Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.452
Filtrar
Mais filtros

Intervalo de ano de publicação
1.
Cell ; 159(7): 1578-90, 2014 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-25525876

RESUMO

Proteasomes and lysosomes constitute the major cellular systems that catabolize proteins to recycle free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPPII) is a large cytosolic proteolytic complex that functions in tandem with the proteasome-ubiquitin protein degradation pathway. We found that autosomal recessive TPP2 mutations cause recurrent infections, autoimmunity, and neurodevelopmental delay in humans. We show that a major function of TPPII in mammalian cells is to maintain amino acid levels and that TPPII-deficient cells compensate by increasing lysosome number and proteolytic activity. However, the overabundant lysosomes derange cellular metabolism by consuming the key glycolytic enzyme hexokinase-2 through chaperone-mediated autophagy. This reduces glycolysis and impairs the production of effector cytokines, including IFN-γ and IL-1ß. Thus, TPPII controls the balance between intracellular amino acid availability, lysosome number, and glycolysis, which is vital for adaptive and innate immunity and neurodevelopmental health.


Assuntos
Imunidade Adaptativa , Aminopeptidases/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Glicólise , Imunidade Inata , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Proteólise , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Aminopeptidases/química , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Feminino , Humanos , Síndromes de Imunodeficiência/imunologia , Lisossomos/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Alinhamento de Sequência , Serina Endopeptidases/química
2.
Nature ; 611(7935): 312-319, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36261521

RESUMO

Infectious diseases are among the strongest selective pressures driving human evolution1,2. This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis3. This pandemic devastated Afro-Eurasia, killing up to 30-50% of the population4. To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease.


Assuntos
DNA Antigo , Predisposição Genética para Doença , Imunidade , Peste , Seleção Genética , Yersinia pestis , Humanos , Aminopeptidases/genética , Aminopeptidases/imunologia , Peste/genética , Peste/imunologia , Peste/microbiologia , Peste/mortalidade , Yersinia pestis/imunologia , Yersinia pestis/patogenicidade , Seleção Genética/imunologia , Europa (Continente)/epidemiologia , Europa (Continente)/etnologia , Imunidade/genética , Conjuntos de Dados como Assunto , Londres/epidemiologia , Dinamarca/epidemiologia
3.
Mol Cell ; 77(6): 1163-1175.e9, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31995729

RESUMO

Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates.


Assuntos
Aminopeptidases/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Receptores de Superfície Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Aminopeptidases/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Citoplasma/metabolismo , Mutação , Ligação Proteica , Transporte Proteico , Receptores de Superfície Celular/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade , Proteínas de Transporte Vesicular/genética
4.
Am J Hum Genet ; 110(4): 691-702, 2023 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-36889308

RESUMO

ERAP2 is an aminopeptidase involved in immunological antigen presentation. Genotype data in human samples from before and after the Black Death, an epidemic due to Yersinia pestis, have marked changes in allele frequency of the single-nucleotide polymorphism (SNP) rs2549794, with the T allele suggested to be deleterious during this period, while ERAP2 is also implicated in autoimmune diseases. This study explored the association between variation at ERAP2 and (1) infection, (2) autoimmune disease, and (3) parental longevity. Genome-wide association studies (GWASs) of these outcomes were identified in contemporary cohorts (UK Biobank, FinnGen, and GenOMICC). Effect estimates were extracted for rs2549794 and rs2248374, a haplotype tagging SNP. Additionally, cis expression and protein quantitative trait loci (QTLs) for ERAP2 were used in Mendelian randomization (MR) analyses. Consistent with decreased survival in the Black Death, the T allele of rs2549794 showed evidence of association with respiratory infection (odds ratio; OR for pneumonia 1.03; 95% CI 1.01-1.05). Effect estimates were larger for more severe phenotypes (OR for critical care admission with pneumonia 1.08; 95% CI 1.02-1.14). In contrast, opposing effects were identified for Crohn disease (OR 0.86; 95% CI 0.82-0.90). This allele was shown to associate with decreased ERAP2 expression and protein levels, independent of haplotype. MR analyses suggest that ERAP2 expression may be mediating disease associations. Decreased ERAP2 expression is associated with severe respiratory infection with an opposing association with autoimmune diseases. These data support the hypothesis of balancing selection at this locus driven by autoimmune and infectious disease.


