final PAM-Plg assembly.〈/p〉〈/div〉 〈/div〉 〈div xml:lang="en"〉 〈h5〉Graphical Abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0022283619304243-ga1.jpg" width="402" alt="Unlabelled Image" title="Unlabelled Image"〉〈/figure〉〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: September 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Fish & Shellfish Immunology, Volume 92〈/p〉 〈p〉Author(s): Yang Hu, Wei-Chao Chen, Yu-Feng Shen, Bin Zhu, Gao-Xue Wang〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Viral diseases in aquaculture were challenging because there are few preventative measures and/or treatments. Our previous study indicated that imidazole arctigenin derivatives possessed antiviral activities against infectious hematopoietic necrosis virus (IHNV). Based on the structure-activity relationship in that study, a new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), was designed, synthesized and its anti-IHNV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC〈sub〉50〈/sub〉), we found that EOA (IC〈sub〉50〈/sub〉 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole arctigenin derivatives in our previous study. Besides, EOA could significantly decrease cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum cyprinid (EPC) cells. In addition, EOA significantly inhibited apoptosis induced by IHNV in EPC cells. Further data verified that EOA inhibited IHNV replication in rainbow trout, with reducing 32.0% mortality of IHNV-infected fish. The results suggested that EOA was more stable with a prolonged inhibitory half-life in the early stage of virus infection (1–4 days). Consistent with above results, EOA repressed IHNV glycoprotein gene expression in virus sensitive tissues (kidney and spleen) in the early stage of virus infection. Moreover, histopathological evaluation showed that tissues from the spleen and kidney of fish infected with IHNV exhibited pathological changes. But there were no lesions in any of the tissues from the control group and EOA-treaten group. In accordance with the histopathological assay, EOA could elicited anti-inflammation response in non-viral infected rainbow trout by down-regulating the expression of cytokine genes (〈em〉IL-8〈/em〉, 〈em〉IL-12p40〈/em〉, and 〈em〉TNF-α〈/em〉). Altogether, EOA was expected to be a therapeutic agent against IHNV infection in the field of aquaculture.〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: September 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Fish & Shellfish Immunology, Volume 92〈/p〉 〈p〉Author(s): Junjun He, Haiying Liang, Jiaping Zhu, Xiaochen Fang〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Antibacterial peptides (AMPs) constitute an important part of the body's innate immune system and are responsible for a wide range of inhibitory effects against pathogens such as bacteria, fungi, and viruses. In this study, multi-step high performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify proteins with antibacterial activity from the serum of 〈em〉Pinctada fucata martensii〈/em〉 (〈em〉P.f. Martensii〈/em〉) and obtain a component named 〈em〉P.f. Martensii〈/em〉 antimicrobial peptide-1 (PmAMP-1). 〈em〉PmAMP-1〈/em〉 cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) and mRNA expression of was analyzed by quantitative real-time PCR (qRT-PCR). From the results of this study, full-length 〈em〉PmAMP-1 c〈/em〉DNA was shown to be 700 base pairs (bp) long with an open reading frame (ORF) of 294 bp, encoding 97 amino acids with a predicted structure that is mostly α-helices. 〈em〉PmAMP-〈/em〉1 mRNA was constitutively expressed in all tested tissues including the adductor muscle, mantle, hepatopancreas, gill, gonads and hemocytes. The highest level of 〈em〉PmAMP-〈/em〉1 transcription was observed at 8 h and 2 h after bacterial challenge in hemocytes and adductor muscle (p 〈 0.01), respectively. Furthermore, PmAMP-1 caused significant morphological alterations in 〈em〉E. coli,〈/em〉 as shown by transmission electron microscopy (TEM). The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: Available online 6 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Journal of Molecular Biology〈/p〉 〈p〉Author(s): Timothy A. Wencewicz〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The biosynthesis of antibiotics and self-protection mechanisms employed by antibiotic producers are an integral part of the growing antibiotic resistance threat. The origins of clinically relevant antibiotic resistance genes found in human pathogens have been traced to ancient microbial producers of antibiotics in natural environments. Widespread and frequent antibiotic use amplifies environmental pools of antibiotic resistance genes and increases the likelihood for the selection of a resistance event in human pathogens. This perspective will provide an overview of the origins of antibiotic resistance to highlight the crossroads of antibiotic biosynthesis and producer self-protection that result in clinically relevant resistance mechanisms. Some case studies of synergistic antibiotic combinations, adjuvants, and hybrid antibiotics will also be presented to show how native antibiotic producers manage the emergence of antibiotic resistance.