RESUMO
Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.
Assuntos
Gasderminas , Lipoilação , Proteínas de Ligação a Fosfato , Espécies Reativas de Oxigênio , Animais , Feminino , Humanos , Masculino , Camundongos , Aciltransferases/metabolismo , Microscopia Crioeletrônica , Cisteína/metabolismo , Gasderminas/química , Gasderminas/metabolismo , Inflamassomos/metabolismo , Lipossomos/metabolismo , Lipossomos/química , Mitocôndrias/metabolismo , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Células THP-1RESUMO
All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.
Assuntos
Bactérias , Infecções Bacterianas , Membrana Celular , Proteínas de Ligação ao GTP , Reconhecimento da Imunidade Inata , Humanos , Citocinas/química , Tomografia com Microscopia Eletrônica , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/química , Hidrólise , Imunidade Celular , Microscopia Crioeletrônica , Gasderminas/química , Proteínas de Ligação a Fosfato/química , Conformação Proteica , Membrana Celular/química , Membrana Celular/imunologia , Caspases Iniciadoras/química , Infecções Bacterianas/imunologia , Bactérias/imunologiaRESUMO
Metabolic reprogramming is a key attribute of cancer progression. An altered expression of pyruvate kinase M2 (PKM2), a phosphotyrosine-binding protein is observed in many human cancers. PKM2 plays a vital role in metabolic reprogramming, transcription and cell cycle progression and thus is deliberated as an attractive target in anticancer drug development. The expression of PKM2 is essential for aerobic glycolysis and cell proliferation, especially in cancer cells, facilitating selective targeting of PKM2 in cell metabolism for cancer therapeutics. We have screened a virtual library of phytochemicals from the IMPPAT (Indian Medicinal Plants, Phytochemistry and Therapeutics) database of Indian medicinal plants to identify potential activators of PKM2. The initial screening was carried out for the physicochemical properties of the compounds, and then structure-based molecular docking was performed to select compounds based on their binding affinity towards PKM2. Subsequently, the ADMET (absorption, distribution, metabolism, excretion and toxicity) properties, PAINS (Pan-assay interference compounds) patterns, and PASS evaluation were carried out to find more potent hits against PKM2. Here, Tuberosin was identified from the screening process bearing appreciable binding affinity toward the PKM2-binding pocket and showed a worthy set of drug-like properties. Finally, molecular dynamics simulation for 100 ns was performed, which showed decent stability of the protein-ligand complex and relatival conformational dynamics throughout the trajectory. The study suggests that modulating PKM2 with natural compounds is an attractive approach in treating human malignancy after required validation.
Assuntos
Ativadores de Enzimas , Isoflavonas , Neoplasias , Piruvato Quinase , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Glicosídeos/farmacologia , Glicosídeos/uso terapêutico , Isoflavonas/farmacologia , Isoflavonas/uso terapêutico , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Piruvato Quinase/metabolismoRESUMO
Gasdermin D (GSDMD) is a recently identified pore-forming protein that is crucial for the execution of pyroptosis, a highly inflammatory form of cell death. GSDMD contains an N-terminal and a C-terminal domain that are separated by a proteolysis-sensitive linker. Upon cleavage of this linker by inflammasome-activated caspases, the N-terminal domain of GSDMD oligomerizes and forms pores at the plasma membrane, allowing cell swelling and subsequently membrane rupture to mediate pyroptosis. GSDMD is a key substrate of inflammatory caspases downstream of inflammasome activation and is driving various pathologies. Here, we describe a simple method to study GSDMD cleavage following canonical inflammasome activation in murine primary macrophages and neutrophils and human cell lines using immunoblotting.
Assuntos
Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular , Animais , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato/química , PiroptoseRESUMO
Endosome capture is a major physiological barrier to the successful delivery of nanomedicine. Here, we found a strategy to deliver ultrasmall nanoparticles (<10 nm) to the cytosolic compartment of pyroptotic cells with spontaneous endosomal escape. To mimic pathological pyroptotic cells, J774A.1 macrophages were stimulated with lipopolysaccharide (LPS) plus nigericin (Nig) or adenosine triphosphate (ATP) to form specific gasdermin D protein-driven membrane pores at an N-terminal domain (GSDMDNterm). Through GSDMDNterm membrane pores, both anionic and cationic nanoparticles (NPs) with diameters less than 10 nm were accessed into the cytosolic compartment of pyroptotic cells in an energy- and receptor-independent manner, while NPs larger than the size of GSDMDNterm membrane pores failed to enter pyroptotic cells. NPs pass through GSDMDNterm membrane pores via free diffusion and then access into the cytoplasm of pyroptotic cells in a microtubule-independent manner. Interestingly, we found that LPS-primed NPs may act as Trojan horse, deliver extracellular LPS into normal cells through endocytosis, and in turn induce GSDMDNterm membrane pores, which facilitate further internalization of NPs. This study presented a straightforward method of distinguishing normal and pyroptotic cells through GSDMD membrane pores, implicating their potential application in monitoring the delivery of desired nanomedicines in pyroptosis-related diseases and conditions.