Assuntos
Doenças Autoimunes , Peste , Humanos , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos/genética , Doenças Autoimunes/genética , Polimorfismo de Nucleotídeo Único/genética , Predisposição Genética para Doença , Aminopeptidases/genética , Aminopeptidases/metabolismo
5.
PLoS Pathog ; 20(7): e1012359, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38980912

RESUMO

A strong genetic predictor of outcome following untreated HIV-1 infection is the carriage of specific alleles of human leukocyte antigens (HLAs) that present viral epitopes to T cells. Residual variation in outcome measures may be attributed, in part, to viral adaptation to HLA-restricted T cell responses. Variants of the endoplasmic reticulum aminopeptidases (ERAPs) influence the repertoire of T cell epitopes presented by HLA alleles as they trim pathogen-derived peptide precursors to optimal lengths for antigen presentation, along with other functions unrelated to antigen presentation. We investigated whether ERAP variants influence HLA-associated HIV-1 adaptation with demonstrable effects on overall HIV-1 disease outcome. Utilizing host and viral data of 249 West Australian individuals with HIV-1 subtype B infection, we identified a novel association between two linked ERAP2 single nucleotide polymorphisms (SNPs; rs2248374 and rs2549782) with plasma HIV RNA concentration (viral load) (P adjusted = 0.0024 for both SNPs). Greater HLA-associated HIV-1 adaptation in the HIV-1 Gag gene correlated significantly with higher viral load, lower CD4+ T cell count and proportion; P = 0.0103, P = 0.0061, P = 0.0061, respectively). When considered together, there was a significant interaction between the two ERAP2 SNPs and HLA-associated HIV-1 adaptation on viral load (P = 0.0111). In a comprehensive multivariate model, addition of ERAP2 haplotypes and HLA associated adaptation as an interaction term to known HLA and CCR5 determinants and demographic factors, increased the explanatory variance of population viral load from 17.67% to 45.1% in this dataset. These effects were not replicated in publicly available datasets with comparably sized cohorts, suggesting that any true global epistasis may be dependent on specific HLA-ERAP allelic combinations. Our data raises the possibility that ERAP2 variants may shape peptide repertoires presented to HLA class I-restricted T cells to modulate the degree of viral adaptation within individuals, in turn contributing to disease variability at the population level. Analyses of other populations and experimental studies, ideally with locally derived ERAP genotyping and HLA-specific viral adaptations are needed to elucidate this further.


Assuntos
Aminopeptidases , Epistasia Genética , Infecções por HIV , HIV-1 , Polimorfismo de Nucleotídeo Único , Humanos , Aminopeptidases/genética , Infecções por HIV/imunologia , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/genética , Austrália , Masculino , Feminino , Antígenos HLA/genética , Carga Viral , Adulto , Pessoa de Meia-Idade
6.
Mol Cell ; 72(6): 1013-1020.e6, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30576652

RESUMO

Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one of the largest ESs, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L have increased amino acid misincorporation, as well as readthrough and frameshifting errors. By employing quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.


Assuntos
Caulobacter crescentus/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , RNA Bacteriano/metabolismo , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminopeptidases/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sítios de Ligação , Caulobacter crescentus/genética , Linhagem Celular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Camundongos , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Ribossomos/química , Ribossomos/genética , Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
7.
Hum Mol Genet ; 32(6): 1048-1060, 2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36444934

RESUMO

Diabetic kidney disease (DKD) is recognized as an important public health challenge. However, its genomic mechanisms are poorly understood. To identify rare variants for DKD, we conducted a whole-exome sequencing (WES) study leveraging large cohorts well-phenotyped for chronic kidney disease and diabetes. Our two-stage WES study included 4372 European and African ancestry participants from the Chronic Renal Insufficiency Cohort and Atherosclerosis Risk in Communities studies (stage 1) and 11 487 multi-ancestry Trans-Omics for Precision Medicine participants (stage 2). Generalized linear mixed models, which accounted for genetic relatedness and adjusted for age, sex and ancestry, were used to test associations between single variants and DKD. Gene-based aggregate rare variant analyses were conducted using an optimized sequence kernel association test implemented within our mixed model framework. We identified four novel exome-wide significant DKD-related loci through initiating diabetes. In single-variant analyses, participants carrying a rare, in-frame insertion in the DIS3L2 gene (rs141560952) exhibited a 193-fold increased odds [95% confidence interval (CI): 33.6, 1105] of DKD compared with noncarriers (P = 3.59 × 10-9). Likewise, each copy of a low-frequency KRT6B splice-site variant (rs425827) conferred a 5.31-fold higher odds (95% CI: 3.06, 9.21) of DKD (P = 2.72 × 10-9). Aggregate gene-based analyses further identified ERAP2 (P = 4.03 × 10-8) and NPEPPS (P = 1.51 × 10-7), which are both expressed in the kidney and implicated in renin-angiotensin-aldosterone system modulated immune response. In the largest WES study of DKD, we identified novel rare variant loci attaining exome-wide significance. These findings provide new insights into the molecular mechanisms underlying DKD.


Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Insuficiência Renal Crônica , Humanos , Aminopeptidases , Nefropatias Diabéticas/genética , Sequenciamento do Exoma , Rim , Insuficiência Renal Crônica/genética
8.
PLoS Pathog ; 19(7): e1011507, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37440595

RESUMO

Pore-forming toxins (PFTs) are effective tools for pathogens infection. By disrupting epithelial barriers and killing immune cells, PFTs promotes the colonization and reproduction of pathogenic microorganisms in their host. In turn, the host triggers defense responses, such as endocytosis, exocytosis, or autophagy. Bacillus thuringiensis (Bt) bacteria produce PFT, known as crystal proteins (Cry) which damage the intestinal cells of insects or nematodes, eventually killing them. In insects, aminopeptidase N (APN) has been shown to act as an important receptor for Cry toxins. Here, using the nematode Caenorhabditis elegans as model, an extensive screening of APN gene family was performed to analyze the potential role of these proteins in the mode of action of Cry5Ba against the nematode. We found that one APN, MNP-1, participate in the toxin defense response, since the mnp-1(ok2434) mutant showed a Cry5Ba hypersensitive phenotype. Gene expression analysis in mnp-1(ok2434) mutant revealed the involvement of two protease genes, F19C6.4 and R03G8.6, that participate in Cry5Ba degradation. Finally, analysis of the transduction pathway involved in F19C6.4 and R03G8.6 expression revealed that upon Cry5Ba exposure, the worms up regulated both protease genes through the activation of the FOXO transcription factor DAF-16, which was translocated into the nucleus. The nuclear location of DAF-16 was found to be dependent on mnp-1 under Cry5Ba treatment. Our work provides evidence of new host responses against PFTs produced by an enteric pathogenic bacterium, resulting in activation of host intestinal proteases that degrade the PFT in the intestine.


Assuntos
Bacillus thuringiensis , Proteínas de Caenorhabditis elegans , Animais , Caenorhabditis elegans/microbiologia , Peptídeo Hidrolases/metabolismo , Aminopeptidases/metabolismo , Endotoxinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Hemolisinas/metabolismo , Intestinos , Endopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Bacillus thuringiensis/metabolismo , Fatores de Transcrição Forkhead/metabolismo
9.
Nat Chem Biol ; 19(9): 1158-1166, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37386135

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.


Assuntos
Aminopeptidases , Pseudomonas aeruginosa , Aminopeptidases/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Peptídeos Cíclicos/metabolismo , Biofilmes , Peptídeo Hidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
10.
Mol Cell ; 68(5): 970-977.e11, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29220658

RESUMO

Mitoproteases are becoming recognized as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Through multi-omic profiling of diverse Saccharomyces cerevisiae mitoprotease deletion strains, we predicted numerous associations between mitoproteases and distinct mitochondrial processes. These include a strong association between the mitochondrial matrix octapeptidase Oct1p and coenzyme Q (CoQ) biosynthesis-a pathway essential for mitochondrial respiration. Through Edman sequencing and in vitro and in vivo biochemistry, we demonstrated that Oct1p directly processes the N terminus of the CoQ-related methyltransferase, Coq5p, which markedly improves its stability. A single mutation to the Oct1p recognition motif in Coq5p disrupted its processing in vivo, leading to CoQ deficiency and respiratory incompetence. This work defines the Oct1p processing of Coq5p as an essential post-translational event for proper CoQ production. Additionally, our data visualization tool enables efficient exploration of mitoprotease profiles that can serve as the basis for future mechanistic investigations.


Assuntos
Aminopeptidases/metabolismo , Metabolismo Energético , Metabolômica/métodos , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquinona/biossíntese , Aminopeptidases/genética , Estabilidade Enzimática , Genótipo , Metiltransferases/genética , Mutação , Fenótipo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Ubiquinona/genética
11.
Mol Cell ; 65(5): 801-817.e4, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28216226

RESUMO

Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2TRFH domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas Nucleares/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Aminopeptidases/genética , Aminopeptidases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Exodesoxirribonucleases , Fase G1 , Fase G2 , Células HCT116 , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fase S , Serina Proteases/genética , Serina Proteases/metabolismo , Complexo Shelterina , Relação Estrutura-Atividade , Telômero/genética , Telômero/patologia , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genética
12.
PLoS Genet ; 18(1): e1010025, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35081133

RESUMO

Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1's role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.