〈/p〉〈/div〉 〈/div〉 〈div xml:lang="en"〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0022283619304310-ga1.jpg" width="411" alt="Unlabelled Image" title="Unlabelled Image"〉〈/figure〉〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: Available online 7 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Journal of Molecular Biology〈/p〉 〈p〉Author(s): Tim Rasmussen, Vanessa J. Flegler, Akiko Rasmussen, Bettina Böttcher〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Since life has emerged, gradients of osmolytes over the cell membrane cause pressure changes in the cell and require tight regulation to prevent cell rupture. The mechanosensitive channel of small conductance (MscS) releases solutes and water when a hypo-osmotic shock raises the pressure in the cell. It is a member of a large family of MscS-like channels found in bacteria, archaea, fungi and plants and model for mechanosensation. MscS senses the increase of tension in the membrane directly by the force from the lipids, but the molecular mechanism is still elusive. We determined the lipid interactions of MscS by resolving the structure of 〈em〉Escherichia coli〈/em〉 MscS embedded in membrane discs to 2.9-Å resolution using cryo-electron microscopy. The membrane is attached only to parts of the sensor paddles of MscS, but phospholipid molecules move through grooves into remote pockets on the cytosolic side. On the periplasmic side, a lipid bound by R88 at the pore entrance is separated from the membrane by TM1 helices. The N-terminus interacts with the periplasmic membrane surface. We demonstrate that the unique membrane domain of MscS promotes deep penetration of lipid molecules and shows multimodal interaction with the membrane to fine-tune tension sensing.〈/p〉〈/div〉 〈/div〉 〈div xml:lang="en"〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0022283619304279-ga1.jpg" width="301" alt="Unlabelled Image" title="Unlabelled Image"〉〈/figure〉〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: Available online 6 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Journal of Molecular Biology〈/p〉 〈p〉Author(s): Ivonne Bernal, Jonathan Börnicke, Johannes Heidemann, Dmitri Svergun, Julia A. Horstmann, Marc Erhardt, Anne Tuukkanen, Charlotte Uetrecht, Michael Kolbe〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Many medically relevant Gram‐negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multi-protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components are unknown. Here we reconstitute soluble complexes of the 〈em〉Salmonella〈/em〉 Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaO〈sub〉C〈/sub〉, which we show to be essential for the solubility of SpaO. We establish domain–domain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X‐ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaO〈sub〉C〈/sub〉/OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in 〈em〉in situ〈/em〉 cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.〈/p〉〈/div〉 〈/div〉 〈div xml:lang="en"〉 〈h5〉Graphical Abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0022283619304255-ga1.jpg" width="500" alt="Unlabelled Image" title="Unlabelled Image"〉〈/figure〉〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: Available online 6 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Journal of Molecular Biology〈/p〉 〈p〉Author(s): Jacob Gordon, Monica C. Pillon, Robin E. Stanley〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The ribosome plays a universal role in translating the cellular proteome. Defects in the ribosome assembly factor Las1L are associated with congenital lethal motor neuron disease and X-linked intellectual disability disorders, yet its role in processing precursor ribosomal RNA (pre-rRNA) is largely unclear. The Las1L endoribonuclease associates with the Nol9 polynucleotide kinase to form the internal transcribed spacer 2 (ITS2) pre-rRNA endonuclease–kinase machinery. Together, Las1L–Nol9 catalyzes RNA cleavage and phosphorylation to mark the ITS2 for degradation. While ITS2 processing is critical for the production of functional ribosomes, the regulation of mammalian Las1L–Nol9 remains obscure. Here we characterize the human Las1L–Nol9 complex and identify critical molecular features that regulate its assembly and spatial organization. We establish that Las1L and Nol9 form a higher-order complex and identify the regions responsible for orchestrating this intricate architecture. Structural analysis by high-resolution imaging defines the intricate spatial pattern of Las1L–Nol9 within the nucleolar sub-structure linked with late pre-rRNA processing events. Furthermore, we uncover a Nol9-encoded nucleolar localization sequence that is responsible for nucleolar transport of the assembled Las1L–Nol9 complex. Together, these data provide a mechanism for the assembly and nucleolar localization of the human ITS2 pre-rRNA endonuclease–kinase complex.