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1.
Proc Natl Acad Sci U S A ; 119(23): e2202799119, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35648833

RESUMO

SignificanceThe Smc5/6 complex plays multiple roles in DNA replication and repair. Its genome-protecting functions rely on its interaction with DNA; however, how this complex engages DNA is poorly understood. We report on a cryogenic electron microscopy structure of DNA-bound budding yeast Smc5/6 complex, revealing that its subunits form a clamp to encircle a double-helical DNA. We define the multi-subunit interactions forming the DNA clamp and the DNA binding sites distributed among subunits. We identify subunit transformations upon DNA capture and functional effects conferred by its multiple DNA contact sites. Our findings, in conjunction with studies on other structural maintenance of chromosomes (SMC) complexes, suggest a common SMC DNA-clamp mechanism with individual complex specific features that enable diverse genome organization and protection functions by SMC family complexes.


Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , DNA Fúngico/química , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
2.
Dis Markers ; 2022: 1313359, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35686033

RESUMO

Hepatocellular carcinoma (HCC) is one of the malignancies with an extremely inferior prognosis in the abdominal cavity, making it essential to develop more effective biomarkers for HCC. Although GNG5 has been linked to increased patient survival in a variety of human malignancies, no evidence has been found for its involvement in the development of HCC yet. Our study first analyzed the expression and prognosis of GNG5 in HCC using The Cancer Genome Atlas database (TCGA database) with the Gene Expression Omnibus database (GEO database) and found that GNG5 has a potential oncogenic role. Based on survival analysis, the clinical importance and prognostic value of the GNG5 gene were studied. Relying on tumor Immune Estimation Resource database (TIMER database), we analyzed the correlation between the GNG5 gene and HCC Immune infiltration cells. GNG5 expression levels were significantly higher in HCC tissues compared to normal liver tissues. HCC patients with high GNG5 expression had significantly reduced overall survival time and affected multiple immune cell infiltrates. Additionally, KEGG functional enrichment analysis indicated the PI3K-Akt signaling pathway as the most promising carcinogenic pathway associated with GNG5. This is the first comprehensive revelation of GNG5 as a possible new biological marker associated with immune infiltration in HCC. Additionally, it holds promise as an emerging target for HCC immunotherapy.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Biomarcadores Tumorais/genética , Carcinogênese , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/genética , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Subunidades Proteicas
3.
Science ; 376(6598): eabm9326, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35679401

RESUMO

INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the Xenopus laevis oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of X. laevis NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of X. laevis Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text].


Assuntos
Inteligência Artificial , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Proteínas de Xenopus , Animais , Microscopia Crioeletrônica , Citosol/metabolismo , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Oócitos , Conformação Proteica , Subunidades Proteicas/metabolismo , Software , Proteínas de Xenopus/química , Xenopus laevis/metabolismo
4.
Nat Commun ; 13(1): 3272, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35672408

RESUMO

The activity of V-ATPase is well-known to be regulated by reversible dissociation of its V1 and Vo domains in response to growth factor stimulation, nutrient sensing, and cellular differentiation. The molecular basis of its regulation by an endogenous modulator without affecting V-ATPase assembly remains unclear. Here, we discover that a lysosome-anchored protein termed (mammalian Enhancer-of-Akt-1-7 (mEAK7)) binds to intact V-ATPase. We determine cryo-EM structure of human mEAK7 in complex with human V-ATPase in native lipid-containing nanodiscs. The structure reveals that the TLDc domain of mEAK7 engages with subunits A, B, and E, while its C-terminal domain binds to subunit D, presumably blocking V1-Vo torque transmission. Our functional studies suggest that mEAK7, which may act as a V-ATPase inhibitor, does not affect the activity of V-ATPase in vitro. However, overexpression of mEAK7 in HCT116 cells that stably express subunit a4 of V-ATPase represses the phosphorylation of ribosomal protein S6. Thus, this finding suggests that mEAK7 potentially links mTOR signaling with V-ATPase activity.