Assuntos
Aminopeptidases/genética , Caenorhabditis elegans/genética , Instabilidade Genômica , Aminopeptidases/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular , Proliferação de Células , Replicação do DNA , Prolina/metabolismo
13.
Immunology ; 171(1): 131-145, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37858978

RESUMO

Endoplasmic reticulum aminopeptidase 1 (ERAP1) belongs to the oxytocinase subfamily of M1 aminopeptidases (M1APs), which are a diverse family of metalloenzymes involved in a wide range of functions and have been implicated in various chronic and infectious diseases of humans. ERAP1 trims antigenic precursors into correct sizes (8-10 residues long) for Major Histocompatibility Complex (MHC) presentation, by a unique molecular ruler mechanism in which it makes concurrent bindings to substrate N- and C-termini. We have previously determined four crystal structures of ERAP1 C-terminal regulatory domain (termed ERAP1_C domain) in complex with peptide carboxyl (PC)-ends that carry various anchor residues, and identified a specificity subsite for recognizing the PC anchor side chain, denoted as the SC subsite to follow the conventional notations: S1 site for P1, S2 site for P2, and so forth. In this study, we report studies on structure-guided mutational and hydrolysis kinetics, and peptide trimming assays to further examine the functional roles of this SC subsite. Most strikingly, a point mutation V737R results in a change of substrate preference from a hydrophobic to a negatively charged PC anchor residue; the latter is presumed to be a poor substrate for WT ERAP1. These studies validate the crystallographic observations that this SC subsite is directly involved in binding and recognition of the substrate PC anchor and presents a potential target to modulate MHC-restricted immunopeptidomes.


Assuntos
Aminopeptidases , Antígenos , Humanos , Aminopeptidases/genética , Aminopeptidases/química , Aminopeptidases/metabolismo , Antígenos/metabolismo , Peptídeos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Apresentação de Antígeno
14.
J Cell Sci ; 135(10)2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-35466366

RESUMO

Tripeptidyl peptidase II (TPPII or TPP2) degrades N-terminal tripeptides from proteins and peptides. Studies in both humans and mice have shown that TPPII deficiency is linked to cellular immune-senescence, lifespan regulation and the aging process. However, the mechanism of how TPPII participates in these processes is less clear. In this study, we established a chemical probe-based assay and found that although the mRNA and protein levels of TPPII were not altered during senescence, its enzymatic activity was reduced in senescent human fibroblasts. We also showed that elevation of the levels of the serine protease inhibitor serpinB2 reduced TPPII activity in senescent cells. Moreover, suppression of TPPII led to elevation in the amount of lysosomal contents as in well as TPPI (TPP1) and ß-galactosidase activities, suggesting that lysosome biogenesis is induced to compensate for the reduction of TPPII activity in senescent cells. Together, this study discloses a critical role of the serpinB2-TPPII signaling pathway in proteostasis during senescence. Since serpinB2 levels can be increased by a variety of cellular stresses, reduction of TPPII activity through activation of serpinB2 might represent a common pathway for cells to respond to different stress conditions. This article has an associated First Person interview with the first author of the paper.


Assuntos
Aminopeptidases , Senescência Celular , Dipeptidil Peptidases e Tripeptidil Peptidases , Peptídeos e Proteínas de Sinalização Intracelular , Aminopeptidases/genética , Aminopeptidases/metabolismo , Senescência Celular/genética , Senescência Celular/fisiologia , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteostase/genética , Proteostase/fisiologia , Serina Endopeptidases/metabolismo , Transdução de Sinais
15.
Gastroenterology ; 164(3): 454-466, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36496055

