RESUMO
BACKGROUND: Epithelial damage, repair and remodelling are critical features of chronic airway diseases including chronic obstructive pulmonary disease (COPD). Interleukin (IL)-33 released from damaged airway epithelia causes inflammation via its receptor, serum stimulation-2 (ST2). Oxidation of IL-33 to a non-ST2-binding form (IL-33ox) is thought to limit its activity. We investigated whether IL-33ox has functional activities that are independent of ST2 in the airway epithelium. METHODS: In vitro epithelial damage assays and three-dimensional, air-liquid interface (ALI) cell culture models of healthy and COPD epithelia were used to elucidate the functional role of IL-33ox. Transcriptomic changes occurring in healthy ALI cultures treated with IL-33ox and COPD ALI cultures treated with an IL-33-neutralising antibody were assessed with bulk and single-cell RNA sequencing analysis. RESULTS: We demonstrate that IL-33ox forms a complex with receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR) expressed on airway epithelium. Activation of this alternative, ST2-independent pathway impaired epithelial wound closure and induced airway epithelial remodelling in vitro. IL-33ox increased the proportion of mucus-producing cells and reduced epithelial defence functions, mimicking pathogenic traits of COPD. Neutralisation of the IL-33ox pathway reversed these deleterious traits in COPD epithelia. Gene signatures defining the pathogenic effects of IL-33ox were enriched in airway epithelia from patients with severe COPD. CONCLUSIONS: Our study reveals for the first time that IL-33, RAGE and EGFR act together in an ST2-independent pathway in the airway epithelium and govern abnormal epithelial remodelling and muco-obstructive features in COPD.
Assuntos
Interleucina-33 , Doença Pulmonar Obstrutiva Crônica , Humanos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33/genética , Interleucina-33/metabolismo , Oxirredução , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Receptor para Produtos Finais de Glicação Avançada/metabolismoRESUMO
TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Glicoproteínas de Membrana/fisiologia , Proteínas Mutantes/metabolismo , Mutação , Neurônios/patologia , Síndromes Neurotóxicas/patologia , Receptores Imunológicos/fisiologia , Doença de Alzheimer , Amiloide/metabolismo , Animais , Camundongos , Camundongos Knockout , Proteínas Mutantes/genética , Neurônios/metabolismo , Síndromes Neurotóxicas/etiologiaRESUMO
Partial loss-of-function variants in the TREM2 immune receptor are associated with increased risk for Alzheimer's disease (AD) and other forms of neurodegenerative disease, but the molecular bases for these connections are unknown. Three new structures of WT and R47H mutant TREM2 immunoglobulin-like (Ig-like) domain now reveal that R47 functions to correctly position elements of the ligand-binding surface. Intriguingly, the authors also demonstrate a disruption of receptor oligomerization by the R47H mutation, suggesting a role for ligand-induced clustering in receptor signaling and resultant plaque clearance.
Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Glicoproteínas de Membrana/química , Proteínas Mutantes/química , Receptores Imunológicos/química , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Genótipo , Humanos , Glicoproteínas de Membrana/genética , Proteínas Mutantes/genética , Mutação , Placa Amiloide/química , Placa Amiloide/genética , Receptores Imunológicos/genéticaRESUMO
CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The â¼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located â¼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.
Assuntos
Biocatálise , Proteínas de Membrana/química , Mathanococcus/enzimologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Prenilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Sequência Conservada , Cristalografia por Raios X , Cisteína/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/classificação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Transdução de Sinais , Especificidade por SubstratoRESUMO
The posttranslational modification of C-terminal CAAX motifs in proteins such as Ras, most Rho GTPases, and G protein γ subunits, plays an essential role in determining their subcellular localization and correct biological function. An integral membrane methyltransferase, isoprenylcysteine carboxyl methyltransferase (ICMT), catalyzes the final step of CAAX processing after prenylation of the cysteine residue and endoproteolysis of the -AAX motif. We have determined the crystal structure of a prokaryotic ICMT ortholog, revealing a markedly different architecture from conventional methyltransferases that utilize S-adenosyl-L-methionine (SAM) as a cofactor. ICMT comprises a core of five transmembrane α helices and a cofactor-binding pocket enclosed within a highly conserved C-terminal catalytic subdomain. A tunnel linking the reactive methyl group of SAM to the inner membrane provides access for the prenyl lipid substrate. This study explains how an integral membrane methyltransferase achieves recognition of both a hydrophilic cofactor and a lipophilic prenyl group attached to a polar protein substrate.
Assuntos
Proteínas Metiltransferases/química , Proteínas Metiltransferases/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Metabolismo dos Lipídeos , Methanosarcina/enzimologia , Metilação , Modelos Moleculares , Mutação , Proteínas Metiltransferases/genética , Estrutura Terciária de Proteína , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how Protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.
Assuntos
Ligação Proteica , Proteínas , Termodinâmica , Proteínas/metabolismo , Proteínas/química , Proteínas/genética , Mutação , Mutação Puntual , Conformação Proteica , Biologia Computacional/métodos , Modelos MolecularesRESUMO
Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.
RESUMO
Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.