Assuntos
ATPases Vacuolares Próton-Translocadoras , Humanos , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
5.
Int Immunopharmacol ; 109: 108906, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35671640

RESUMO

BACKGROUND: High speed of COVID-19 vaccination has raised some concerns about the safety of the new vaccines. It is of a great importance to perform a review of the safety and efficacy of the COVID-19 vaccines. METHODS: Two International electronic databases (PubMed, ISI) were searched for clinical trials reporting efficacy and safety of COVID-19 vaccines compared to control group. Pooled risk ratio (RR) for total, systemic and local adverse events following immunization was calculated for different vaccine modalities. RESULTS: The pooled RRs of total adverse reactions for Inactivated, mRNA, and vector vaccines were 1.46 (95% CI: 1.19-1.78), 2.01 (95% CI: 1.82 - 2.23), and 1.65 (95% CI: 1.31 - 2.32) respectively. The pooled RR for occurrence of systemic adverse reactions following immunization for different vaccine modalities was 1.13 (95% CI: 0.79 - 1.61), 1.53 (95% CI 1.08 - 2.16), 1.58 (95% CI: 1.13 - 1.90), 0.72 (95% CI: 0.34 - 1.55), and 1.62 (95% CI: 1.39 - 1.89) for inactivated vaccine, mRNA, vector, DNA, and protein subunit vaccines respectively. The pooled RR of local adverse event following immunization with inactivated vaccine, mRNA vaccine, vector vaccine, DNA vaccine, and protein subunit vaccine was 2.18 (95% CI: 1.32 - 3.59), 4.96 (95% CI: 4.02 - 6.11), 1.48 (95% CI: 0.88-2.50) 1.04 (95% CI: 0.12-8.75), and 4.09 (95% CI: 2.63-6.35) respectively. CONCLUSION: mRNA vaccines are associated with greater risk of adverse events following immunization. However, at the present moment the benefits of all types of vaccines approved by WHO, still outweigh the risks of them and vaccination if available, is highly recommended.


Assuntos
Vacinas contra COVID-19 , COVID-19 , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Humanos , Subunidades Proteicas , RNA Mensageiro , Vacinação/efeitos adversos , Vacinas de Produtos Inativados/efeitos adversos , Vacinas Sintéticas
6.
J Virol ; 96(12): e0049422, 2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35604143

RESUMO

G protein subunit ß1 (GNB1), the beta subunit of the G protein family, plays an important role in regulating transmembrane signal transduction. Although a recent study has demonstrated that GNB1 can bind the matrix protein 1 (M1) to facilitate M1 transport to budding sites and promote the release of progeny influenza A virus (IAV), whether the GNB1 protein has other functions in IAV replication requires further study. Here, we found that GNB1 promoted IAV replication, as virus yield decreased in GNB1 knockdown or knockout cells. GNB1 interacted with polymerase subunits PB2, PB1, and PA. Overexpressed GNB1 facilitated PB2 binding to importin α3, α5, and α7 promoting the nuclear import of PB2, enhancing viral RNA synthesis and polymerase activity. Altogether, our results demonstrated that GNB1 positively regulates virus replication by interacting with polymerase subunits and facilitating the nuclear import of PB2, which provide novel insights into the molecular mechanism of IAV. IMPORTANCE Until now, there has been only one article on the role of GNB1 in IAV budding. No study has investigated the role of GNB1 in IAV replication. In this study, our research demonstrated that GNB1 could increase the interaction between PB2 and the importin α isoform and mediate the nuclear import of PB2. Therefore, GNB1 could promote viral replication and transcription. Our results provide a better understanding of the molecular mechanisms of viral replication and provide potential antiviral drug targets.


Assuntos
Vírus da Influenza A , Influenza Humana , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Influenza Humana/genética , Subunidades Proteicas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genética
7.
Front Immunol ; 13: 875236, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35514964

