AlphaFind is a web-based search engine that provides fast structure-based retrieval in the entire set of AlphaFold DB structures. Unlike other protein processing tools, AlphaFind is focused entirely on tertiary structure, automatically extracting the main 3D features of each protein chain and using a machine learning model to find the most similar structures. This indexing approach and the 3D feature extraction method used by AlphaFind have both demonstrated remarkable scalability to large datasets as well as to large protein structures. The web application itself has been designed with a focus on clarity and ease of use. The searcher accepts any valid UniProt ID, Protein Data Bank ID or gene symbol as input, and returns a set of similar protein chains from AlphaFold DB, including various similarity metrics between the query and each of the retrieved results. In addition to the main search functionality, the application provides 3D visualizations of protein structure superpositions in order to allow researchers to instantly analyze the structural similarity of the retrieved results. The AlphaFind web application is available online for free and without any registration at https://alphafind.fi.muni.cz.
- MeSH
- databáze proteinů * MeSH
- internet MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- proteiny chemie genetika metabolismus MeSH
- proteom * chemie genetika MeSH
- sbalování proteinů MeSH
- software * MeSH
- strojové učení MeSH
- strukturní homologie proteinů MeSH
- vyhledávač MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny MeSH
- proteom * MeSH
Kleisin NSE4 and circular form of SMC5/6 is indispensable for DSB repair and necessary for gene targeting but is not enough for recovery of cells from DNA damage in Physcomitrella. Structural maintenance of chromosomes (SMC) complexes are involved in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play a prominent role in DNA stabilization during repair by tethering DNA at the site of lesion by a heteroduplex of SMC5 and SMC6 encircled with non-SMC components NSE1, NSE3 and kleisin NSE4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to DNA damage, kinetics of DSB repair and gene targeting. In the moss Physcomitrella (Physcomitrium patens), SMC6 and NSE4 are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight reduction of transcript levels by dCas9 binding was enough to obtain stable lines with severe DSB repair defects and specific bleomycin sensitivity. We show that survival after bleomycin or MMS treatment fully depends on active SMC6, whereas attenuation of NSE4 has little or negligible effect. We conclude that circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the DSB repair, nevertheless there are other functions associated with the SMC5/6 complex, which are critical to survive DNA damage.
- Klíčová slova
- Comet assay, DNA repair, Gene targeting, NSE4 kleisin, Physcomitrella, Physcomitrium patens, SMC5/6 complex, dCas9,
- MeSH
- DNA rostlinná genetika metabolismus MeSH
- dvouřetězcové zlomy DNA * MeSH
- fylogeneze MeSH
- genotyp MeSH
- mechy genetika metabolismus MeSH
- multiproteinové komplexy genetika metabolismus MeSH
- mutace MeSH
- oprava DNA * MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- poškození DNA * MeSH
- proteiny buněčného cyklu klasifikace genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny klasifikace genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA rostlinná MeSH
- multiproteinové komplexy MeSH
- proteiny buněčného cyklu MeSH
- rostlinné proteiny MeSH
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.
- MeSH
- adenosindifosfát farmakologie MeSH
- adenosintrifosfát farmakologie MeSH
- Caenorhabditis elegans metabolismus MeSH
- druhová specificita MeSH
- fluorescence MeSH
- interferometrie MeSH
- jednovláknová DNA metabolismus MeSH
- nukleotidy metabolismus MeSH
- proteiny Caenorhabditis elegans metabolismus MeSH
- rekombinasa Rad51 chemie metabolismus MeSH
- sekvenční homologie aminokyselin * MeSH
- stabilita proteinů účinky léků MeSH
- vazba proteinů účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenosindifosfát MeSH
- adenosintrifosfát MeSH
- jednovláknová DNA MeSH
- nukleotidy MeSH
- proteiny Caenorhabditis elegans MeSH
- rekombinasa Rad51 MeSH
Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane, is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and super-resolution imaging and flow cytometry, we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics are further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of nonpalmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.
- Klíčová slova
- LAT, MD simulations, T cells, microscopy, transmembrane domain,
- MeSH
- adaptorové proteiny signální transdukční chemie genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- glycin genetika metabolismus MeSH
- interferenční mikroskopie MeSH
- Jurkat buňky MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- membránové proteiny chemie genetika metabolismus MeSH
- mutace MeSH
- prolin genetika metabolismus MeSH
- proteinové domény MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- T-lymfocyty metabolismus MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- glycin MeSH
- LAT protein, human MeSH Prohlížeč
- membránové proteiny MeSH
- prolin MeSH
- vápník MeSH
The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.
- MeSH
- biologické modely MeSH
- Eukaryota klasifikace genetika metabolismus MeSH
- fylogeneze MeSH
- gramnegativní bakterie klasifikace genetika metabolismus MeSH
- konzervovaná sekvence MeSH
- mitochondriální proteiny klasifikace genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- molekulární evoluce * MeSH
- molekulární modely MeSH
- Naegleria klasifikace genetika metabolismus MeSH
- peroxizomy metabolismus MeSH
- protozoální proteiny klasifikace genetika metabolismus MeSH
- sekreční systém typu II klasifikace genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mitochondriální proteiny MeSH
- protozoální proteiny MeSH
- sekreční systém typu II MeSH
Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.
