RIG-I-like receptors detect viral RNA in infected cells and promote oligomerization of the outer mitochondrial membrane protein MAVS to induce innate immunity to viral infection through type I interferon production. Mitochondrial reactive oxygen species (mROS) have been shown to enhance anti-viral MAVS signalling, but the mechanisms have remained obscure. Using a biochemical oligomerization-reporter fused to the transmembrane domain of MAVS, we found that mROS inducers promoted lipid-dependent MAVS transmembrane domain oligomerization in the plane of the outer mitochondrial membrane. These events were mirrored by Sendai virus infection, which similarly induced lipid peroxidation and promoted lipid-dependent MAVS transmembrane domain oligomerization. Our observations point to a role for mROS-induced changes in lipid bilayer properties in modulating antiviral innate signalling by favouring the oligomerization of MAVS transmembrane domain in the outer-mitochondrial membrane. ; This work was supported by a Wellcome Trust Intermediate Clinical Fellowship (093764 o RV), a Wellcome Trust Principal Research Fellowship (084812 to DR), a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (100140) and European Union Seventh Framework Programme Grant (Beta Bat, 277713). T
Picornavirus 2B, a non-structural protein required for effective viral replication, has been implicated in cell membrane permeabilization during the late phases of infection. Here, we have approached the molecular mechanism of this process by assessing the pore-forming activity of an overlapping peptide library that spanned the complete 2B sequence. At non-cytopathic concentrations, only the P3 peptide, spanning 2B residues 35–55, effectively assembled hydrophilic pores that allowed diffusion of low molecular mass solutes across the cell plasma membrane (IC50 ≈ 4 × 10−7 M) and boundary liposome bilayers (starting at peptide to lipid molar ratios > 1:104). Circular dichroism data were consistent with its capacity to fold as a helix in a membrane-like environment. Furthermore, addition of this peptide to a sealed plasma-membrane model, consisting of retinal rod outer segments patch-clamped in a whole-cell configuration, induced ion channel activity within seconds at concentrations as low as 10−8 M. Thus, we have established a "one-helix" 2B version that possesses the intrinsic pore-forming activity required to directly and effectively permeabilize the cell plasma membrane. We conclude that 2B viroporin can be classified as a genuine pore-forming toxin of viral origin, which is produced intracellularly at certain times post infection ; We acknowledge the financial support of the DGICYT project numbers BFU2006-02182/BMC (to V.M. and L.C.) and BFU2005-06095/BMC (to S.S.M. and J.L.N.). Further support to J.L.N. was obtained from the Basque Government (AE2004-1-2) and the University of the Basque Country (042.310-13552). The CBM was awarded an institutional grant by the Fundación Ramón Areces. Financial support to G.R. included grants from the Ministero del'Università e della Ricerca (MIUR), Roma and from the "Comitato dei sostenitori dell'Università di Ferrara" (Project "Trasporto di carica fotoindotto in materiali funzionali"). ; Peer reviewed
Funding: Work in J.T, lab is supported by grant BB/M007200/1 from the U.K. Biotechnology and Biomedical Sciences Research Council (BBSRC) and by the Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS). ; In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)–plasma membrane (PM) contacts coincide with regulation of cell‐to‐cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell‐to‐cell signalling in plants, act as ER‐PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER‐PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER‐PM contacts and cell‐to‐cell signalling. ; Publisher PDF ; Peer reviewed