RESUMO

A variety of methods have been explored to increase delivery efficiencies for DNA vaccine. However, the immunogenicity of DNA vaccines has not been satisfactorily improved. Unlike most of the previous attempts, we provided evidence suggesting that changing the injection site successively (successively site-translocated inoculation, SSTI) could significantly enhance the immunogenicity of DNA vaccines in a previous study. To simplify the strategy and to evaluate its impact on candidate SARS-CoV-2 vaccines, we immunized mice with either a SARS-CoV-2 spike-based DNA vaccine or a spike protein subunit vaccine via three different inoculation strategies. Our data demonstrated that S protein specific antibody responses elicited by the DNA vaccine or the protein subunit vaccine showed no significant difference among different inoculation strategies. Of interest, compared with the conventional site fixed inoculation (SFI), both successive site-translocating inoculation (SSTI) and the simplified translocating inoculation (STI) strategy improved specific T cell responses elicited by the DNA vaccine. More specifically, the SSTI strategy significantly improved both the monofunctional (IFN-γ+IL-2-TNF-α-CD8+) and the multifunctional (IFN-γ+IL-2-TNF-α+CD8+, IFN-γ+IL-2-TNF-α+CD4+, IFN-γ+IL-2+TNF-α+CD4+) T cell responses, while the simplified translocating inoculation (STI) strategy significantly improved the multifunctional CD8+ (IFN-γ+IL-2-TNF-α+CD8+, IFN-γ+IL-2+TNF-α+CD8+) and CD4+ (IFN-γ+IL-2-TNF-α+CD4+, IFN-γ+IL-2+TNF-α+CD4+) T cell responses. The current study confirmed that changing the site of intra muscular injection can significantly improve the immunogenicity of DNA vaccines.


Assuntos
COVID-19 , Doenças Sexualmente Transmissíveis , Vacinas de DNA , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Interleucina-2 , Camundongos , Subunidades Proteicas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Fator de Necrose Tumoral alfa
8.
Protein Sci ; 31(6): e4313, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35634769

RESUMO

Pigeon iron-sulfur (Fe-S) cluster assembly 1 homolog (clISCA1) is a target protein for research into the biomagnetoreception mechanism, as the clCRY4/clISCA1 oligomer, a complex composed of the columnar clISCA1 oligomer and the magnetosensor candidate protein cryptochrome-4 (clCRY4) oligomer, tends to orient itself along weak magnetic fields, such as geomagnetic fields, under blue light. To obtain insight into the magnetic orientation mechanism of the clCRY4/clISCA1 oligomer, we inspected magnetic field effects on the structure and molecular behavior of clISCA1 by small angle X-ray scattering analysis. The results indicated that the clISCA1 protomer took the Fe-S cluster-bound globular form and unbound rod-like form. The globular clISCA1 protomer assembled to form columnar oligomers, which allowed for the binding of many Fe-S clusters at the interface between clISCA1 protomers. Moreover, the translational diffusion and the columnar oligomerization of clISCA1 were controlled by the external static magnetic field and Fe-S clusters bound to clISCA1. However, the columnar clISCA1 oligomer was not oriented along the external static magnetic field (~1 T) when clCRY4 was not bound to clISCA1. This result indicated that clCRY4 has a function to enhance the magnetic orientational property of clCRY4/clISCA1 oligomer.


Assuntos
Proteínas Ferro-Enxofre , Animais , Columbidae/metabolismo , Proteínas Ferro-Enxofre/genética , Campos Magnéticos , Subunidades Proteicas/metabolismo , Enxofre
9.
Bioorg Chem ; 124: 105755, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35551043

RESUMO

Drug repositioning is one of the most effective approaches towards drug discovery and development. It involves the identification of new therapeutic indications of existing drugs. The present study evaluated several drugs for their ability to modulate activity of the p8 subunit of TFIIH complex. Negative modulation of p8 subunit activity disrupts protein-protein interactions (PPIs) among the subunits of TFIIH complex, and thereby the TFIIH-associated functions. TFIIH complex has key role in the transcription and nucleotide excision repair activity in cancerous cells. TFIIH complex has emerged as a privileged drug target in anticancer research. Out of 60 drugs, amlopipine (13), diltiazem (16), gemfibrozil (19), levocitrizine dihydrochloride (20), losartan potassium (22), clorthalidone (24), and escitalopram (28) showed interactions with subunit p8 in the ligand-protein binding and chemical shift perturbation studies. The Kd values were found to be between 0.25 and 1 mM. These drugs also caused thermal destabilization of the subunit p8 by negatively shifting the melting temperature by ≥ 2 °C. Molecular docking studies indicated the interaction of these drugs with important residues of p8-p52 complex, such as Glu48, Lys51, Glu496, and Glu455 via non-covalent interactions. This study has thereby identified 7 drugs that can be investigated further as potential anticancer drugs.


