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1.
Cell ; 150(2): 339-50, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22817896

RESUMO

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of ß-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.


Assuntos
Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Amiloide/química , Humanos , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Alinhamento de Sequência
2.
Immunity ; 38(5): 896-905, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23602765

RESUMO

A20 is an anti-inflammatory protein linked to multiple human autoimmune diseases and lymphomas. A20 possesses a deubiquitinating motif and a zinc finger, ZF4, that binds ubiquitin and supports its E3 ubiquitin ligase activity. To understand how these activities mediate A20's physiological functions, we generated two lines of gene-targeted mice, abrogating either A20's deubiquitinating activity (Tnfaip3(OTU) mice) or A20's ZF4 (Tnfaip3(ZF4) mice). Both Tnfaip3(OTU) and Tnfaip3(ZF4) mice exhibited increased responses to TNF and sensitivity to colitis. A20's C103 deubiquitinating motif restricted both K48- and K63-linked ubiquitination of receptor interacting protein 1 (RIP1). A20's ZF4 was required for recruiting A20 to ubiquitinated RIP1. A20(OTU) proteins and A20(ZF4) proteins complemented each other to regulate RIP1 ubiquitination and NFκB signaling normally in compound mutant Tnfaip3(OTU/ZF4) cells. This complementation involved homodimerization of A20 proteins, and we have defined an extensive dimerization interface in A20. These studies reveal how A20 proteins collaborate to restrict TNF signaling.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Cisteína Endopeptidases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Multimerização Proteica , Transdução de Sinais/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Dedos de Zinco/genética
3.
Nature ; 529(7587): 537-40, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26789246

RESUMO

Cellular immunity against viral infection and tumour cells depends on antigen presentation by major histocompatibility complex class I (MHC I) molecules. Intracellular antigenic peptides are transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) and then loaded onto the nascent MHC I molecules, which are exported to the cell surface and present peptides to the immune system. Cytotoxic T lymphocytes recognize non-self peptides and program the infected or malignant cells for apoptosis. Defects in TAP account for immunodeficiency and tumour development. To escape immune surveillance, some viruses have evolved strategies either to downregulate TAP expression or directly inhibit TAP activity. So far, neither the architecture of TAP nor the mechanism of viral inhibition has been elucidated at the structural level. Here we describe the cryo-electron microscopy structure of human TAP in complex with its inhibitor ICP47, a small protein produced by the herpes simplex virus I. Here we show that the 12 transmembrane helices and 2 cytosolic nucleotide-binding domains of the transporter adopt an inward-facing conformation with the two nucleotide-binding domains separated. The viral inhibitor ICP47 forms a long helical hairpin, which plugs the translocation pathway of TAP from the cytoplasmic side. Association of ICP47 precludes substrate binding and prevents nucleotide-binding domain closure necessary for ATP hydrolysis. This work illustrates a striking example of immune evasion by persistent viruses. By blocking viral antigens from entering the endoplasmic reticulum, herpes simplex virus is hidden from cytotoxic T lymphocytes, which may contribute to establishing a lifelong infection in the host.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Microscopia Crioeletrônica , Herpesvirus Humano 1/imunologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/ultraestrutura , Evasão da Resposta Imune , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Retículo Endoplasmático/metabolismo , Herpesvirus Humano 1/química , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestrutura , Proteínas Imediatamente Precoces/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica
4.
Sci Rep ; 11(1): 14397, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257348

RESUMO

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.


Assuntos
Anticorpos Biespecíficos , Complexo CD3 , Citocinas , Citocinas/metabolismo , Ativação Linfocitária , Distribuição Tecidual
5.
Sci Rep ; 9(1): 12031, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427700

RESUMO

Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.


Assuntos
Anticorpos Biespecíficos/imunologia , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Ativação do Complemento , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Ligação Proteica , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia
6.
Cytokine Growth Factor Rev ; 25(2): 175-83, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24411492

RESUMO

The CBM signalosome plays a pivotal role in mediating antigen-receptor induced NF-κB signaling to regulate lymphocyte functions. The CBM complex forms filamentous structure and recruits downstream signaling components to activate NF-κB. MALT1, the protease component in the CBM complex, cleaves key proteins in the feedback loop of the NF-κB signaling pathway and enhances NF-κB activation. The aberrant activity of the CBM complex has been linked to aggressive lymphoma. Recent years have witnessed dramatic progresses in understanding the assembly mechanism of the CBM complex, and advances in the development of targeted therapy for aggressive lymphoma. Here, we will highlight these progresses and give an outlook on the potential translation of this knowledge from bench to bedside for aggressive lymphoma patients.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Caspases/imunologia , Guanilato Ciclase/imunologia , NF-kappa B/imunologia , Proteínas de Neoplasias/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/genética , Caspases/genética , Guanilato Ciclase/genética , Linfoma , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Estrutura Terciária de Proteína , Receptores de Antígenos/imunologia , Transdução de Sinais
7.
Nat Struct Mol Biol ; 17(11): 1324-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20935634

RESUMO

The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.


Assuntos
Proteína de Domínio de Morte Associada a Fas/química , Mutação , Receptor fas/química , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Síndrome Linfoproliferativa Autoimune/genética , Caspase 8/química , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Receptor fas/genética
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