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1.
Nat Immunol ; 21(1): 54-64, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31819256

RESUMO

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.


Assuntos
Apoptose/imunologia , Caspase 8/imunologia , Neutrófilos/imunologia , Proteínas Quinases/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Animais , Caspase 8/genética , Células Cultivadas , Deleção de Genes , Inflamação/imunologia , Interleucina-1/imunologia , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores Tipo I de Interleucina-1/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Mol Syst Biol ; 16(12): e9310, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33438817

RESUMO

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large-scale peptide-phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM-ligand complexes, indicating that a large majority of the phage-derived peptides are likely to target natural peptide-binding sites and could thus act as inhibitors of natural protein-protein interactions. The complete dataset has been assembled in an online database (http://www.prm-db.org) that will enable many structural, functional, and biological studies of PRMs and SLiMs.


Assuntos
Bases de Dados de Proteínas , Peptídeos/metabolismo , Inquéritos e Questionários , Sequência de Aminoácidos , Bacteriófagos/metabolismo , Humanos , Ligantes , Peptídeos/química
3.
Biochem J ; 475(2): 429-440, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29259080

RESUMO

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.


Assuntos
Domínio B30.2-SPRY/genética , Domínio de Ativação e Recrutamento de Caspases/genética , Proteína DEAD-box 58/química , Receptores Citoplasmáticos e Nucleares/química , Proteínas Recombinantes de Fusão/química , Deleção de Sequência , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Histidina/genética , Histidina/metabolismo , Humanos , Camundongos , Modelos Moleculares , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Imunológicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Immunol Cell Biol ; 95(2): 146-151, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27826146

RESUMO

Immunological responses activated by pathogen recognition come in many guises. The proliferation, differentiation and recruitment of immune cells, and the production of inflammatory cytokines and chemokines are central to lifelong immunity. Cell death serves as a key function in the resolution of innate and adaptive immune responses. It also coordinates cell-intrinsic effector functions to restrict infection. Necrosis was formally considered a passive form of cell death or a consequence of pathogen virulence factor expression, and necrotic tissue is frequently associated with infection. However, there is now emerging evidence that points to a role for regulated forms of necrosis, such as pyroptosis and necroptosis, driving inflammation and shaping the immune response.


Assuntos
Apoptose , Citotoxicidade Imunológica , Animais , Bactérias/metabolismo , Caspases/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Inflamassomos/metabolismo
5.
Biochem J ; 456(2): 231-40, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24015671

RESUMO

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen ß-strands arranged into two ß-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp(488) and Trp(621)) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.


Assuntos
Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Ligação de Hidrogênio , Imunidade Inata , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Viroses/imunologia
6.
J Immunol ; 187(7): 3798-805, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21876038

RESUMO

The mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens via the TLRs. TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing IFN-ß (TRIF), which results in the transcription of a small array of genes, including IFN-ß. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli, including cytokines and microbes. NO is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. We have recently identified iNOS as a target of the mammalian SPSB2 protein. The SOCS box is a peptide motif, which, in conjunction with elongins B and C, recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin ligase complex. In this study, we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterize the interaction between SPSB1 and iNOS. Through the use of SPSB1 transgenic mouse macrophages and short hairpin RNA knockdown of SPSB1, we show that SPSB1 controls both the induction of iNOS and the subsequent production of NO downstream of TLR3 and TLR4. Further, we demonstrate that regulation of iNOS by SPSB1 is dependent on the proteasome. These results suggest that SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production.


Assuntos
Regulação da Expressão Gênica/imunologia , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/metabolismo , Animais , Western Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Imunoprecipitação , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
7.
Methods Mol Biol ; 2523: 253-263, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35759202

RESUMO

Neutrophil extracellular traps (NETs) are networks of chromatin and microbicidal proteins released by neutrophils in response to infection and tissue damage. Although classically viewed as a discrete biochemical and cellular process in neutrophils, the effector pathways integrating diverse upstream activating signals to control the formation of NETs (NETosis) are poorly defined. Cell death is one such common unifying endpoint of neutrophils, with several bona fide non-apoptotic cell death agonists now described to initiate NETosis. Integrating these new genetic findings into our existing knowledge of NETosis will likely reveal varied cellular and biochemical processes controlling NET release and specific anti-microbial and inflammatory effector functions of NETs triggered by specific non-apoptotic cell death. To facilitate investigation of regulated cell death pathways in NETosis, we offer a detailed protocol for neutrophil purification from mouse bone marrow and human blood, analysis of NETs by flow cytometry, and validation by immunogold electron microscopy. Future studies may better define cell death-specific forms of NETosis and their influence on inflammation and autoimmunity.


Assuntos
Armadilhas Extracelulares , Morte Celular Regulada , Animais , Morte Celular , Armadilhas Extracelulares/metabolismo , Inflamação/metabolismo , Camundongos , Neutrófilos/metabolismo
8.
Sci Signal ; 11(546)2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30181240

RESUMO

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.


Assuntos
Apoptose , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Proteínas Quinases/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Células Cultivadas , Armadilhas Extracelulares/genética , Histonas/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Neutrófilos/microbiologia , Neutrófilos/ultraestrutura , Proteínas Quinases/genética , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
9.
Oncotarget ; 8(35): 57948-57963, 2017 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-28938529

RESUMO

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis.

10.
Nat Commun ; 6: 6282, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25693118

RESUMO

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1ß inflammatory responses independent of MLKL and necroptotic cell death.


Assuntos
Células da Medula Óssea/citologia , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Autoanticorpos/química , Caspase 8/metabolismo , Ativação Enzimática , Feminino , Inflamação , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/química , Fígado/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
11.
Protein Sci ; 22(1): 1-10, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23139046

RESUMO

The SPRY domain is a protein interaction module found in 77 murine and ~100 human proteins, and is implicated in important biological pathways, including those that regulate innate and adaptive immunity. The current definition of the SPRY domain is based on a sequence repeat discovered in the splA kinase and ryanodine receptors. The greater SPRY family is divided into the B30.2 (which contains a PRY extension at the N-terminus) and "SPRY-only" sub-families. In this brief review, we examine the current structural and biochemical literature on SPRY/B30.2 domain involvement in key immune processes and highlight a PRY-like 60 amino acid region in the N-terminus of "SPRY-only" proteins. Phylogenetic, structural, and functional analyses suggest that this N-terminal region is related to the PRY region of B30.2 and should be characterized as part of an extended SPRY domain. Greater understanding of the functional importance of the N-terminal region in "SPRY only" proteins will enhance our ability to interrogate SPRY interactions with their respective binding partners.


Assuntos
Imunidade Inata/imunologia , Proteínas/imunologia , Animais , Humanos , Proteínas/química , Proteínas/metabolismo
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