Assuntos
Membrana Celular/metabolismo , Citosol/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Nanopartículas/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Membrana Celular/química , Células Cultivadas , Citosol/química , Sistemas de Liberação de Medicamentos , Lipopolissacarídeos/química , Macrófagos/química , Camundongos , Nanopartículas/química , Proteínas de Ligação a Fosfato/química , Proteínas Citotóxicas Formadoras de Poros/química , PiroptoseRESUMO
Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.
Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Autofagossomos/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Cristalografia , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genética , Fosfatidilinositol 3-Quinase/metabolismo , Mutação Puntual , Conformação Proteica em alfa-Hélice , Transporte Proteico , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Inhibition of ruminal microbial urease is of particular interest due to its crucial role in regulating urea-N utilization efficiency and nitrogen pollution in the livestock industry. Acetohydroxamic acid (AHA) is currently the only commercially available urease inhibitor, but it has adverse side effects. The urease accessory protein UreG, which facilitates the functional incorporation of the urease nickel metallocentre, has been proposed in developing urease inhibitor through disrupting urease maturation. The objective of this study was to screen natural compounds as potential urease inhibitors by targeting UreG in a predominant ruminal microbial urease. In silico screening and in vitro tests for potential inhibitors were performed using molecular docking and an assay for the GTPase activity of UreG. Chelerythrine chloride was selected as a potential urease inhibitor of UreG with an inhibition concentration IC50 value of 18.13 µM. It exhibited mixed inhibition, with the Ki value being 26.28 µM. We further explored its inhibition mechanism using isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy, and we found that chelerythrine chloride inhibited the binding of nickel to UreG and induced changes in the secondary structure, especially the α-helix and ß-sheet of UreG. Chelerythrine chloride formed a pi-anion interaction with the Asp41 residue of UreG, which is an important residue in initiating the conformational changes of UreG. In conclusion, chelerythrine chloride exhibited a potential inhibitory effect on urease, which provided new evidence for strategies to develop novel urease inhibitors targeting UreG to reduce nitrogen excretion from ruminants.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Benzofenantridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Rúmen/microbiologia , Amônia/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzofenantridinas/química , Sítios de Ligação , Bovinos , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Ligação ProteicaRESUMO
The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Multimerização Proteica , Piroptose , Transdução de Sinais , Aminoácidos/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Testes Genéticos , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Macrófagos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas de Ligação a Fosfato/química , Domínios Proteicos , RNA Guia de Cinetoplastídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such "elimination damage" generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically.
Assuntos
Alanina/análogos & derivados , Aminobutiratos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteoma , Receptores Acoplados a Proteínas G/metabolismo , Alanina/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Feminino , Glutationa/metabolismo , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genética , Fosforilação , Domínios Proteicos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Sulfetos/metabolismoRESUMO
Stroke is a major cause of death and disability worldwide that triggers a variety of neuropathological conditions, leading to the initiation of several pro-inflammatory mediators and neuronal damage. Neuroinflammation has been considered the potential therapeutic target and contributes to the pathology of ischemia and reperfusion. Pyroptosis is an inflammatory form of programmed cell death that plays an important role in immune protection against stroke. Gasdermin D (GSDMD) is the final executor of pyroptosis upon cleavage by caspases-1/4/5/11, followed by canonical and noncanonical inflammasome activation, leading to a series of inflammatory responses. GSDMD N-terminal domain assembles plasma membrane as well as organelle membrane pores to induce cytolysis, thereby triggering cytokine release and inflammatory-related cell death. In our review, we concisely summarized and highlighted the potential role of GSDMD-regulated pyroptosis and the biological characteristic of GSDMD as a therapeutic target in ischemic stroke. A better understanding of the roles of GSDMD may provide a theoretical basis for the design of novel therapeutic interventions for the treatment of ischemic stroke.
