RESUMO
Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.
Assuntos
Cisteína , Dissulfetos , Biblioteca de Peptídeos , Dissulfetos/química , Cisteína/química , Motivos de Aminoácidos , Descoberta de Drogas/métodos , Sequência de Aminoácidos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Humanos , Oxirredução , Dobramento de ProteínaRESUMO
MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase âºC-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a ß5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.
Assuntos
Proteínas Proto-Oncogênicas c-met , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Humanos , Domínios Proteicos/genética , Mutação , Motivos de Aminoácidos , Análise Mutacional de DNARESUMO
In eukaryotes, the origin recognition complex (ORC) faciliates the assembly of pre-replicative complex (pre-RC) at origin DNA for replication licensing. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit is crucial for this process. Removing a segment (residues 176-200) from Orc2-IDR or mutating a key isoleucine (194) significantly inhibits replication initiation across the genome. These Orc2-IDR mutants are capable of assembling the ORC-Cdc6-Cdt1-Mcm2-7 intermediate, which exhibits impaired ATP hydrolysis and fails to be convered into the subsequent Mcm2-7-ORC complex and pre-RC. These defects can be partially rescued by the Orc2-IDR peptide. Moreover, the phosphorylation of this Orc2-IDR region by S cyclin-dependent kinase blocks its binding to Mcm2-7 complex, causing a defective pre-RC assembly. Our findings provide important insights into the multifaceted roles of ORC in supporting origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.
Assuntos
Replicação do DNA , Complexo de Reconhecimento de Origem , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fosforilação , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Origem de Replicação/genética , Ligação Proteica , Mutação , Fase G1 , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/química , Motivos de AminoácidosRESUMO
The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-ß in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.
Assuntos
Motivos de Aminoácidos , Vírus da Influenza A Subtipo H1N1 , Infecções por Orthomyxoviridae , Proteínas não Estruturais Virais , Replicação Viral , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/química , Animais , Cães , Virulência , Camundongos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/fisiologia , Infecções por Orthomyxoviridae/virologia , Humanos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/patogenicidade , FemininoRESUMO
The protein S100B is a part of the S100 protein family, which consists of at least 25 calcium-binding proteins. S100B is highly conserved across different species, supporting important biological functions. The protein was shown to play a role in gut microbiota eubiosis and is secreted in human breast milk, suggesting a physiological trophic function in newborn development. This study explores the possible presence of the S100B motif in plant genomes, and of S100B-like immunoreactive material in different plant extracts, opening up potential botanical uses for dietary supplementation. To explore the presence of the S100B motif in plants, a bioinformatic workflow was used. In addition, the immunoreactivity of S100B from vegetable and fruit samples was tested using an ELISA assay. The S100B motif was expected in silico in the genome of different edible plants belonging to the Viridiplantae clade, such as Durio zibethinus or Malus domestica and other medicinal species. S100B-like immunoreactive material was also detected in samples from fruits or leaves. The finding of S100B-like molecules in plants sheds new light on their role in phylogenesis and in the food chain. This study lays the foundation to elucidate the possible beneficial effects of plants or derivatives containing the S100B-like principle and their potential use in nutraceuticals.
Assuntos
Suplementos Nutricionais , Alimento Funcional , Plantas Comestíveis , Subunidade beta da Proteína Ligante de Cálcio S100 , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Plantas Comestíveis/química , Simulação por Computador , Motivos de Aminoácidos , Fitoterapia/métodos , Biologia Computacional/métodos , Humanos , Frutas/química , Frutas/metabolismo , Filogenia , Proteínas de Plantas/metabolismoRESUMO
To support PTM proteomic analysis and annotation in different species, we developed PTMoreR, a user-friendly tool that considers the surrounding amino acid sequences of PTM sites during BLAST, enabling a motif-centric analysis across species. By controlling sequence window similarity, PTMoreR can map phosphoproteomic results between any two species, perform site-level functional enrichment analysis, and generate kinase-substrate networks. We demonstrate that the majority of real P-sites in mice can be inferred from experimentally derived human P-sites with PTMoreR mapping. Furthermore, the compositions of 129 mammalian phosphoproteomes can also be predicted using PTMoreR. The method also identifies cross-species phosphorylation events that occur on proteins with an increased tendency to respond to the environmental factors. Moreover, the classic kinase motifs can be extracted across mammalian species, offering an evolutionary angle for refining current motifs. PTMoreR supports PTM proteomics in non-human species and facilitates quantitative phosphoproteomic analysis.
