RESUMO
The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.
Assuntos
Síndromes de Imunodeficiência , Proteínas do Tecido Nervoso , Ubiquitinas , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Feminino , Masculino , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/genética , Inflamação/imunologia , Inflamação/genética , Linfócitos B/imunologia , Mutação com Perda de Função , Fibroblastos/metabolismo , Fibroblastos/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Camundongos , AlelosRESUMO
Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteína-Arginina N-Metiltransferases/química , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.
RESUMO
BACKGROUND: Due to the importance of both prostaglandins (PGs) and leukotrienes (LTs) as pro-inflammatory mediators, and the potential for eicosanoid shunting in the presence of pathway target inhibitors, we have investigated an approach to inhibiting the formation of both PGs and LTs as part of a multi-targeted drug discovery effort. METHODS: We generated ligand-protein X-ray crystal structures of known inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) and the 5-Lipoxygenase Activating Protein (FLAP), with their respective proteins, to understand the overlapping pharmacophores. We subsequently used molecular modeling and structure-based drug design (SBDD) to identify hybrid structures intended to inhibit both targets. RESULTS: This work enabled the preparation of compounds 4 and 5, which showed potent in vitro inhibition of both targets. SIGNIFICANCE: Our findings enhance the structural understanding of mPGES-1 and FLAP's unique ligand binding pockets and should accelerate the discovery of additional dual inhibitors for these two important integral membrane protein drug targets.
Assuntos
Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Descoberta de Drogas , Eicosanoides/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Prostaglandina-E Sintases/antagonistas & inibidores , Inibidores da Proteína Ativadora de 5-Lipoxigenase/química , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Eicosanoides/metabolismo , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Prostaglandina-E Sintases/metabolismo , Relação Estrutura-AtividadeRESUMO
Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry. The catalytic PRMT5 subunits form a core tetramer and the MEP50 subunits are arranged peripherally in complex with the PRMT5 N-terminal domain. The cryo-EM reconstruction shows good side chain definition and shows a well-resolved peak for a bound dehydrosinefungin inhibitor molecule. These results demonstrate the applicability of cryo-EM in determining structures of human protein complexes of biomedical significance and suggests cryo-EM could be further utilized to understand PRMT5 interactions with other biologically important binding proteins and ligands.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Microscopia Crioeletrônica , Proteína-Arginina N-Metiltransferases/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Imageamento Tridimensional , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Dendritic cells (DCs) are a phenotypically and functionally heterogenous population of leukocytes with distinct subsets serving a different set of specialized immune functions. Here we applied an in vitro whole cell panning approach using antibody phage display technology to identify cell-surface epitopes specifically expressed on human blood BDCA3(+) DCs. A single-chain antibody fragment (anti-1F12 scFv) was isolated that recognizes a conserved surface antigen expressed on both human BDCA3(+) DCs and mouse CD8alpha(+) DCs. We demonstrate that anti-1F12 scFv binds Nectin-like protein 2 (Necl2, Tslc1, SynCaM, SgIGSF, or Igsf4), an adhesion molecule involved in tumor suppression, synapse formation, and spermatogenesis. Thus, Necl2 defines a specialized subset of DCs in both mouse and human. We further show that Necl2 binds Class-I-restricted T-cell-associated molecule (CRTAM), a receptor primarily expressed on activated cytotoxic lymphocytes. When present on antigen presenting cells, Necl2 regulates IL-22 expression by activated CD8(+) T-cells. We propose that Necl2/CRTAM molecular pair could regulate a large panel of cell/cell interactions both within and outside of the immune system.
Assuntos
Células Dendríticas/citologia , Imunoglobulinas/metabolismo , Imunoglobulinas/fisiologia , Proteínas de Membrana/fisiologia , Linfócitos T/metabolismo , Animais , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Adesão Celular , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Agregação Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Separação Celular , Técnicas de Cocultura , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Humanos , Sistema Imunitário/fisiologia , Immunoblotting , Imunoprecipitação , Interleucinas/biossíntese , Lentivirus/genética , Leucócitos/metabolismo , Ligantes , Linfócitos/citologia , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Octoxinol/farmacologia , Biblioteca de Peptídeos , Fenótipo , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Baço/metabolismo , Interleucina 22RESUMO
Fluorogenic peptide substrates with fluorophore/quencher-capped ends have found extensive use in monitoring protease activity in the screening of small-molecule libraries for protease inhibitors. We report here the identification and characterization of a fluorogenic substrate for tumor necrosis factor-alpha converting enzyme (TACE). This substrate is a 10-amino-acid peptide (LAQAVRSSSR) capped with an o-aminobenzoyl group on the N-terminal end and with a 3-(2,4-dinitrophenyl)-L-2,3-diaminopropionic amide group on the C-terminal end. Exhaustive enzymatic conversion of the substrate to products resulted in a fluorescence enhancement of -11-fold. A single cleavage occurred at the A-V scissile bond of the peptide. The validity of this fluorimetric assay for TACE was corroborated by an independent HPLC method. Interestingly, the hydrolysis of the substrate displayed positive cooperativity with a Hill coefficient of 1.5, while the hydrolysis of the corresponding uncapped peptide displayed Michaelis-Menten kinetics. A k(cat) value of 21.6 s(-1) and an S(0.5) value of 342 microM were obtained for the fluorogenic substrate. The addition of the two capping groups on the two ends of the peptide enhanced the k(cat) value by 64-fold. Nine additional decapeptides that contained the same capping groups on the two ends and substitutions at the P1 and P1' sites were also tested. TACE appears to slightly prefer the A-V scissile bond. The enzyme also cleaves scissile bonds such as F-V, A-I, and A-L efficiently.
Assuntos
Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/metabolismo , Fluorometria/métodos , Metaloendopeptidases/análise , Oligopeptídeos/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animais , Células CHO , Cromatografia Líquida de Alta Pressão/métodos , Cricetinae , Corantes Fluorescentes/síntese química , Humanos , Hidrólise , Cinética , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Oligopeptídeos/síntese química , Especificidade por Substrato/fisiologiaRESUMO
We describe the isolation, cloning, and characterization of human Nek8, a new mammalian NIMA-related kinase, and its candidate substrate Bicd2. Nek8 was isolated as a beta-casein kinase activity in rabbit lung and has an N-terminal catalytic domain homologous to the Nek family of protein kinases. Nek8 also contains a central domain with homology to RCC1, a guanine nucleotide exchange factor for the GTPase Ran, and a C-terminal coiled-coil domain. Like Nek2, Nek8 prefers beta-casein over other exogenous substrates, has shared biochemical requirements for kinase activity, and is capable of autophosphorylation and oligomerization. Nek8 activity is not cell cycle regulated, but like Nek3, levels are consistently higher in G(0)-arrested cells. During the purification of Nek8 a second protein co-chromatographed with Nek8 activity. This protein, Bicd2, is a human homolog of the Drosophila protein Bicaudal D, a coiled-coil protein. Bicd2 is phosphorylated by Nek8 in vitro, and the endogenous proteins associate in vivo. Bicd2 localizes to cytoskeletal structures, and its subcellular localization is dependent on microtubule morphology. Treatment of cells with nocodazole leads to dramatic reorganization of Bicd2, and correlates with Nek8 phosphorylation. This may be indicative of a role for Nek8 and Bicd2 associated with cell cycle independent microtubule dynamics.