Assuntos
Antineoplásicos , Reposicionamento de Medicamentos , Antineoplásicos/farmacologia , Simulação de Acoplamento Molecular , Subunidades Proteicas/química , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição Genética
11.
Signal Transduct Target Ther ; 7(1): 146, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35504917

RESUMO

With the constantly mutating of SARS-CoV-2 and the emergence of Variants of Concern (VOC), the implementation of vaccination is critically important. Existing SARS-CoV-2 vaccines mainly include inactivated, live attenuated, viral vector, protein subunit, RNA, DNA, and virus-like particle (VLP) vaccines. Viral vector vaccines, protein subunit vaccines, and mRNA vaccines may induce additional cellular or humoral immune regulations, including Th cell responses and germinal center responses, and form relevant memory cells, greatly improving their efficiency. However, some viral vector or mRNA vaccines may be associated with complications like thrombocytopenia and myocarditis, raising concerns about the safety of these COVID-19 vaccines. Here, we systemically assess the safety and efficacy of COVID-19 vaccines, including the possible complications and different effects on pregnant women, the elderly, people with immune diseases and acquired immunodeficiency syndrome (AIDS), transplant recipients, and cancer patients. Based on the current analysis, governments and relevant agencies are recommended to continue to advance the vaccine immunization process. Simultaneously, special attention should be paid to the health status of the vaccines, timely treatment of complications, vaccine development, and ensuring the lives and health of patients. In addition, available measures such as mix-and-match vaccination, developing new vaccines like nanoparticle vaccines, and optimizing immune adjuvant to improve vaccine safety and efficacy could be considered.


Assuntos
Vacinas contra COVID-19 , Idoso , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/uso terapêutico , Feminino , Humanos , Gravidez , Subunidades Proteicas , SARS-CoV-2/genética , Vacinas de Partículas Semelhantes a Vírus
12.
Genes (Basel) ; 13(4)2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35456499

RESUMO

BACKGROUND: GNG4, a member of the G-protein γ family, is a marker of poor overall survival (OS) rates in some malignancies. However, the potential role of GNG4 in bladder cancer (BLCA) is unknown. It is also unclear whether GNG4 may be utilized as a marker to guide chemotherapy or immunotherapy. METHODS: Single-cell RNA sequencing data were used to explore the expression of GNG4 in tumor microenvironment of BLCA. Bulk RNA sequencing data from TCGA were used to evaluate the relationship between GNG4 expression and biological features, such as immune cell infiltrations and gene mutations. The associations between GNG4 expression and survival in BLCA patients under or not under immunotherapy were evaluated using seven BLCA cohorts. RESULTS: GNG4 was specifically expressed in exhausted CD4+ T cells. And the high expression of the GNG4 was associated with high level of immune cell infiltration. The high-GNG4-expression group displayed a better response to immunotherapy, whereas patients in the low-GNG4-expression group often benefited from chemotherapy. Moreover, the high-GNG4 group was more similar to the basal group, whereas the low-GNG4 group was similar to the luminal group. CONCLUSIONS: GNG4 may be a potential biomarker for the prediction of the response to therapy in BLCA. Higher GNG4 expression can be used as a predictor of response to immunotherapy, and lower GNG4 expression can be used as a predictor of response to chemotherapy.


Assuntos
Neoplasias da Bexiga Urinária , Biomarcadores Tumorais/genética , Feminino , Humanos , Imunoterapia , Masculino , Prognóstico , Subunidades Proteicas , Microambiente Tumoral/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética
13.
J Am Soc Mass Spectrom ; 33(5): 851-858, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35467879