Assuntos
Mamíferos , Fosfoproteínas , Proteômica , Animais , Proteômica/métodos , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Camundongos , Fosforilação , Mamíferos/metabolismo , Especificidade da Espécie , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Software , Sequência de Aminoácidos , Proteoma/metabolismoRESUMO
BRCA2 is essential for DNA repair by homologous recombination in mitosis and meiosis. It interacts with recombinases RAD51 and DMC1 to facilitate the formation of nucleoprotein filaments on resected DNA ends that catalyse recombination-mediated repair. BRCA2's BRC repeats bind and disrupt RAD51 and DMC1 filaments, whereas its PhePP motifs bind recombinases and stabilise their nucleoprotein filaments. However, the mechanism of filament stabilisation has hitherto remained unknown. Here, we report the crystal structure of a BRCA2-DMC1 complex, revealing how core interaction sites of PhePP motifs bind to recombinases. The interaction mode is conserved for RAD51 and DMC1, which selectively bind to BRCA2's two distinct PhePP motifs via subtly divergent binding pockets. PhePP motif sequences surrounding their core interaction sites protect nucleoprotein filaments from BRC-mediated disruption. Hence, we report the structural basis of how BRCA2's PhePP motifs stabilise RAD51 and DMC1 nucleoprotein filaments for their essential roles in mitotic and meiotic recombination.
Assuntos
Proteína BRCA2 , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Ligação Proteica , Rad51 Recombinase , Rad51 Recombinase/metabolismo , Rad51 Recombinase/química , Proteína BRCA2/metabolismo , Proteína BRCA2/química , Proteína BRCA2/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleoproteínas/metabolismo , Nucleoproteínas/química , Nucleoproteínas/genética , Cristalografia por Raios X , Meiose , Sítios de Ligação , Motivos de Aminoácidos , Modelos Moleculares , MitoseRESUMO
INTRODUCTION: Dual-expressing lymphocytes (DEs) are unique immune cells that express both B cell receptors (BCRs, surface antibody) and T cell receptors (TCRs). In type 1 diabetes, DE antibodies are predominated by one antibody (x-mAb), an IgM monoclonal antibody with a germline-encoded CDR3 that recognizes self-reactive TCRs. We explored if x-mAb and its interacting TCRs have distinct structural features. METHODS: Using bioinformatics, we compared x-mAb and its most common interacting TCRαß to billions of antigen receptor sequences to determine if they were unique or randomly generated. RESULTS: X-mAb represents a unique class of human antibodies with a conserved CDR3 sequence (CARx1-4DTAMVYYFYDW), consisting of a fixed DJH motif (DTAMVYYFDYW) paired with various VH genes. A public TCRß clonotype (CASSPGTEAFF) associated with x-mAb on DEs features two invariant segments, VßD (CASSPGT) and DJß (PGTEAFF), key to two large families of public TCRß clonotypes-CASSPGT-Jßx and CASSPGT-Jßx-formed by recombining the VßD motif with Jß genes and the DJß motif with Vß genes. B cells also use CASSPGT as a VHD motif for public IGH clonotypes (CASSPGT-Jßx). DISCUSSION: DEs, unlike conventional T and B cells, use invariant motifs to create public antibodies and TCRs, a trait previously seen only in cartilaginous fish.
Assuntos
Anticorpos Monoclonais , Humanos , Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/genética , Biologia Computacional/métodos , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Motivos de Aminoácidos , Imunoglobulina M/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de AminoácidosRESUMO
Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.
