RESUMO
Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Transcrição Gênica/imunologia , Doença Aguda , Imunidade Adaptativa/genética , Imunidade Adaptativa/imunologia , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/imunologia , Variação Genética/imunologia , Hepacivirus/fisiologia , Hepatite C Crônica/genética , Hepatite C Crônica/virologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Fatores de Tempo , Adulto JovemRESUMO
Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes. To address this challenge, we developed novel chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. First, we confirmed that PARKIN does not require an E2 enzyme for substrate ubiquitination, lysine selection, and polyubiquitin chain formation. Second, we confirmed that UbFluor quantitatively detects naturally occurring activation states of PARKIN caused by Ser65 phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb). Third, we showed that both pUb and the ubiquitin-accepting substrate contribute to maximal pPARKIN ubiquitin conjugation turnover. pUb enhances the transthiolation step, whereas the substrate clears the pPARKINâ¼Ub thioester intermediate. Finally, we established that UbFluor can quantify activation or inhibition of PARKIN by structural mutations. These results demonstrate the feasibility of using UbFluor for quantitative studies of the biochemistry of RBR E3s and for high-throughput screening of small-molecule activators or inhibitors of PARKIN and other RBR E3 ligases.
Assuntos
Sondas Moleculares/química , Poliubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitinação , Regulação Alostérica , Animais , Polarização de Fluorescência/métodos , Humanos , Mutação , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.
Assuntos
Antígenos HLA-B/imunologia , Hepacivirus/imunologia , Mutação de Sentido Incorreto , Supressão Genética , Linfócitos T Citotóxicos/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Linfócitos T Citotóxicos/virologia , Proteínas não Estruturais Virais/genéticaRESUMO
Herpesviral deubiquitinating enzymes (DUBs) were discovered in 2005, are highly conserved across the family, and are proving to be increasingly important players in herpesviral infection. EBV's DUB, BPLF1, is known to regulate both cellular and viral target activities, yet remains largely unstudied. Our work has implicated BPLF1 in a wide range of processes including infectivity, viral DNA replication, and DNA repair. Additionally, knockout of BPLF1 delays and reduces human B-cell immortalization and lymphoma formation in humanized mice. These findings underscore the importance of BPLF1 in viral infectivity and pathogenesis and suggest that inhibition of EBV's DUB activity may offer a new approach to specific therapy for EBV infections. We set out to discover and characterize small molecule inhibitors of BPLF1 deubiquitinating activity through high-throughput screening. An initial small pilot screen resulted in discovery of 10 compounds yielding >80% decrease in BPLF1 DUB activity at a 10⯵M concentration. Follow-up dose response curves of top hits identified several compounds with an IC50 in the low micromolar range. Four of these hits were tested for their ability to cleave ubiquitin chains as well as their effects on viral infectivity and cell viability. Further characterization of the top hit, commonly known as suramin was found to not be selective yet decreased viral infectivity by approximately 90% with no apparent effects on cell viability. Due to the conserved nature of Herpesviral deubiquitinating enzymes, identification of an inhibitor of BPLF1 may prove to be an effective and promising new avenue of therapy for EBV and other herpesviral family members.
Assuntos
Antivirais/farmacologia , Enzimas Desubiquitinantes/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/enzimologia , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Sobrevivência Celular , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Bibliotecas de Moléculas Pequenas , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
PARKIN is a RING-Between-RING (RBR) E3 ligase, which ubiquitinates mitochondrial proteins in response to mitochondrial damage. Ser65 of PARKIN is phosphorylated by kinase PINK1 (pPARKIN), which causes partial PARKIN activation. PINK1 also phosphorylates Ser65 of ubiquitin (pUb), which further activates pPARKIN. Due to the lack of precise and quantitative assays to quantify the activity of PARKIN, there were many conflicting reports on the role of pUb as a PARKIN activator, whether S65E PARKIN is a true phosphomimetic of pPARKIN, and the effect of substrate of PARKIN turnover was also not known. This protocol provides a step-by-step guide on the use of the UbFluor probe to precisely quantitate changes in the activity of PARKIN in response to phosphorylation, allosteric activation by pUb, protein substrates, and activating structural mutations. These results pave the way to discover PARKIN activators and to precisely quantify the activity of other RBR E3s. © 2018 by John Wiley & Sons, Inc.
Assuntos
Corantes Fluorescentes/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Regulação Alostérica , Ativação Enzimática , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Fosforilação , Ubiquitina/químicaRESUMO
HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases. The release of fluorophore upon transthiolation allows fluorescence polarization detection of HECT E3 activity. In the presence of inhibitors, HECT E3 activity is ablated, and thus no reaction and no change in FP are observed. This assay has been adapted for high-throughput screening of small molecules against HECT E3 ligases, and its utility has been proven in the discovery of HECT E3 ligase inhibitors. © 2017 by John Wiley & Sons, Inc.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/análise , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ésteres/análise , Ésteres/química , Ésteres/metabolismo , Fluorescência , Humanos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidoresRESUMO
HECT E3 ubiquitin ligases (â¼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1â¼Ub, E2â¼Ub, and HECT E3â¼Ub (where â¼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s. These probes undergo a direct transthiolation reaction with the catalytic cysteine of HECT E3s, producing the catalytically active HECT E3â¼Ub thioester accompanied by fluorophore release. Thus, a fluorescence polarization assay can continuously monitor UbFluor consumption by HECT E3s, and changes in UbFluor consumption rendered by biochemical point mutations or small molecule modulation of HECT E3 activity. © 2017 by John Wiley & Sons, Inc.
Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Humanos , Modelos Moleculares , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
We describe the characterization of the temperature and strain responses of fiber Bragg grating sensors by use of an interferometric interrogation technique to provide an absolute measurement of the grating wavelength. The fiber Bragg grating temperature response was found to be nonlinear over the temperature range -70 degrees C to 80 degrees C. The nonlinearity was observed to be a quadratic function of temperature, arising from the linear dependence on temperature of the thermo-optic coefficient of silica glass over this range, and is in good agreement with a theoretical model.