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The tripartite motif (TRIM) protein family members have been implicated in a multitude of physiologies and pathologies in different tissues. With diverse functions in cellular processes including regulation of signaling pathways, protein degradation, and transcriptional control, the impact of TRIM dysregulation can be multifaceted and complex. Here, we focus on the cellular and molecular roles of TRIMs identified in the brain in the context of a selection of pathologies including cancer and neurodegeneration. By examining each disease in parallel with described roles in brain development, we aim to highlight fundamental common mechanisms employed by TRIM proteins and identify opportunities for therapeutic intervention.
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Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors.
Assuntos
Cisteína , Fluoretos , Cisteína/química , Ligantes , Aminoácidos , EnxofreRESUMO
The ubiquitin system contains a wealth of potential drug targets for many diseases and conditions, including neurodegenerative, immune, metabolic and developmental diseases, as well as multiple cancers. Despite years of research, relatively few clinical inhibitors or specific chemical probes for proteins within the ubiquitin system exist, with many interesting target proteins yet to be explored. Fragment-based drug discovery (FBDD) offers efficient and broad coverage of chemical space with small libraries, using covalent and non-covalent approaches. Coupled with advances in structural biology and proteomics, FBDD now provides a thorough screening platform for inhibitor discovery within the ubiquitin system. In this mini review, we summarise the current scope of FBDD and how it has been applied to ubiquitin-activating (E1), ubiquitin-conjugating (E2), ubiquitin ligase (E3) and deubiquitinating (DUB) enzymes. We also discuss the newest frontiers of FBDD and how they could be applied to enable inhibitor and novel chemical probe discovery and provide functional insight into the ubiquitin system.
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The Parkin-coregulated gene (PACRG), which encodes a protein of unknown function, shares a bidirectional promoter with Parkin (PRKN), which encodes an E3 ubiquitin ligase. Because PRKN is important in mitochondrial quality control and protection against stress, we tested whether PACRG also affected these pathways in various cultured human cell lines and in mouse embryonic fibroblasts. PACRG did not play a role in mitophagy but did play a role in tumor necrosis factor (TNF) signaling. Similarly to Parkin, PACRG promoted nuclear factor κB (NF-κB) activation in response to TNF. TNF-induced nuclear translocation of the NF-κB subunit p65 and NF-κB-dependent transcription were decreased in PACRG-deficient cells. Defective canonical NF-κB activation in the absence of PACRG was accompanied by a decrease in linear ubiquitylation mediated by the linear ubiquitin chain assembly complex (LUBAC), which is composed of the two E3 ubiquitin ligases HOIP and HOIL-1L and the adaptor protein SHARPIN. Upon TNF stimulation, PACRG was recruited to the activated TNF receptor complex and interacted with LUBAC components. PACRG functionally replaced SHARPIN in this context. In SHARPIN-deficient cells, PACRG prevented LUBAC destabilization, restored HOIP-dependent linear ubiquitylation, and protected cells from TNF-induced apoptosis. This function of PACRG in positively regulating TNF signaling may help to explain the association of PACRG and PRKN polymorphisms with an increased susceptibility to intracellular pathogens.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Chaperonas Moleculares/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Mitofagia/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Protein ubiquitination plays a key role in the regulation of cellular processes, and misregulation of the ubiquitin system is linked to many diseases. So far, development of tool compounds that target enzymes of the ubiquitin system has been slow and only a few specific inhibitors are available. Here, we report the selection of single-domain antibodies (single-dAbs) based on a human scaffold that recognize the catalytic domain of HOIP, a subunit of the multi-component E3 LUBAC and member of the RBR family of E3 ligases. Some of these dAbs affect ligase activity and provide mechanistic insight into the ubiquitin transfer mechanism of different E2-conjugating enzymes. Furthermore, we show that the co-crystal structure of a HOIP RBR/dAb complex serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.
Assuntos
Inibidores Enzimáticos/farmacologia , Chaperonas Moleculares/química , Anticorpos de Domínio Único/química , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Cristalização , Inibidores Enzimáticos/química , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
Assuntos
Inibidores Enzimáticos/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Ligantes , Células MCF-7 , Modelos Moleculares , Estrutura Secundária de Proteína , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/químicaRESUMO
The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.
Assuntos
Escherichia coli/química , Metaloproteases/química , Infecções por Salmonella/genética , Salmonella enterica/química , Sequência de Aminoácidos/genética , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/patogenicidade , Células HeLa , Humanos , Imunidade Celular , Metaloproteases/genética , NF-kappa B/química , Conformação Proteica , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Transdução de Sinais , Relação Estrutura-Atividade , Fator de Transcrição RelA/química , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/genética , Zinco/químicaRESUMO
RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1.
Assuntos
Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/imunologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Células Cultivadas , Proteína DEAD-box 58/imunologia , Células HEK293 , Humanos , Interferons/biossíntese , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Viral/imunologia , Receptores Imunológicos , Transdução de Sinais , Fatores de Transcrição/química , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
RING-between-RING (RBR) ubiquitin ligases work with multiple E2 enzymes and function through an E3-ubiquitin thioester intermediate. The RBR module comprises three domains, RING1, IBR and RING2 that collaborate to transfer ubiquitin from the E2~Ub conjugate, recognised by RING1, onto a catalytic cysteine in RING2 and finally onto the substrate in a multi-step reaction. Recent studies have shown that RING1 domains bind E2~Ub conjugates in an open conformation to supress ubiquitin transfer onto lysine residues and promote formation of the E3 thioester intermediate. However, how the nature of the E2 influences the ubiquitin transfer process is currently unclear. We report here a detailed characterization of the RBR/E2-conjugate recognition step that indicates that this mechanism depends on the nature of the E2 enzyme and differs between UbcH5 and UbcH7. In the case of UbcH5~Ub an interaction with ubiquitin is necessary to stabilize the transfer complex while recognition of UbcH7~Ub is driven primarily by E2-RING1 contacts. Furthermore our analysis suggests that RBRs, in isolation and in complex with ubiquitin-loaded E2s, are dynamic species and that their intrinsic flexibility might be a key aspect of their catalytic mechanism.