- Klíčová slova
- cryo-electron microscopy, genome release, receptor, structure, virus,
- MeSH
- aktivace viru fyziologie MeSH
- elektronová kryomikroskopie MeSH
- enterovirové infekce metabolismus virologie MeSH
- genom virový genetika MeSH
- HeLa buňky MeSH
- kapsida metabolismus MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- mezibuněčná adhezivní molekula-1 chemie genetika metabolismus MeSH
- molekulární modely MeSH
- Rhinovirus genetika metabolismus fyziologie MeSH
- RNA virová chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- svlékání virového obalu fyziologie MeSH
- vazba proteinů MeSH
- virion genetika metabolismus ultrastruktura MeSH
- virové plášťové proteiny chemie genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ICAM1 protein, human MeSH Prohlížeč
- mezibuněčná adhezivní molekula-1 MeSH
- RNA virová MeSH
- virové plášťové proteiny MeSH
Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.
- MeSH
- analýza přežití MeSH
- epitopy imunologie MeSH
- imunoglobulin G aplikace a dávkování imunologie MeSH
- klíšťová encefalitida imunologie prevence a kontrola virologie MeSH
- kohortové studie MeSH
- kultivované buňky MeSH
- lidé MeSH
- monoklonální protilátky aplikace a dávkování genetika imunologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- neutralizující protilátky aplikace a dávkování genetika imunologie MeSH
- proteiny virového obalu genetika imunologie MeSH
- protilátky virové aplikace a dávkování genetika imunologie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- viry klíšťové encefalitidy účinky léků imunologie fyziologie MeSH
- zkřížené reakce imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- epitopy MeSH
- imunoglobulin G MeSH
- monoklonální protilátky MeSH
- neutralizující protilátky MeSH
- proteiny virového obalu MeSH
- protilátky virové MeSH
CATH (https://www.cathdb.info) identifies domains in protein structures from wwPDB and classifies these into evolutionary superfamilies, thereby providing structural and functional annotations. There are two levels: CATH-B, a daily snapshot of the latest domain structures and superfamily assignments, and CATH+, with additional derived data, such as predicted sequence domains, and functionally coherent sequence subsets (Functional Families or FunFams). The latest CATH+ release, version 4.3, significantly increases coverage of structural and sequence data, with an addition of 65,351 fully-classified domains structures (+15%), providing 500 238 structural domains, and 151 million predicted sequence domains (+59%) assigned to 5481 superfamilies. The FunFam generation pipeline has been re-engineered to cope with the increased influx of data. Three times more sequences are captured in FunFams, with a concomitant increase in functional purity, information content and structural coverage. FunFam expansion increases the structural annotations provided for experimental GO terms (+59%). We also present CATH-FunVar web-pages displaying variations in protein sequences and their proximity to known or predicted functional sites. We present two case studies (1) putative cancer drivers and (2) SARS-CoV-2 proteins. Finally, we have improved links to and from CATH including SCOP, InterPro, Aquaria and 2DProt.
- MeSH
- anotace sekvence MeSH
- COVID-19 epidemiologie prevence a kontrola virologie MeSH
- databáze proteinů statistika a číselné údaje MeSH
- epidemie MeSH
- internet MeSH
- lidé MeSH
- proteinové domény * MeSH
- proteiny chemie genetika metabolismus MeSH
- SARS-CoV-2 genetika metabolismus fyziologie MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů metody MeSH
- sekvenční homologie aminokyselin MeSH
- virové proteiny chemie genetika metabolismus MeSH
- výpočetní biologie metody statistika a číselné údaje MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny MeSH
- virové proteiny MeSH
Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Despite genomic databases growing exponentially, classical biochemical characterization techniques are time-demanding, cost-ineffective and low-throughput. Therefore, computational methods are being developed to explore the unmapped sequence space efficiently. Selection of putative enzymes for biochemical characterization based on rational and robust analysis of all available sequences remains an unsolved problem. To address this challenge, we have developed EnzymeMiner-a web server for automated screening and annotation of diverse family members that enables selection of hits for wet-lab experiments. EnzymeMiner prioritizes sequences that are more likely to preserve the catalytic activity and are heterologously expressible in a soluble form in Escherichia coli. The solubility prediction employs the in-house SoluProt predictor developed using machine learning. EnzymeMiner reduces the time devoted to data gathering, multi-step analysis, sequence prioritization and selection from days to hours. The successful use case for the haloalkane dehalogenase family is described in a comprehensive tutorial available on the EnzymeMiner web page. EnzymeMiner is a universal tool applicable to any enzyme family that provides an interactive and easy-to-use web interface freely available at https://loschmidt.chemi.muni.cz/enzymeminer/.
- MeSH
- biokatalýza MeSH
- enzymy chemie metabolismus MeSH
- hydrolasy chemie MeSH
- rozpustnost MeSH
- sekvenční analýza proteinů MeSH
- sekvenční homologie aminokyselin MeSH
- software * MeSH
- stabilita enzymů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- enzymy MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.
- Klíčová slova
- avian retrovirus, chicken, restriction factor, tetherin,
- MeSH
- antigen stromálních buněk kostní dřeně genetika imunologie MeSH
- buněčné linie MeSH
- fibroblasty imunologie virologie MeSH
- Galliformes genetika imunologie virologie MeSH
- genové produkty gag - virus lidské imunodeficience genetika imunologie MeSH
- HEK293 buňky MeSH
- HIV-1 genetika imunologie MeSH
- interakce hostitele a patogenu genetika imunologie MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- Passeriformes genetika imunologie virologie MeSH
- ptačí proteiny genetika imunologie MeSH
- ptačí sarkom genetika imunologie virologie MeSH
- regulace genové exprese MeSH
- replikace viru MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- selekce (genetika) MeSH
- signální transdukce MeSH
- uvolnění viru z buňky MeSH
- viry ptačího sarkomu genetika imunologie patogenita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
- Názvy látek
- antigen stromálních buněk kostní dřeně MeSH
- genové produkty gag - virus lidské imunodeficience MeSH
- ptačí proteiny MeSH