RESUMO

BACKGROUND & AIMS: Drug-induced liver injury (DILI) due to amoxicillin-clavulanate (AC) has been associated with HLA-A∗02:01, HLA-DRB1∗15:01, and rs2476601, a missense variant in PTPN22. The aim of this study was to identify novel risk factors for AC-DILI and to construct a genetic risk score (GRS). METHODS: Transcriptome-wide association study and genome-wide association study analyses were performed on 444 AC-DILI cases and 10,397 population-based controls of European descent. Associations were confirmed in a validation cohort (n = 133 cases and 17,836 population-based controls). Discovery and validation AC-DILI cases were also compared with 1358 and 403 non-AC-DILI cases. RESULTS: Transcriptome-wide association study revealed a significant association of AC-DILI risk with reduced liver expression of ERAP2 (P = 3.7 × 10-7), coding for an aminopeptidase involved in antigen presentation. The lead eQTL single nucleotide polymorphism, rs1363907 (G), was associated with AC-DILI risk in the discovery (odds ratio [OR], 1.68; 95% CI, 1.23-1.66; P = 1.7 × 10-7) and validation cohorts (OR, 1.2; 95% CI, 1.04-2.05; P = .03), following a recessive model. We also identified HLA-B∗15:18 as a novel AC-DILI risk factor in both discovery (OR, 4.19; 95% CI, 2.09-8.36; P = 4.9 × 10-5) and validation (OR, 7.78; 95% CI, 2.75-21.99; P = .0001) cohorts. GRS, incorporating rs1363907, rs2476601, HLA-B∗15:18, HLA-A∗02:01, and HLA-DRB1∗15:01, was highly predictive of AC-DILI risk when cases were analyzed against both general population and non-AC-DILI control cohorts. GRS was the most significant predictor in a regression model containing known AC-DILI clinical risk characteristics and significantly improved the predictive model. CONCLUSIONS: We identified novel associations of AC-DILI risk with ERAP2 low expression and with HLA-B∗15:18. GRS based on the 5 risk variants may assist AC-DILI causality assessment and risk management.


Assuntos
Antibacterianos , Doença Hepática Induzida por Substâncias e Drogas , Humanos , Antibacterianos/efeitos adversos , Alelos , Cadeias HLA-DRB1/genética , Estudo de Associação Genômica Ampla , Combinação Amoxicilina e Clavulanato de Potássio , Fígado , Fatores de Risco , Antígenos HLA-A/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Aminopeptidases/genética
16.
Eur J Immunol ; 53(8): e2350449, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37134263

RESUMO

ER aminopeptidase 1 (ERAP1) is an ER-resident aminopeptidase that excises N-terminal residues of peptides that then bind onto Major Histocompatibility Complex I molecules (MHC-I) and indirectly modulates adaptive immune responses. ERAP1 contains an allosteric regulatory site that accommodates the C-terminus of at least some peptide substrates, raising questions about its exact influence on antigen presentation and the potential of allosteric inhibition for cancer immunotherapy. We used an inhibitor that targets this regulatory site to study its effect on the immunopeptidome of a human cancer cell line. The immunopeptidomes of allosterically inhibited and ERAP1 KO cells contain high-affinity peptides with sequence motifs consistent with the cellular HLA class I haplotypes but are strikingly different in peptide composition. Compared to KO cells, allosteric inhibition did not affect the length distribution of peptides and skewed the peptide repertoire both in terms of sequence motifs and HLA allele utilization, indicating significant mechanistic differences between the two ways of disrupting ERAP1 function. These findings suggest that the regulatory site of ERAP1 plays distinct roles in antigenic peptide selection, which should be taken into consideration when designing therapeutic interventions targeting the cancer immunopeptidome.


Assuntos
Aminopeptidases , Peptídeos , Humanos , Aminopeptidases/genética , Apresentação de Antígeno , Antígenos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo
17.
Am J Gastroenterol ; 119(8): 1496-1505, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38314748

RESUMO

INTRODUCTION: The aim of this study is to describe the presenting features, genetic factors, and outcomes of 23 adults who developed liver injury after coronavirus disease 2019 (COVID-19) mRNA vaccination. METHODS: Patients with suspected COVID-19 vaccine hepatitis were enrolled into the Drug-Induced Liver Injury Network. Causality was assessed using the Drug-Induced Liver Injury Network expert opinion score. High-resolution HLA sequencing was undertaken using Illumina platform. RESULTS: Amongst the 16 high causality cases, median time to onset was 16 days, median age was 63 years, and 75% were female. The injury was hepatocellular in 75% with a median alanine aminotransferase of 497 U/L, and 37% had jaundice. An antinuclear antibody and smooth muscle antibody were detectable in 27% and 36%, but only 12% had an elevated immunoglobulin G level. During follow-up, 37% received a short course of corticosteroids, and 88% fully recovered by 6 months with no deaths observed. HLA alleles associated with autoimmune hepatitis were not overrepresented compared with controls, but an ERAP-2 variant (rs1263907) and the ERAP-1 Hap6 haplotype were significantly overrepresented in the high causality cases vs controls ( P = 0.026 and 5 × 10 -5 , respectively). DISCUSSION: Acute liver injury may arise within 8 weeks of COVID-19 mRNA vaccination that is generally mild and self-limited in most patients. The absence of an association with the AIH HLA alleles combined with the significant ERAP-2 and ERAP-1 Hap6 haplotype associations implicates a unique but very rare host immune response to vaccine-derived antigens in the pathogenesis of COVID-19 vaccine hepatotoxicity.