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Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Calorimetria , Catálise , Humanos , Complexos Multiproteicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Termodinâmica , Ubiquitina/química , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2â¼ubiquitin (E2â¼Ub) via their RINGs but canonical RING E3s promote closed E2â¼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2â¼Ub to favor Ub transfer to the E3 active site. This different RING/E2â¼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2â¼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.
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Proteínas de Transporte/química , Enzimas de Conjugação de Ubiquitina/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Zinco/química , Zinco/metabolismoRESUMO
Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases.
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Ubiquitina/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Humanos , Polimorfismo Genético , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais , Termotolerância , Fatores de Necrose Tumoral/metabolismo , Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Reguladoras de Apoptose/química , Espalhamento a Baixo Ângulo , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas NLR , Nucleotídeos/química , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Difração de Raios XRESUMO
Recruitment of the deubiquitinase CYLD to signaling complexes is mediated by its interaction with HOIP, the catalytically active component of the linear ubiquitin chain assembly complex (LUBAC). Here, we identify SPATA2 as a constitutive direct binding partner of HOIP that bridges the interaction between CYLD and HOIP. SPATA2 recruitment to TNFR1- and NOD2-signaling complexes is dependent on HOIP, and loss of SPATA2 abolishes CYLD recruitment. Deficiency in SPATA2 exerts limited effects on gene activation pathways but diminishes necroptosis induced by tumor necrosis factor (TNF), resembling loss of CYLD. In summary, we describe SPATA2 as a previously unrecognized factor in LUBAC-dependent signaling pathways that serves as an adaptor between HOIP and CYLD, thereby enabling recruitment of CYLD to signaling complexes.
Assuntos
Macrófagos/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Enzima Desubiquitinante CYLD , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Plasmídeos , Cultura Primária de Células , Ligação Proteica , Proteínas/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Supressoras de Tumor/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Domínio Catalítico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/química , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Ubiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes. LUBAC-generated linear (M1) ubiquitin is important for signaling via various immune receptors. We show here that the deubiquitinases CYLD and A20, but not OTULIN, are recruited to the TNFR1- and NOD2-associated signaling complexes (TNF-RSC and NOD2-SC), at which they cooperate to limit gene activation. Whereas CYLD recruitment depends on its interaction with LUBAC, but not on LUBAC's M1-chain-forming capacity, A20 recruitment requires this activity. Intriguingly, CYLD and A20 exert opposing effects on M1 chain stability in the TNF-RSC and NOD2-SC. While CYLD cleaves M1 chains, and thereby sensitizes cells to TNF-induced death, A20 binding to them prevents their removal and, consequently, inhibits cell death. Thus, CYLD and A20 cooperatively restrict gene activation and regulate cell death via their respective activities on M1 chains. Hence, the interplay between LUBAC, M1-ubiquitin, CYLD, and A20 is central for physiological signaling through innate immune receptors.
Assuntos
Morte Celular/fisiologia , Ativação Transcricional/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Enzima Desubiquitinante CYLD , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Transdução Genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Proteínas Supressoras de Tumor/metabolismoRESUMO
Linear ubiquitin chains are important regulators of cellular signalling pathways that control innate immunity and inflammation through nuclear factor (NF)-κB activation and protection against tumour necrosis factor-α-induced apoptosis. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multisubunit E3 ligase. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or 'donor' ubiquitin. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The carboxy-terminal portion of HOIP adopts a novel fold that, together with a zinc-finger, forms a ubiquitin-binding platform that orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3â¼ubiquitin thioester. The C-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine, providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-κB pathway in vivo, and they explain the determinants of linear ubiquitin chain specificity by LUBAC.
Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Células HeLa , Humanos , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4(+) and CD8(+) T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3(B)) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3(B) domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.
Assuntos
Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Humanos , Sinapses Imunológicas/metabolismo , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Transporte Proteico/imunologia , Proteínas Proto-Oncogênicas c-vav/química , Domínios de Homologia de srcRESUMO
The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP--the catalytic subunit of LUBAC--is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.
Assuntos
Poliubiquitina/biossíntese , Ubiquitina-Proteína Ligases/química , Animais , Domínio Catalítico , Bovinos , Ésteres/química , Poliubiquitina/química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Compostos de Sulfidrila/químicaRESUMO
SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.
Assuntos
Apoptose , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Dermatite/genética , Dermatite/metabolismo , Dermatite/patologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
We have addressed complex formation between the death domain (DD) of the death receptor CD95 (Fas/APO-1) with the DD of immediate adaptor protein FADD using nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and size-exclusion chromatography with in-line light scattering. We find complexation to be independent of the C-terminal 12 residues of CD95 and insensitive to mutation of residues that engage in the high-order clustering of CD95-DD molecules in a recently reported crystal structure obtained at pH 4. Differential NMR linewidths indicate that the C-terminal region of the CD95 chains remains in a disordered state and (13)C-methyl TROSY data are consistent with a lack of high degree of symmetry for the complex. The overall molecular mass of the complex is inconsistent with that in the crystal structure, and the complex dissociates at pH 4. We discuss these findings using sequence analysis of CD95 orthologs and the effect of FADD mutations on the interaction with CD95.