This work aimed at studying the potentiality of interactions between kaolinite surfaces and a protein-fragment (350–370 amino acid units) extracted from the glycoprotein E1 in the transmembrane domain (TMD) of hepatitis C virus capsid. A computational work was performed for locating the potential electrostatic interaction sites between kaolinite aluminol and siloxane surfaces and the residues of this protein-fragment ligand, monitoring the possible conformational changes. This hydrated neutralized kaolinite/protein-fragment system was simulated by means of molecular modeling based on atomistic force fields based on empirical interatomic potentials and molecular dynamic (MD) simulations. The MD calculations indicated that the studied protein-fragment interacted with the kaolinite surfaces with an exothermic process and structural distortions were observed, particularly with the hydrophilic aluminol surface by favorable adsorption energy. The viral units isolation or trapping by the adsorption on the kaolinite nanoparticles producing structural distortion of the peptide ligands could lead to the blockage of the entry on the receptor and hence a lack of viral activity would be produced. Therefore, these findings with the proposed insights could be an useful information for the next experimental and development studies in the area of discovering inhibitors of the global challenged hepatitis and other pathogenic viruses based on the phyllosilicate surface activity. These MD studies can be extended to other viruses like the COVID-19 interacting with silicate minerals surfaces. ; Authors would like to acknowledge the contribution of the European COST Action CA17120 supported by the EU Framework Program Horizon 2020 and the financial support of the Andalusia Government projects [RNM1897 and RNM363, and CTS-946]; the MINECO and FEDER projects [FIS2016-77692-C2-2-P, PCIN-2017-098]. ; Peer reviewed
A coding polymorphism of human ATG16L1 (rs2241880; T300A) increases the risk of Crohn's disease and it has been shown to enhance susceptibility of ATG16L1 to caspase cleavage. Here we show that T300A also alters the ability of the C-terminal WD40-repeat domain of ATG16L1 to interact with an amino acid motif that recognizes this region. Such alteration impairs the unconventional autophagic activity of TMEM59, a transmembrane protein that contains the WD40 domain-binding motif, and disrupts its normal intracellular trafficking and its ability to engage ATG16L1 in response to bacterial infection. TMEM59-induced autophagy is blunted in cells expressing the fragments generated by caspase processing of the ATG16L1-T300A risk allele, whereas canonical autophagy remains unaffected. These results suggest that the T300A polymorphism alters the function of motif-containing molecules that engage ATG16L1 through the WD40 domain, either by influencing this interaction under non-stressful conditions or by inhibiting their downstream autophagic signalling after caspase-mediated cleavage. ; This work was funded by grants from the Ministerio de Ciencia e Innovación (Ref. SAF2011-23714) and the Ministerio de Economía y Competitividad (Ref. SAF2014-53320-R) of the Spanish Government, the Broad Medical Research Program at Crohn's and Colitis Foundation of America (CCFA; Ref. IBD-0369), the Junta de Castilla y León local government (Department of Education (Refs. CSI001A10-2, FIC016U14) and Department of Health (Ref. SAN11-FXP)) and the Fundación Solorzano (Refs. FS/1-2009 and FS/18-2014). R.J.X. is funded by the NIH (grants R01DK097485, P30DK043351 and U19AI109725) and the Helmsley Trust. Additional funding comes from the FEDER programme of the European Union. E.B.R. and I.S.G. are graduate students funded by predoctoral fellowships from the FPU programme (Ministerio de Educación, MEC, Spanish Government) and the Fundación Moraza, respectively. ; Peer Reviewed
Retrotransposons and retroviruses share similar intracellular life cycles and major encoded proteins, but retrotransposons lack the envelope (env) critical for infectivity. Retrotransposons are ubiquitous and abundant in plants and active retroviruses are known in animals. Although a few env-containing retroelements,gypsy-like Athila, Cyclops, andCalypso and copia-like SIRE-1, have been identified in plants, the general presence and functionality of the domain remains unclear. We show here that env-class elements are present throughout the flowering plants and are widely transcribed. Within the grasses, we show the transcription of the envdomain itself for Bagy-2 and related retrotransposons, all members of the Athila