〈/p〉〈/div〉 〈/div〉 〈div xml:lang="en"〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0022283619304309-ga1.jpg" width="281" alt="Unlabelled Image" title="Unlabelled Image"〉〈/figure〉〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: September 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Fish & Shellfish Immunology, Volume 92〈/p〉 〈p〉Author(s): Bin Zhong, Zeyin Jiang, Zhenhuang Chen, Kazue Ishihara, Huilin Mao, Shanghong Wang, Gang Lin, Chengyu Hu〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Recently, studies have shown that IκB kinase β (IKKβ), a critical kinase in the nucleus factor kappa-B (NF-κB) pathway, participates in inflammatory responses associated with unfolded protein response (UPR) and plays an important role in ER stress-induced cell death. The unfolded protein response (UPR), which is a regulatory system to restore cellular homeostasis in the endoplasmic reticulum (ER), such as oxidative stress, bacterial infection, and virus invasion. The UPR pathways have been reported to be involved in immune responses in mammals, including the classical NF-κB pathway. However, the molecular mechanism of their crosstalk remains to be elucidated. Previously, we demonstrated that IKKβ also has some conserved functions between fish and human, as grass carp (〈em〉Ctenopharyngodon idella〈/em〉) IKKβ (CiIKKβ) can activate NF-κB pathway. In this study, we found that CiIKKβ level in nucleus was elevated under ER stress and CiIKKβ can interact with grass carp X-box-binding protein 1 (CiXBP1S), a key transcription factor in UPR. Consistently, fluorescent histochemical analysis of grass carp kidney (CIK) cells indicated that CiIKKβ and CiXBP1S colocalized under ER stress. Furthermore, overexpression of CiIKKβ in CIK cells enhanced ER stress tolerance by regulating UPR signaling and resulted in the significant increase of cell viability.〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: September 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Fish & Shellfish Immunology, Volume 92〈/p〉 〈p〉Author(s): Lu-Yun Ni, Qing Han, Hong-Ping Chen, Xiao-Chun Luo, An-Xing Li, Xue-Ming Dan, Yan-Wei Li〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Macrophage expressed gene 1 (Mpeg1) is a molecule that can form pores and destroy the cell membrane of invading pathogens. In this study, we identified two Mpeg1 isoforms from the orange-spotted grouper (〈em〉Epinephelus coioides〈/em〉) and named them EcMpeg1a and EcMpeg1b. Predicted proteins of the two EcMpeg1s contained a signal peptide, a conserved membrane attack complex/perforin (MACPF) domain, a transmembrane segment, and an intracellular region. Sequence alignment demonstrated that two EcMpeg1 proteins share a high sequence identity with that of other teleosts. Tissue distribution analysis showed that EcMpeg1s were expressed in all tissues tested in healthy grouper, with the highest expression in the head kidney and spleen. After infection with the ciliate parasite 〈em〉Cryptocaryon irritans〈/em〉, expression of the two EcMpeg1s was significantly upregulated in the spleen and gills. Furthermore, the recombinant EcMpeg1a showed antiparasitic and antibacterial activity against Gram-negative and -positive bacteria, whereas EcMpeg1b had an inhibitory effect only against Gram-positive bacteria. These results indicated that EcMpeg1s play an important role in the host response against invading pathogens.〈/p〉〈/div〉 〈/div〉

Description: 〈p〉Publication date: September 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Fish & Shellfish Immunology, Volume 92〈/p〉 〈p〉Author(s): Pengfei Chu, Libo He, Cheng Yang, Wencheng Zeng, Rong Huang, Lanjie Liao, Yongming Li, Zuoyan Zhu, Yaping Wang〈/p〉 〈div xml:lang="en"〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Autophagy is an essential and conserved process that plays an important role in physiological homeostasis, adaptive response to stress and the immune response. Autophagy-related proteins (ATGs) are key components of the autophagic machinery. In the study, grass carp (〈em〉Ctenopharyngodon idella〈/em〉) autophagy-related gene 5 (〈em〉ATG5〈/em〉) and 12 (〈em〉ATG12〈/em〉) were identified. In the gill and intestine, 〈em〉ATG5〈/em〉 and 〈em〉ATG12〈/em〉 were highly expressed, but after grass carp reovirus (GCRV) infection, they were decreased significantly. In 〈em〉Ctenopharyngodon idella〈/em〉 kidney (CIK) cells, the sharp variation of 〈em〉ATG5〈/em〉 and 〈em〉ATG12〈/em〉 expression was observed after poly(I:C) infection. Subcellular localisation showed that ATG5 and ATG12 were evenly distributed in the cytoplasm and nucleus. However, the interaction between ATG5 and ATG12 was only found in cytoplasm in both 293T cells and CIK cells. In addition, the overexpression of ATG5 or ATG12 in 293T cells showed enhanced autophagy, and autophagic process was facilitated when ATG5 and ATG12 were simultaneously overexpressed. Dual-luciferase activity assay indicated that both ATG5 and ATG12 remarkably suppressed the promoter activity of 〈em〉IRF3〈/em〉, 〈em〉IRF7〈/em〉, and 〈em〉IFN-I〈/em〉. Further, ATG5 and ATG12 conjugate showed far stronger inhibitory affection on the expression of 〈em〉IFN-I〈/em〉 than either ATG5 or ATG12 in response to poly(I:C) or GCRV infection. Taken together, the results demonstrate that grass carp ATG5 and ATG12 play an important role in innate immunity and autophagy.〈/p〉〈/div〉 〈/div〉