Assuntos
Motivos de Aminoácidos , ATPases Translocadoras de Prótons , Humanos , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Lactoferrina/farmacologia , Lactoferrina/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Cisteína/metabolismo , Cisteína/química , Candida albicans/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacosRESUMO
Glutamate dehydrogenases (GDH) serve as the key regulated enzyme that links protein and carbohydrate metabolism. Combined with motif reassembly and mutation, novel GDHs were designed. Motif reassembly of thermophilic GDH and malate dehydrogenase aims to overcome stability and activity tradeoff in nonaqueous systems. Structural compatibility and dynamic cooperation of the designed AaDHs were studied by molecular dynamics simulation. Furthermore, multipoint mutations improved its catalytic activity for unnatural substrates. Amino acid interaction network analysis indicated that the high density of hydrogen-bonded salt bridges is beneficial to the stability. Finally, the experimental verification determines the kinetics of AaDHs in a nonaqueous system. The activity of Aa05 was increased by 1.78-fold with ionic liquid [EMIM]BF4. This study presents the strategy of a combination of rigid motif assembly and mutations of active sites for robust dehydrogenases with high activity in the nonaqueous system, which overcomes the activity-stability tradeoff effect.
Assuntos
Glutamato Desidrogenase , Simulação de Dinâmica Molecular , Glutamato Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Glutamato Desidrogenase/genética , Cinética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia de Proteínas , Estabilidade Enzimática , Domínio Catalítico , Motivos de Aminoácidos , MutaçãoRESUMO
The inversion of substrate size specificity is an evolutionary roadblock for proteins. The Duf4243 dioxygenases GedK and BTG13 are known to catalyze the aromatic cleavage of bulky tricyclic hydroquinone. In this study, we discover a Duf4243 dioxygenase PaD that favors small monocyclic hydroquinones from the penicillic-acid biosynthetic pathway. Sequence alignments between PaD and GedK and BTG13 suggest PaD has three additional motifs, namely motifs 1-3, distributed at different positions in the protein sequence. X-ray crystal structures of PaD with the substrate at high resolution show motifs 1-3 determine three loops (loops 1-3). Most intriguing, loops 1-3 stack together at the top of the pocket, creating a lid-like tertiary structure with a narrow channel and a clearly constricted opening. This drastically changes the substrate specificity by determining the entry and binding of much smaller substrates. Further genome mining suggests Duf4243 dioxygenases with motifs 1-3 belong to an evolutionary branch that is extensively involved in the biosynthesis of natural products and has the ability to degrade diverse monocyclic hydroquinone pollutants. This study showcases how natural enzymes alter the substrate specificity fundamentally by incorporating new small motifs, with a fixed overall scaffold-architecture. It will also offer a theoretical foundation for the engineering of substrate specificity in enzymes and act as a guide for the identification of aromatic dioxygenases with distinct substrate specificities.
Assuntos
Motivos de Aminoácidos , Dioxigenases , Especificidade por Substrato , Dioxigenases/metabolismo , Dioxigenases/genética , Dioxigenases/química , Cristalografia por Raios X , Hidroquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Sequência de Aminoácidos , Modelos Moleculares , Alinhamento de SequênciaRESUMO
The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKß and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBß) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/ß dimerization in substrate motif recognition.
Assuntos
Motivos de Aminoácidos , Quinase I-kappa B , NF-kappa B , Multimerização Proteica , Quinase I-kappa B/metabolismo , Quinase I-kappa B/química , Quinase I-kappa B/genética , Humanos , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/genética , Simulação de Acoplamento Molecular , Células HEK293 , Especificidade por SubstratoRESUMO
Fifty-eight million individuals worldwide are affected by chronic hepatitis C virus (HCV) infection, a primary driver of liver cancer for which no vaccine is available1. The HCV envelope proteins E1 and E2 form a heterodimer (E1/E2), which is the target for neutralizing antibodies2. However, the higher-order organization of these E1/E2 heterodimers, as well as that of any Hepacivirus envelope protein complex, remains unknown. Here we determined the cryo-electron microscopy structure of two E1/E2 heterodimers in a homodimeric arrangement. We reveal how the homodimer is established at the molecular level and provide insights into neutralizing antibody evasion and membrane fusion by HCV, as orchestrated by E2 motifs such as hypervariable region 1 and antigenic site 412, as well as the organization of the transmembrane helices, including two internal to E1. This study addresses long-standing questions on the higher-order oligomeric arrangement of Hepacivirus envelope proteins and provides a critical framework in the design of novel HCV vaccine antigens.
Assuntos
Hepacivirus , Multimerização Proteica , Proteínas do Envelope Viral , Humanos , Motivos de Aminoácidos , Anticorpos Neutralizantes/imunologia , Microscopia Crioeletrônica , Hepacivirus/química , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepacivirus/ultraestrutura , Evasão da Resposta Imune/imunologia , Fusão de Membrana , Modelos Moleculares , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/ultraestrutura , Internalização do Vírus , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologiaRESUMO
Sorting signals are crucial for the anchoring of proteins to the cell surface in archaea and bacteria. These proteins often feature distinct motifs at their C-terminus, cleaved by sortase or sortase-like enzymes. Gram-positive bacteria exhibit the LPXTGX consensus motif, cleaved by sortases, while Gram-negative bacteria employ exosortases recognizing motifs like PEP. Archaea utilize exosortase homologs known as archaeosortases for signal anchoring. Traditionally identification of such C-terminal sorting signals was performed with profile Hidden Markov Models (pHMMs). The Cell-Wall PREDiction (CW-PRED) method introduced for the first time a custom-made class HMM for proteins in Gram-positive bacteria that contain a cell wall sorting signal which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues. Here we present a new and updated version of CW-PRED for predicting C-terminal sorting signals in Archaea, Gram-positive, and Gram-negative bacteria. We used a large training set and several model enhancements that improve motif identification in order to achieve better discrimination between C-terminal signals and other proteins. Cross-validation demonstrates CW-PRED's superiority in sensitivity and specificity compared to other methods. Application of the method in reference proteomes reveals a large number of potential surface proteins not previously identified. The method is available for academic use at http://195.251.108.230/apps.compgen.org/CW-PRED/ and as standalone software.
Assuntos
Proteínas Arqueais , Proteínas de Bactérias , Sinais Direcionadores de Proteínas , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Archaea/metabolismo , Archaea/genética , Biologia Computacional/métodos , Parede Celular/metabolismo , Parede Celular/química , Cadeias de Markov , Motivos de Aminoácidos , Software , Bactérias/metabolismo , Bactérias/genética , AlgoritmosRESUMO
The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Coesinas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Ligação Proteica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Motivos de Aminoácidos , Mitose , Cromátides/metabolismo , Proteínas de Transporte , Proteínas Proto-OncogênicasRESUMO
Introduction. Cytotoxin-associated gene A (CagA) from Helicobacter pylori is highly related to chronic gastritis. Tyrosine phosphorylation of Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs from CagA determines the pathogenicity of H. pylori.Gap statement. The precise amino acid variations surrounding the EPIYA motifs and their correlation with clinical outcomes have been poorly explored.Aim. The purpose of this study was to examine the CagA 3' region polymorphism of H. pylori and its association with chronic gastritis in the Chinese population.Method. A total of 86 cagA-positive H. pylori strains were isolated from patients with chronic gastritis in two different hospitals in Beijing, PR China. Genomic DNA was extracted commercial kits, and the cagA 3' variable region of H. pylori was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analysed using the CLC Sequence Viewer, BioEdit, and WebLogo 3.Results. Two hundred and fifty-nine EPIYA motifs were identified from cagA-positive H. pylori strains. Notably, EPIYA-B exhibited a higher frequency of variation in comparison to EPIYA-A, EPIYA-C, and EPIYA-D. The prevalent sequences for East-Asian-type CagA were QVNK and TIDF, while KVNK and TIDD were most commonly observed for Western-type CagA. The CRPIA motifs of East-Asian-type CagA and Western-type CagA varied at positions 4, 6, 7, 8, and 10. CagA-ABD (73.2%) was the most prevalent type, followed by CagA-ABC (18.6%) and CagA-AB (3.4%). The ratio of CagA-ABD was observed to be higher in cases of chronic non-atrophic gastritis with erosive (NAGE) or chronic atrophic gastritis (AG) compared to chronic non-atrophic gastritis (NAG), and the difference was found to be statistically significant (χ2=59.000/64.000, P<0.001).Conclusions. The EPIYA segments of Western-type CagA and East-Asian-type CagA differ significantly and the presence of CagA-ABD may be associated with severe chronic gastritis from this study.