Assuntos
Aminopeptidases , Vacinas contra COVID-19 , COVID-19 , Doença Hepática Induzida por Substâncias e Drogas , Antígenos de Histocompatibilidade Menor , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Aminopeptidases/genética , COVID-19/prevenção & controle , Idoso , Vacinas contra COVID-19/efeitos adversos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Estados Unidos/epidemiologia , SARS-CoV-2 , Adulto , Vacina de mRNA-1273 contra 2019-nCoV , Vacinação/efeitos adversos , Vacina BNT162/efeitos adversos , Vacinas Sintéticas/efeitos adversos
18.
J Transl Med ; 22(1): 750, 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-39123229

RESUMO

BACKGROUND: ERAP2 is an aminopeptidase involved in antigen processing and presentation, and harbor genetic variants linked to several inflammatory diseases such as Inflammatory Bowel Disease (IBD). The lack of an ERAP2 gene homologue in mice has hampered functional studies, and most human studies have focused on cells of hematopoietic origin. Using an IBD biobank as vantage point, this study explores how genetic variation in ERAP2 affects gene expression in human-derived epithelial organoids upon proinflammatory stimulation. METHODS: An IBD patient cohort was genotyped with regards to two single nucleotide polymorphisms (SNP) (rs2910686/rs2248374) associated with ERAP2 expression levels, and we examined the correlation between colon gene expression and genotype, specifically aiming to establish a relationship with ERAP2 expression proficiency. Human-derived colon organoids (colonoids) with known ERAP2 genotype were established and used to explore differences in whole genome gene expression between ERAP2-deficient (n = 4) and -proficient (n = 4) donors upon pro-inflammatory encounter. RESULTS: When taking rs2910686 genotype into account, ERAP2 gene expression is upregulated in the inflamed colon of IBD patients. Colonoids upregulate ERAP2 upon IFNÉ£ stimulation, and ERAP2 expression proficiency is dependent on rs2910686 genotype. Colonoid genotyping confirms that mechanisms independent of the frequently studied SNP rs2248374 can cause ERAP2-deficiency. A total of 586 genes involved in various molecular mechanisms are differentially expressed between ERAP2 proficient- and deficient colonoids upon proinflammatory stimulation, including genes encoding proteins with the following molecular function: catalytic activity (AOC1, CPE, ANPEP and MEP1A), regulator activity (TNFSF9, MDK, GDF15, ILR6A, LGALS3 and FLNA), transmembrane transporter activity (SLC40A1 and SLC5A1), and extracellular matrix structural constituents (FGL2, HMCN2, and MUC17). CONCLUSIONS: ERAP2 is upregulated in the inflamed IBD colon mucosa, and expression proficiency is highly correlated with genotype of rs2910686. While the SNP rs2248374 is commonly used to determine ERAP2 expressional proficiency, our data confirms that mechanisms independent of this SNP can lead to ERAP2 deficiency. Our data demonstrates that epithelial ERAP2 presence affects the inflammatory response in colonoids, suggesting a pleiotropic role of ERAP2 beyond MHC class I antigen processing.


Assuntos
Aminopeptidases , Colo , Inflamação , Doenças Inflamatórias Intestinais , Polimorfismo de Nucleotídeo Único , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Aminopeptidases/genética , Aminopeptidases/metabolismo , Inflamação/genética , Inflamação/patologia , Colo/patologia , Colo/metabolismo , Organoides/metabolismo , Genótipo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Feminino , Masculino , Estudos de Coortes , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células Epiteliais/metabolismo
19.
Nat Chem Biol ; 18(5): 565-574, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35165443

RESUMO

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.


Assuntos
Proteínas Adaptadoras de Sinalização CARD , Inflamassomos , Aminopeptidases/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Neoplasias , Prolina
20.
EMBO Rep ; 23(10): e54136, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-35912982

RESUMO

N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.


Assuntos
Reparo do DNA , Dipeptidil Peptidases e Tripeptidil Peptidases , Aminopeptidases , DNA , Dano ao DNA , Dipeptídeos , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Prolina , Rad51 Recombinase/genética , Serina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA