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1.
Cell ; 161(6): 1252-65, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-26046436

RESUMO

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.


Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , National Institutes of Health (U.S.) , Estados Unidos
2.
Nat Immunol ; 15(4): 343-53, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24531343

RESUMO

The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Infecções por Vírus de DNA/imunologia , Vírus de DNA/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Caspase 1/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Imunidade/genética , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon gama/genética , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Transgenes/genética , Proteínas Virais/genética , Homólogo LST8 da Proteína Associada a mTOR
3.
Trends Immunol ; 43(2): 148-162, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35033428

RESUMO

Ubc13-catalyzed K63 ubiquitination is a major control point for immune signaling. Recent evidence has shown that the control of multiple immune functions, including chronic inflammation, pathogen responses, lymphocyte activation, and regulatory signaling, is altered by K63 ubiquitination. In this review, we detail the novel cellular sensors that are dependent on K63 ubiquitination for their function in the immune signaling network. Many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can target K63 ubiquitination to inhibit pathogen immune responses; we describe novel details of the pathways involved and summarize recent clinically relevant SARS-CoV-2-specific responses. We also discuss recent evidence that regulatory T cell (Treg) versus T helper (TH) 1 and TH17 cell subset regulation might involve K63 ubiquitination. Knowledge gaps that merit future investigation and clinically relevant pathways are also addressed.


Assuntos
COVID-19 , Lisina , Humanos , Lisina/metabolismo , SARS-CoV-2 , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
5.
Cell ; 149(5): 963-5, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22632964
6.
Genes Dev ; 26(10): 1041-54, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22588718

RESUMO

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²âº mediated by inositol triphosphate receptors (IP3Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP3R-dependent manner. By reducing steady-state levels of ER Ca²âº via IP3Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1's ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²âº signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/imunologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo Energético , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Linhagem Celular Tumoral , Endorribonucleases/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Consumo de Oxigênio , Proteínas Serina-Treonina Quinases/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus/imunologia , Estresse Fisiológico , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Biol Chem ; 293(49): 18864-18878, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30291141

RESUMO

The inflammasome is a critical molecular complex that activates interleukin-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate the human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death, and secretion of cleaved interleukin-1ß. Mechanistically, DPP9 interacts with a unique autoproteolytic domain (Function to Find Domain (FIIND)) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in the NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyperactivation seen in the Mendelian autoinflammatory disease Autoinflammation with Arthritis and Dyskeratosis. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multifunctional inflammasome regulator involved in human autoinflammatory diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Inflamassomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Ácidos Borônicos/farmacologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Dipeptídeos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mutação em Linhagem Germinativa , Células HEK293 , Humanos , Inflamação/genética , Mutação de Sentido Incorreto , Proteínas NLR , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos
8.
Mol Cell ; 41(1): 107-16, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21185211

RESUMO

ARTS (apoptosis-related protein in the TGF-ß signaling pathway) is a mitochondrial protein that binds XIAP (X-linked inhibitor of apoptosis protein) upon entering the cytosol, thus promoting cell death. Expression of ARTS is lost in some malignancies. Here, we show that ARTS binds to XIAP at BIR1, a domain distinct from the caspase-binding sites. Furthermore, ARTS interacts with the E3 ligase Siah-1 (seven in absentia homolog 1) to induce ubiquitination and degradation of XIAP. Cells lacking either Siah or ARTS contain higher steady-state levels of XIAP. Thus, ARTS serves as an adaptor to bridge Siah-1 to XIAP, targeting it for destruction.


Assuntos
Proteínas Nucleares/fisiologia , Septinas/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Apoptose , Sítios de Ligação , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Septinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
Biochem J ; 474(21): 3643-3657, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-29061914

RESUMO

Members of the B-cell lymphoma 2 (BCL-2) gene family are attractive targets for cancer therapy as they play a key role in promoting cell survival, a long-since established hallmark of cancer. Clinical utility for selective inhibition of specific anti-apoptotic Bcl-2 family proteins has recently been realized with the Food and Drug Administration (FDA) approval of venetoclax (formerly ABT-199/GDC-0199) in relapsed chronic lymphocytic leukemia (CLL) with 17p deletion. Despite the impressive monotherapy activity in CLL, such responses have rarely been observed in other B-cell malignancies, and preclinical data suggest that combination therapies will be needed in other indications. Additional selective antagonists of Bcl-2 family members, including Bcl-XL and Mcl-1, are in various stages of preclinical and clinical development and hold the promise of extending clinical utility beyond CLL and overcoming resistance to venetoclax. In addition to direct targeting of Bcl-2 family proteins with BH3 mimetics, combination therapies that aim at down-regulating expression of anti-apoptotic BCL-2 family members or restoring expression of pro-apoptotic BH3 family proteins may provide a means to deepen responses to venetoclax and extend the utility to additional indications. Here, we review recent progress in direct and selective targeting of Bcl-2 family proteins for cancer therapy and the search for rationale combinations.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Hematológicas/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Animais , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
10.
J Biol Chem ; 291(27): 14072-14084, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27129202

RESUMO

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.


Assuntos
Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química
11.
Blood ; 126(3): 363-72, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26045609

RESUMO

Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan-Bcl-2 inhibitor (-)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (-)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan-Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (-)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (-)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan-Bcl-2 inhibition by an Mcl-1-targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gossipol/análogos & derivados , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Naftoquinonas/farmacologia , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Gossipol/farmacologia , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nature ; 474(7349): 96-9, 2011 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-21552281

RESUMO

Innate immunity is a fundamental defence response that depends on evolutionarily conserved pattern recognition receptors for sensing infections or danger signals. Nucleotide-binding and oligomerization domain (NOD) proteins are cytosolic pattern-recognition receptors of paramount importance in the intestine, and their dysregulation is associated with inflammatory bowel disease. They sense peptidoglycans from commensal microorganisms and pathogens and coordinate signalling events that culminate in the induction of inflammation and anti-microbial responses. However, the signalling mechanisms involved in this process are not fully understood. Here, using genome-wide RNA interference, we identify candidate genes that modulate the NOD1 inflammatory response in intestinal epithelial cells. Our results reveal a significant crosstalk between innate immunity and apoptosis and identify BID, a BCL2 family protein, as a critical component of the inflammatory response. Colonocytes depleted of BID or macrophages from Bid(-/-) mice are markedly defective in cytokine production in response to NOD activation. Furthermore, Bid(-/-) mice are unresponsive to local or systemic exposure to NOD agonists or their protective effect in experimental colitis. Mechanistically, BID interacts with NOD1, NOD2 and the IκB kinase (IKK) complex, impacting NF-κB and extracellular signal-regulated kinase (ERK) signalling. Our results define a novel role of BID in inflammation and immunity independent of its apoptotic function, furthering the mounting evidence of evolutionary conservation between the mechanisms of apoptosis and immunity.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/imunologia , Células Epiteliais/imunologia , Imunidade Inata/genética , Inflamação/genética , Mucosa Intestinal/imunologia , Animais , Apoptose/imunologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Colite/genética , Colite/imunologia , Células HEK293 , Células HT29 , Humanos , Quinase I-kappa B/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Interferência de RNA , Transdução de Sinais/genética , Transdução de Sinais/imunologia
13.
Mol Cell ; 33(6): 679-91, 2009 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-19328063

RESUMO

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1alpha activity in fly and mouse models of ER stress. BI-1-deficient cells displayed hyperactivation of the ER stress sensor IRE1alpha, leading to increased levels of its downstream target X-box-binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1alpha, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a role for BI-1 in early adaptive responses against ER stress that contrasts with its known downstream function in apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Linfócitos B/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endorribonucleases/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imunoglobulina M/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Fatores de Transcrição de Fator Regulador X , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-Box
14.
J Biol Chem ; 290(44): 26549-61, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26378241

RESUMO

Autophagy is a catabolic cellular mechanism for entrapping cellular macromolecules and organelles in intracellular vesicles and degrading their contents by fusion with lysosomes. Important roles for autophagy have been elucidated for cell survival during nutrient insufficiency, eradication of intracellular pathogens, and counteracting aging through clearance of senescent proteins and mitochondria. Autophagic vesicles become decorated with LC3, a protein that mediates their fusion with lysosomes. LC3 is a substrate of the cysteine protease ATG4B (Autophagin-1), where cleavage generates a C-terminal glycine required for LC3 conjugation to lipids in autophagosomes. ATG4B both cleaves pro-LC3 and also hydrolyzes lipids from cleaved LC3. We show here that phosphorylation of ATG4B at Ser-383 and Ser-392 increases its hydrolyase activity as measured using LC3 as a substrate. Reconstituting atg4b(-/-) cells with phosphorylation-deficient ATG4B showed a role of ATG4B phosphorylation in LC3 delipidation and autophagic flux, thus demonstrating that the cellular activity of ATG4B is modulated by phosphorylation. Proteolytic conversion of pro-LC3 to LC3-I was not significantly impacted by ATG4B phosphorylation in cells. Phosphorylation-deficient ATG4B also showed reduced interactions with the lipid-conjugated LC3 but not unconjugated LC3. Taken together, these findings demonstrate a role for Ser-383 and Ser-392 phosphorylation of ATG4B in control of autophagy.


Assuntos
Autofagia/fisiologia , Cisteína Endopeptidases/metabolismo , Lipoilação/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteólise , Animais , Proteínas Relacionadas à Autofagia , Células Cultivadas , Cisteína Endopeptidases/genética , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Fosforilação/fisiologia
16.
Proc Natl Acad Sci U S A ; 110(19): 7808-13, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23603272

RESUMO

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form "inflammasomes" that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1ß and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1ß secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1ß production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.


Assuntos
Regulação Viral da Expressão Gênica , Imunidade Inata , Inflamassomos/metabolismo , Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Caspases/metabolismo , Chlorocebus aethiops , Citocinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fenótipo , Proteínas Recombinantes/metabolismo , Células Vero , Virulência
17.
Gut ; 64(1): 66-76, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24572142

RESUMO

OBJECTIVE: The genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD. DESIGN: Exome sequencing and immunological profiling were performed in a patient with early onset Crohn's disease (CD). The coding region of the gene encoding X-linked inhibitor of apoptosis protein (XIAP) was sequenced in samples of 275 paediatric IBD and 1047 adult-onset CD patients. XIAP genotyping was performed in samples of 2680 IBD patients and 2864 healthy controls. Functional effects of the variants identified were investigated in primary cells and cultured cell lines. RESULTS: Our results demonstrate the frequent occurrence of private variants in XIAP in about four percent of male patients with paediatric-onset CD. While XIAP mutations are known to be associated with the primary immunodeficiency (PID) X-linked lymphoproliferative disease type 2 (XLP2), CD patients described here exhibited intestinal inflammation in the absence of XLP2 and harboured a spectrum of mutations partially distinct from that observed in XLP2. The majority of XIAP variants identified was associated with a selective defect in NOD1/2 signalling, impaired NOD1/2-mediated activation of NF-κB, and altered NF-κB-dependent cytokine production. CONCLUSIONS: This study reveals the unanticipated, frequent occurrence of XIAP variants in male paediatric-onset CD. The link between XIAP and NOD1/2, and the association of XIAP variants with XLP2, support the concept of PID in a subset of IBD patients. Moreover, these studies provide a rationale for the implementation of XIAP sequencing in clinical diagnostics in male patients with severe CD.


Assuntos
Doença de Crohn/genética , Mutação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Adolescente , Humanos , Lactente , Masculino
18.
Retrovirology ; 12: 74, 2015 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-26297639

RESUMO

BACKGROUND: HIV-1 protease (PR) is essential for viral infectivity as it cleaves Gag and Gag-Pol polyprotein precursors during viral maturation. Recent evidence suggests that cellular proteins can also be cleaved by PR, perhaps representing an important viral strategy to counter host defense mechanisms. Receptor-interacting protein kinase 1 (RIPK1) and RIPK2 belong to a family of serine/threonine kinases with conserved domain architecture and important functions in apoptosis, necrosis and innate immunity. RESULTS: We found that RIPK1 and RIPK2 but not other members of the RIP kinase family are cleaved by HIV-1 PR. In RIPK1, we identified a putative PR cleavage site; a mutation at this site rendered RIPK1 resistant to PR cleavage. RIPK1 and RIPK2 were cleaved during HIV-1 infection of T cell lines or primary activated CD4(+) T cells. Interfering with the viral life cycle at different stages by the addition of specific inhibitors against RT, integrase, or PR, completely prevented RIPK1 and RIPK2 cleavage. Cleavage of RIPK1 disrupted RIPK1/RIPK3 complex formation and RIPK1-mediated induction of NF-kB. CONCLUSIONS: These findings indicate that RIPK1 and RIPK2 are targets of HIV-1 PR activity during infection, and their inactivation may contribute to modulation of cell death and host defense pathways by HIV-1.


Assuntos
Protease de HIV/metabolismo , HIV-1/enzimologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Linfócitos T CD4-Positivos/virologia , Morte Celular , Células Cultivadas , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , HIV-1/fisiologia , Humanos , Células Jurkat , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
20.
PLoS Genet ; 8(10): e1003007, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23093945

RESUMO

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.


Assuntos
Autofagia , Infecções Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/mortalidade , Caenorhabditis elegans/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Estabilidade Enzimática , Predisposição Genética para Doença , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Proteólise , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
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