RESUMO

With the recent improvements in ion mobility resolution, it is now possible to separate small protomeric tautomers, called protomers. In larger molecules above 1000 Da such as peptides, a few studies suggest that protomers do exist as well and may contribute to their gas-phase conformational heterogeneity. In this work, we observed a CCS distribution that can be explained by the presence of protomers of surfactin, a small lipopeptide with no basic site. Following preliminary density functional theoretical calculations, several protonation sites in the gas phase were energetically favorable in positive ionization mode. Experimentally, at least three near-resolved IM peaks were observed in positive ionization mode, while only one was detected in negative ionization mode. These results were in good agreement with the DFT predictions. CID breakdown curve analysis after IM separation showed different inflection points (CE50) suggesting that different intramolecular interactions were implied in the stabilization of the structures of surfactin. The fragment ratio observed after collision-induced fragmentation was also different, suggesting different ring-opening localizations. All these observations support the presence of protomers on the cyclic peptide moieties of the surfactin. These data strongly suggest that protomeric tautomerism can still be observed on molecules above 1000 Da if the IM resolving power is sufficient. It also supports that the proton localization involves a change in the 3D structure that can affect the experimental CCS and the fragmentation channels of such peptides.


Assuntos
Peptídeos Cíclicos , Prótons , Lipopeptídeos , Conformação Molecular , Peptídeos Cíclicos/química , Subunidades Proteicas/química
14.
J Bacteriol ; 204(5): e0055521, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35435721

RESUMO

Alpha-pore-forming toxins (α-PFTs) are secreted by many species of bacteria, including Escherichia coli, Aeromonas hydrophila, and Bacillus thuringiensis, as part of their arsenal of virulence factors, and are often cytotoxic. In particular, for α-PFTs, the membrane-spanning channel they form is composed of hydrophobic α-helices. These toxins oligomerize at the surface of target cells and transition from a soluble to a protomer state in which they expose their hydrophobic regions and insert into the membrane to form a pore. The pores may be composed of homooligomers of one component or heterooligomers with two or three components, resulting in bi- or tripartite toxins. The multicomponent α-PFTs are often expressed from a single operon. Recently, motility-associated killing factor A (MakA), an α-PFT, was discovered in Vibrio cholerae. We report that makA is found on the V. cholerae GI-10 genomic island within an operon containing genes for two other potential α-PFTs, MakB and MakE. We determined the X-ray crystal structures for MakA, MakB, and MakE and demonstrated that all three are structurally related to the α-PFT family in the soluble state, and we modeled their protomer state based on the α-PFT AhlB from A. hydrophila. We found that MakA alone is cytotoxic at micromolar concentrations. However, combining MakA with MakB and MakE is cytotoxic at nanomolar concentrations, with specificity for J774 macrophage cells. Our data suggest that MakA, -B, and -E are α-PFTs that potentially act as a tripartite pore-forming toxin with specificity for phagocytic cells. IMPORTANCE The bacterium Vibrio cholerae causes gastrointestinal, wound, and skin infections. The motility-associated killing factor A (MakA) was recently shown to be cytotoxic against colon, prostate, and other cancer cells. However, at the outset of this study, the capacity of MakA to damage cells in combination with other Mak proteins encoded in the same operon had not been elucidated. We determined the structures of three Mak proteins and established that they are structurally related to the α-PFTs. Compared to MakA alone, the combination of all three toxins was more potent specifically in mouse macrophages. This study highlights the idea that the Mak toxins are selectively cytotoxic and thus may function as a tripartite toxin with cell type specificity.


Assuntos
Vibrio cholerae , Animais , Citotoxinas/genética , Citotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ilhas Genômicas , Camundongos , Proteínas Citotóxicas Formadoras de Poros , Subunidades Proteicas/metabolismo , Vibrio cholerae/metabolismo , Fatores de Virulência/metabolismo
15.
J Biol Chem ; 298(5): 101900, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35398357

RESUMO

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.


Assuntos
Bactérias , Toxinas Bacterianas , Especificidade de Hospedeiro , Polissacarídeos , Animais , Bactérias/classificação , Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Evolução Molecular , Mamíferos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Filogenia , Peste/microbiologia , Polissacarídeos/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Yersinia pestis/metabolismo
16.
Proc Natl Acad Sci U S A ; 119(14): e2107994119, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35363566

RESUMO

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix­loop­helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.