group. Furthermore, Bagy-2transcripts undergo splicing to generate a subgenomic envproduct as do those of retroviruses. Transcription and the polymorphism of their insertion sites in closely related barley cultivars suggests that at least some are propagationally active. The putative ENV polypeptides of Bagy-2 and rice Rigy-2 contain predicted leucine zipper and transmembrane domains typical of retroviral ENVs. These findings raise the prospect of active retroviral agents among the plants. ; C.M.V was supported by Academy of Finland Project 44404, R.K. by the European Union Research Directorate under contract QLK5- 1999-01499, and A.H.S. in part by an Academy of Finland Senior Fellowship. ; Peer reviewed
Full length paper ; Melatonin receptor 1A gene is the prime receptor mediating the effect of melatonin at the neuroendocrine level for control of seasonal reproduction in sheep. The aims of this study were to examine the polymorphism pattern of coding sequence of MTNR1A gene in Chokla sheep, a breed of Indian arid tract and to identify new variations in relation to its aseasonal status. Genomic DNAs of 101 Chokla sheep were collected and an 824 bp coding sequence of Exon II was amplified. RFLP was performed with enzyme RsaI and MnlI to assess the presence of polymorphism at position C606T and G612A, respectively. Genotyping revealed significantly higher frequency of M and R alleles than m and r alleles. RR and MM were found to be dominantly present in the group of studied population. Cloning and sequencing of Exon II followed by mutation/polymorphism analysis revealed ten mutations of which three were non-synonymous mutations (G706A, C893A, G931C). G706A leads to substitution of valine by isoleucine Val125I (U14109) in the fifth transmembrane domain. C893A leads to substitution of alanine by aspartic acid in the third extracellular loop. G931C mutation brings about substitution of amino acid alanine by proline in the seventh transmembrane helix, can affect the conformational stability of the molecule. Polyphen-2 analysis revealed that the polymorphism at position 931 is potentially damaging while the mutations at positions 706 and 893 were benign.It is concluded that G931C mutation of MTNR 1A gene, may explain, in part, the importance of melatonin structure integrity in influencing seasonality in sheep. ; Department of Biotechnology, Government of India (Grant/Project No. BT/PR14462/AAQ/01/442/2010)
Biosilicification (the formation of biological structures from silica) occurs in diverse eukaryotic lineages, plays a major role in global biogeochemical cycles, and has significant biotechnological applications. Silicon (Si) uptake is crucial for biosilicification, yet the evolutionary history of the transporters involved remains poorly known. Recent evidence suggests that the SIT family of Si transporters, initially identified in diatoms, may be widely distributed, with an extended family of related transporters (SIT-Ls) present in some nonsilicified organisms. Here, we identify SITs and SIT-Ls in a range of eukaryotes, including major silicified lineages (radiolarians and chrysophytes) and also bacterial SIT-Ls. Our evidence suggests that the symmetrical 10-transmembrane-domain SIT structure has independently evolved multiple times via duplication and fusion of 5-transmembrane-domain SIT-Ls. We also identify a second gene family, similar to the active Si transporter Lsi2, that is broadly distributed amongst siliceous and nonsiliceous eukaryotes. Our analyses resolve a distinct group of Lsi2-like genes, including plant and diatom Si-responsive genes, and sequences unique to siliceous sponges and choanoflagellates. The SIT/SIT-L and Lsi2 transporter families likely contribute to biosilicification in diverse lineages, indicating an ancient role for Si transport in eukaryotes. We propose that these Si transporters may have arisen initially to prevent Si toxicity in the high Si Precambrian oceans, with subsequent biologically induced reductions in Si concentrations of Phanerozoic seas leading to widespread losses of SIT, SIT-L, and Lsi2-like genes in diverse lineages. Thus, the origin and diversification of two independent Si transporter families both drove and were driven by ancient ocean Si levels. ; This work was supported by the EDEN Evo-Dev-Eco Network Research Exchange Fund, the Musgrave Pratt Fund (Department of Zoology, University of Cambridge) and the Parke-Davis Fund (University of Cambridge) to AOM; the European Research Council (Advanced Investigator Grant No. 247333) and the Wellcome Trust Senior Investigator Award to AOM and REG; the European Research Council [starting grant No. 282101 to Paul Curnow under the European Union's Seventh Framework Programme (FP7/2007-2013)] (to SR); the National Environmental Research Council (grant no. NE/J021954/1) to GLW; the Howard Hughes Medical Institute to NK; the Japan Science and Technology Agency-Centre National de la Recherche Scientifique program to FN; and a National Defense Science and Engineering Graduate fellowship from the United States Department of Defense, a National Science Foundation Central Europe Summer Research Institute Fellowship, a Chang-Lin Tien Fellowship in Environmental Sciences and Biodiversity, a postdoctoral fellowship from the Conseil Régional de Bretagne, and the French Government "Investissements d'Avenir" program OCEANOMICS (ANR-11-BTBR-0008) to DJR. ; This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/molbev/msw209
The transient receptor potential melastatin (TRPM) subfamily belongs to the TRP cation channels family. Since the first cloning of TRPM1 in 1989, tremendous progress has been made in identifying novel members of the TRPM subfamily and their functions. The TRPM subfamily is composed of eight members consisting of four six-transmembrane domain subunits, resulting in homomeric or heteromeric channels. From a structural point of view, based on the homology sequence of the coiled-coil in the C-terminus, the eight TRPM members are clustered into four groups: TRPM1/M3, M2/M8, M4/M5 and M6/M7. TRPM subfamily members have been involved in several physiological functions. However, they are also linked to diverse pathophysiological human processes. Alterations in the expression and function of TRPM subfamily ion channels might generate several human diseases including cardiovascular and neurodegenerative alterations, organ dysfunction, cancer and many other channelopathies. These effects position them as remarkable putative targets for novel diagnostic strategies, drug design and therapeutic approaches. Here, we review the current knowledge about the main characteristics of all members of the TRPM family, focusing on their actions in human diseases. ; ANID-FONDECYT 1201039 1200944 Iniciativa Cientifica Milenio ANID, Chile Millennium Institute on Immunology and Immunotherapy P09-016-F National Research and Development Agency (ANID), Government of Chile AFB180001
Background Plastid-encoded RNA polymerase (PEP) plays an essential role in chloroplast development by governing the expression of genes involved in photosynthesis. At least 12 PEP-associated proteins (PAPs), including FSD3/PAP4, regulate PEP activity and chloroplast development by modulating formation of the PEP complex. Results In this study, we identified FSD3S, a splicing variant of FSD3; the FSD3 and FSD3S transcripts encode proteins with identical N-termini, but different C-termini. Characterization of FSD3 and FSD3S proteins showed that the C-terminal region of FSD3S contains a transmembrane domain, which promotes FSD3S localization to the chloroplast membrane but not to nucleoids, in contrast to FSD3, which localizes to the chloroplast nucleoid. We also found that overexpression of FSD3S negatively affects photosynthetic activity and chloroplast development by reducing expression of genes involved in photosynthesis. In addition, FSD3S failed to complement the chloroplast developmental defects in the fsd3 mutant. Conclusion These results suggest FSD3 and FSD3S, with their distinct localization patterns, have different functions in chloroplast development, and FSD3S negatively regulates expression of PEP-dependent chloroplast genes, and development of chloroplasts. ; This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01323901 and PJ01364301) Rural Development Administration, Republic of Korea, and the National Research Foundation of Korea Grant funded by the Korean Government (MOE) [NRF-2019R1A2C1007103].