Assuntos
Hepatite C , Simeprevir , Humanos , Camundongos , Animais , Simeprevir/farmacologia , Simeprevir/uso terapêutico , Hepatite C/tratamento farmacológico , Hepacivirus/metabolismo , Terapia Genética , Apoptose , Antivirais/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Cell surface membrane proteins comprise a wide array of structurally and functionally diverse proteins involved in a variety of important physiological and homeostatic processes. Complex integral membrane proteins, which are embedded in the lipid bilayer by multiple transmembrane-spanning helices, are represented by families of proteins that are important target classes for drug discovery. Such protein families include G-protein-coupled receptors, ion channels and transporters. Although these targets have typically been the domain of small-molecule drugs, the exquisite specificity of monoclonal antibodies offers a significant opportunity to selectively modulate these target proteins. Nevertheless, the isolation of antibodies with desired pharmacological functions has proved difficult because of technical challenges in preparing membrane protein antigens for antibody drug discovery. In this review, we describe recent progress in defining strategies for the generation of membrane protein antigens. We also describe antibody-isolation strategies that identify antibodies that bind the membrane protein and modulate protein function.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Antígenos/imunologia , Descoberta de Drogas/métodos , Proteínas de Membrana/imunologia , Animais , HumanosRESUMO
We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild-type and H157Y human TREM2 and for the wild-type murine orthologue. Crucially, we also show that the Alzheimer's disease-associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer's and other neurological diseases.
Assuntos
Doença de Alzheimer/genética , Glicoproteínas de Membrana/metabolismo , Proteólise , Receptores Imunológicos/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Meios de Cultivo Condicionados , Células HEK293 , Humanos , Cetocolesteróis/farmacologia , Macrófagos/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Imunológicos/genéticaRESUMO
Hypoxia-inducible transcription factors (HIFs) control adaptation to low oxygen environments by activating genes involved in metabolism, angiogenesis, and redox homeostasis. The finding that HIFs are also regulated by small molecule metabolites highlights the need to understand the complexity of their cellular regulation. Here we use a forward genetic screen in near-haploid human cells to identify genes that stabilize HIFs under aerobic conditions. We identify two mitochondrial genes, oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS), which when mutated stabilize HIF1α in a non-hydroxylated form. Disruption of OGDH complex activity in OGDH or LIAS mutants promotes L-2-hydroxyglutarate formation, which inhibits the activity of the HIFα prolyl hydroxylases (PHDs) and TET 2-oxoglutarate dependent dioxygenases. We also find that PHD activity is decreased in patients with homozygous germline mutations in lipoic acid synthesis, leading to HIF1 activation. Thus, mutations affecting OGDHC activity may have broad implications for epigenetic regulation and tumorigenesis.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Lipoilação , Proteínas Mitocondriais/metabolismo , Aerobiose , Linhagem Celular , Testes Genéticos , Mutação em Linhagem Germinativa/genética , Glutaratos/metabolismo , Células HeLa , Homozigoto , Humanos , Hidroxilação , Prolina/metabolismo , Estabilidade Proteica , SulfurtransferasesRESUMO
The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Caenorhabditis elegans/genética , Degeneração Lobar Frontotemporal/genética , Hidrogéis , Atividade Motora/genética , Transição de Fase , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/genética , Animais , Caenorhabditis elegans , Grânulos Citoplasmáticos/metabolismo , Modelos Animais de Doenças , Longevidade , Mutação , Proteína FUS de Ligação a RNA/química , Ribonucleoproteínas/metabolismoRESUMO
Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.
Assuntos
Benzodiazepinonas/química , Inibidores Enzimáticos/química , Peptidomiméticos/química , Presenilina-1/química , Proteínas Recombinantes de Fusão/química , Regulação Alostérica , Secretases da Proteína Precursora do Amiloide/química , Domínio Catalítico , Células HEK293 , Humanos , Ligantes , Glicoproteínas de Membrana/química , Simulação de Acoplamento Molecular , Presenilina-1/antagonistas & inibidores , Presenilina-1/genética , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-AtividadeRESUMO
MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposi's sarcoma-associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3-associated class I by lysine-63-linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine-63-linked polyubiquitination requires both UbcH5b/c and Ubc13-conjugating enzymes for initiating mono- and subsequent polyubiquitination of class I, and the clathrin-dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine-63-linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.
Assuntos
Endocitose , Endossomos/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Lisina/fisiologia , Lisossomos/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/fisiologia , Regulação para Baixo , Células HeLa , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Poliubiquitina/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Virais/metabolismoRESUMO
RING domains are found in a large number of eukaryotic proteins. Most function as E3 ubiquitin-protein ligases, catalyzing the terminal step in the ubiquitination process. Structurally, these domains have been characterized as binding two zinc ions in a stable cross-brace motif. The tumorigenic human gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus encodes a ubiquitin-protein ligase termed K3, which functions as an immune evasion molecule by ubiquitinating major histocompatibility complex class I. K3 possesses at its N terminus a domain related to cellular RING domains but with an altered zinc ligand arrangement. This domain was initially characterized as a plant homeodomain, a structure not previously known to function as an E3. Here, it is conclusively demonstrated that the K3 N-terminal domain is a variant member of the RING domain family and not a plant homeodomain. The domain is found to interact with the cellular ubiquitin-conjugating enzymes UbcH5A to -C and UbcH13, which dock to the equivalent surface as on classical cellular RING domains. Interaction with UbcH13 suggests a possible role for K3 in catalyzing Lys(63)-linked ubiquitination.