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Gastrite , Infecções por Helicobacter , Helicobacter pylori , Polimorfismo Genético , Humanos , Antígenos de Bactérias/genética , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/epidemiologia , Masculino , Feminino , China/epidemiologia , Doença Crônica , Pessoa de Meia-Idade , Adulto , Idoso , Povo Asiático/genética , Motivos de Aminoácidos , População do Leste AsiáticoRESUMO
Chlamydia trachomatis, a leading cause of bacterial sexually transmitted infections, creates a specialized intracellular replicative niche by translocation and insertion of a diverse array of effectors (Incs [inclusion membrane proteins]) into the inclusion membrane. Here, we characterize IncE, a multifunctional Inc that encodes two non-overlapping short linear motifs (SLiMs) within its short cytosolic C terminus. The proximal SLiM, by mimicking just a small portion of an R-N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) motif, binds and recruits syntaxin (STX)7- and STX12-containing vesicles to the inclusion. The distal SLiM mimics the sorting nexin (SNX)5 and SNX6 cargo binding site to recruit SNX6-containing vesicles to the inclusion. By simultaneously binding two distinct vesicle classes, IncE brings these vesicles in close apposition with each other at the inclusion to facilitate C. trachomatis intracellular development. Our work suggests that Incs may have evolved SLiMs to enable rapid evolution in a limited protein space to disrupt host cell processes.
Assuntos
Proteínas de Bactérias , Chlamydia trachomatis , Chlamydia trachomatis/metabolismo , Humanos , Proteínas de Bactérias/metabolismo , Células HeLa , Motivos de Aminoácidos , Transporte Proteico , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Proteínas Qa-SNARE/metabolismo , Ligação ProteicaRESUMO
The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer.
Assuntos
Proteínas do Envelope de Coronavírus , Domínios PDZ , Ligação Proteica , SARS-CoV-2 , Humanos , Virulência , SARS-CoV-2/patogenicidade , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Proteínas do Envelope de Coronavírus/metabolismo , Proteínas do Envelope de Coronavírus/genética , Animais , Proteínas Viroporinas/metabolismo , Proteínas Viroporinas/genética , COVID-19/virologia , Chlorocebus aethiops , Células Vero , Motivos de Aminoácidos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Replicação ViralRESUMO
Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. The ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within the C-terminal region of RNF13 that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by this variant of the dileucine sorting motif of RNF13.
Assuntos
Motivos de Aminoácidos , Endossomos , Transporte Proteico , Ubiquitina-Proteína Ligases , Humanos , Endossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Células HeLa , Ligação Proteica , Complexo de Golgi/metabolismo , Sequência de Aminoácidos , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 1 de Proteínas Adaptadoras/genéticaRESUMO
Tn3 family transposons are a widespread group of replicative transposons, notorious for contributing to the dissemination of antibiotic resistance, particularly the global prevalence of carbapenem resistance. The transposase (TnpA) of these elements catalyzes DNA breakage and rejoining reactions required for transposition. However, the molecular mechanism for target site selection with these elements remains unclear. Here, we identify a QLxxLR motif in N-terminal of Tn3 TnpAs and demonstrate that this motif allows interaction between TnpA of Tn3 family transposon Tn1721 and the host ß-sliding clamp (DnaN), the major processivity factor of the DNA replication machinery. The TnpA-DnaN interaction is essential for Tn1721 transposition. Our work unveils a mechanism whereby Tn3 family transposons can bias transposition into certain replisomes through an interaction with the host replication machinery. This study further expands the diversity of mobile elements that use interaction with the host replication machinery to bias integration.