The infectious disease CoViD-19 is caused by a new severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). A possible infection mechanism includes dual host receptor recognitions by the SARS-CoV-2 transmembrane spike (S) glycoproteins. SARS-CoV-2 S contains two different domains, the receptor-binding domain (RBD) and the N-terminal domain (NTD), which interact with angiotensin-converting enzyme 2 (ACE2) and the ganglioside-rich domain of the plasma membrane at the surface of respiratory cell, respectively. NTD amino acids (111-162) form a functional ganglioside-binding domain (GBD) that is conserved in all clinical isolates. Herein, the single point mutations (SPMs) of GBD residues to which the virus is prone during genetic adaptation are predicted using an in silico protein engineering approach. Consequently, their effects on the attachment of SARS-CoV-2 S to the ganglioside-linked 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using molecular docking simulations. Val120Tyr and Asn122Trp are found to be the most likely SPMs in the GBD of SARS-CoV-2 S being involved in very specific recognition with 9-O-Ac-Sia through electrostatic interactions. Val120Tyr and Asn122Trp are also found to be the most likely SPMs in the GBD of SARS-CoV-2 S that is involved in conspicuously hydrophobic recognition with hidroxychloroquine (Hcq), thereby indicating the ability of Hcq to competitively inhibit GBD interactions with lipid rafts. However, the considerably non-specific binding of Hcq and the micromolar range of the dissociation constants of the SARS-CoV-2 S/Hcq complexes do not support the proposal of treating Hcq as a drug candidate. A usable guideline for the structure-based design of anti-CoViD-19 drugs is given.
Trehalose provides the main energy source for the physiological activities of insects, especially in adverse conditions. Trehalase is the only enzyme that hydrolyzes trehalose, therefore it is important to clarify the distribution and expression of trehalase under adverse conditions such as high temperatures and starvation. Here, we have cloned the trehalase genes and investigated their expression in different tissues, at multiple development stages, and with the treatments of high temperature and starvation in Bombus lantschouensis, which is considered to be one of the most commercially viable native species in China. The results suggest that the membrane-bound (BlTre-2) cDNA has an open reading frame of 1986 nucleotides, which encodes a protein of 662 amino acids, and two putative transmembrane domains. qRT-PCR analysis indicated that BlTre-2 was expressed in 10 tissues and at nine development stages, with the highest expression in general in 30-day-old workers, and in ovarian tissue in particular. The expression of BlTre-1 for 15-day-old workers which were exposed to a pre-treatment of 45°C increased over the first 5 h and subsequently decreased over time. In contrast the expression of BlTre-2 consistently decreased over time. The highest expression levels of BlTre-1 and BlTre-2 were observed the newly emerged adult workers when starved for 16-20 h. These results indicate that BlTre-2 may be part of the carbohydrate metabolism of the bumblebee, and that BlTre-1 is a key gene regulating energy metabolism and providing trehalose when exposed to a high temperature. Both BlTre-1 and BlTre-2 may balance trehalose and provide energy when B. lantschouensis is starved.
To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access. ; Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10(-7), odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain. ; National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) DK064869 DK062432 National Human Genome Research Institute (NHGRI) DK064869 DK043351 HG005923 Crohns and Colitis Foundation 3765 Leona M. & Harry B. Helmsley Charitable Trust 2015PG-IBD001 Amgen 2013583217 CCFA 3765 Cedars-Sinai F. Widjaja Foundation, info:eu-repo/grantAgreement/EC/FP7/305479, European Union DK062413 AI067068 U54DE023789-01 Leona M. and Harry B. Helmsley Charitable Trust Crohn's and Colitis Foundation of America NIH DK062431 U01 DK062429 U01 DK062422 R01 DK092235 U01 DK062420 Medical Research Council, UK MR/J00314X/1 Wellcome Trust WT091310 098051 Inflammatory Bowel Disease Genetic Research Chair at the University of Pittsburgh PO1DK046763
Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. ; The work of M.F-M. is supported by the CNIO (Caja Navarra International Postdoctoral Program) and the Miguel Servet (FIS) grant. The work of V.L. is supported by the French ANR MIRI BLAN08-1335497 Project and the ERC Advanced Grant Sisyphe. This study is supported by the Spanish National Bioinformatics Institute (INB-ISCIII) and by grants from the Spanish Government (CONSOLIDER CSD2007-00050, BIO2007-666855, and BIO2011-26205), European Commission FP7 project ASSET (HEALTH-F4-2010-259348), and the NHGRI-NIH ENCODE grants (U54 HG00455-04 and U54 HG004557)