Assuntos
Acinetobacter baumannii , Sequências Hélice-Alça-Hélice , Manitol , Desidrogenase do Álcool de Açúcar , Acinetobacter baumannii/enzimologia , Manitol/metabolismo , Pressão Osmótica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Estresse Salino , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismo
17.
Nat Struct Mol Biol ; 29(5): 430-439, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35469063

RESUMO

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited on disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the regulator of the ATPase of vacuoles and endosomes (RAVE) complex through an unknown mechanism. We used cryogenic-electron microscopy of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex and the V1 complex lacking subunit C. On separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.


Assuntos
Proteínas de Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Endossomos/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Vacúolos/metabolismo
18.
Nature ; 604(7904): 127-133, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35355010

RESUMO

Many aspects of plant photoperception are mediated by the phytochrome (Phy) family of bilin-containing photoreceptors that reversibly interconvert between inactive Pr and active Pfr conformers1,2. Despite extensive biochemical studies, full understanding of plant Phy signalling has remained unclear due to the absence of relevant 3D models. Here we report a cryo-electron microscopy structure of Arabidopsis PhyB in the Pr state that reveals a topologically complex dimeric organization that is substantially distinct from its prokaryotic relatives. Instead of an anticipated parallel architecture, the C-terminal histidine-kinase-related domains (HKRDs) associate head-to-head, whereas the N-terminal photosensory regions associate head-to-tail to form a parallelogram-shaped platform with near two-fold symmetry. The platform is internally linked by the second of two internal Per/Arnt/Sim domains that binds to the photosensory module of the opposing protomer and a preceding 'modulator' loop that assembles tightly with the photosensory module of its own protomer. Both connections accelerate the thermal reversion of Pfr back to Pr, consistent with an inverse relationship between dimer assembly and Pfr stability. Lopsided contacts between the HKRDs and the platform create profound asymmetry to PhyB that might imbue distinct signalling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate-sensitive surface for conformation-dependent interactions between plant Phy photoreceptors and their signalling partners.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo B , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Microscopia Crioeletrônica , Luz , Fitocromo B/química , Fitocromo B/metabolismo , Domínios Proteicos , Subunidades Proteicas/metabolismo
19.
Nature ; 604(7904): 190-194, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35355020

RESUMO

Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits1,2 and can be modulated by essential medicines including general anaesthetics and benzodiazepines3. Human GABAAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABAARs assembled from α4, ß3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors4-7. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data8,9, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABAARs.


Assuntos
Benzodiazepinas , Receptores de GABA-A , Transdução de Sinais , Benzodiazepinas/farmacologia , Sítios de Ligação , Microscopia Crioeletrônica , Histamina/metabolismo , Humanos , Ligantes , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA-Seq , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Receptores de GABA-A/ultraestrutura , Análise de Célula Única , Ácido gama-Aminobutírico/metabolismo
20.
Emerg Microbes Infect ; 11(1): 1145-1153, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35343384

RESUMO

Analysis of large-scale gene expression post vaccination can provide an overview of immune responses. We used transcriptional approaches to comprehensively analyze the innate immune response signatures elicited by protein subunit (PS) vaccine ZF2001 and an mRNA vaccine named RRV. A fine-grained time-dependent dissection of large-scale gene expression post immunization revealed that ZF001 induced MHC class II-related genes, including cd74 and H2-Aa, more expeditiously than the RRV. Notably, the RRV induced MHC class I-related genes such as Tap1/2, B2m, and H2-D1/K1. At day 21 post immunization, the titres of binding and neutralization antibody (NAb) induced by both vaccines were comparable, which were accordant with the expression level of genes essential to BCR/TCR signalling transduction and B/T cells activation at day 7. However, compared to ZF2001, the early responses of RRV were more robust, including the activation of pattern recognition receptors (PRRs), expression of genes involved in RNA degradation, and transcription inhibition, which are directly related to anti-viral signals. This pattern also coincided with the induction of cytokines by the RRV. Generally, the transcriptomic patterns of two very different vaccines mapped here provide a framework for establishing correlates between the induction of genes and protection, which can be tailored for evoking specific and potent immune responses against SARS-CoV-2.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Humanos , Imunidade Inata , Subunidades Proteicas/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus , Transcriptoma , Vacinação , Vacinas de Subunidades , Vacinas Sintéticas
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