Assuntos
Isquemia Encefálica/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , AVC Isquêmico/metabolismo , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/metabolismo , Isquemia Encefálica/tratamento farmacológico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , AVC Isquêmico/tratamento farmacológico , Proteínas de Ligação a Fosfato/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Piroptose/fisiologiaRESUMO
Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the Pi biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a single turnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.
Assuntos
Cumarínicos/química , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Nucleotídeos/química , Proteínas de Ligação a Fosfato/metabolismo , Fosfatos/análise , Adenosina Trifosfatases/metabolismo , Técnicas Biossensoriais , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrólise , Cinética , Proteínas de Ligação a Fosfato/químicaRESUMO
As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.
Assuntos
Inflamassomos/química , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Macrófagos/metabolismo , Proteínas de Ligação a Fosfato/química , Animais , Caspase 1/metabolismo , Microscopia Crioeletrônica , Humanos , Interleucina-1/metabolismo , Camundongos Endogâmicos C57BL , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Eletricidade EstáticaRESUMO
The DING proteins are ubiquitous in the three domains of life, from mesophiles to thermo- and hyperthermophiles. They belong to a family of more than sixty members and have a characteristic N-terminus, DINGGG, which is considered a "signature" of these proteins. Structurally, they share a highly conserved phosphate binding site, and a three dimensional organization resembling the "Venus Flytrap", both reminding the ones of PstS proteins. They have unusually high sequence conservation, even between distantly related species. Nevertheless despite that the genomes of most of these species have been sequenced, the DING gene has not been reported for all the relative characterized DING proteins. Identity of known DING proteins has been confirmed immunologically and, in some cases, by N-terminal sequence analysis. Only a few of the DING proteins have been purified and biochemically characterized. DING proteins are heterogeneous for their wide range of biological activities and some show different activities not always correlated with each other. Most of them have been originally identified for different biological properties, or rather for binding to phosphate and also to other ligands. Their involvement in pathologies is described. This review is an update of the most recent findings on old and new DING proteins.
Assuntos
Extremófilos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Sequência de Aminoácidos , Archaea/metabolismo , Eucariotos/metabolismo , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genéticaRESUMO
The striatin-interacting phosphatase and kinase (STRIPAK) complex is a large, multisubunit protein phosphatase 2A (PP2A) assembly that integrates diverse cellular signals in the Hippo pathway to regulate cell proliferation and survival. The architecture and assembly mechanism of this critical complex are poorly understood. Using cryo-EM, we determine the structure of the human STRIPAK core comprising PP2AA, PP2AC, STRN3, STRIP1, and MOB4 at 3.2-Å resolution. Unlike the canonical trimeric PP2A holoenzyme, STRIPAK contains four copies of STRN3 and one copy of each the PP2AA-C heterodimer, STRIP1, and MOB4. The STRN3 coiled-coil domains form an elongated homotetrameric scaffold that links the complex together. An inositol hexakisphosphate (IP6) is identified as a structural cofactor of STRIP1. Mutations of key residues at subunit interfaces disrupt the integrity of STRIPAK, causing aberrant Hippo pathway activation. Thus, STRIPAK is established as a noncanonical PP2A complex with four copies of regulatory STRN3 for enhanced signal integration.
Assuntos
Microscopia Crioeletrônica , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/metabolismo , Autoantígenos/ultraestrutura , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/ultraestrutura , Via de Sinalização Hippo , Humanos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/ultraestrutura , Ácido Fítico/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/ultraestrutura , Proteínas Serina-Treonina Quinases/química , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de SinaisRESUMO
The association of Zika virus (ZIKV) infection with a congenital malformation in fetuses, neurological, and other systemic complications in adults have brought significant global health emergency. ZIKV targets nerve cells in the brain and causes cell death, such as pyroptosis, leading to neuroinflammation. Here we described a novel mechanism of pyroptosis caused by ZIKV protease. We found that ZIKV protease directly cleaved the GSDMD into N-terminal fragment (1-249) leading to pyroptosis in a caspase-independent manner, suggesting a direct mechanism of ZIKV-induced cell death and subsequent inflammation. Our findings might shed new light to explore the pathogenesis of ZIKV infections where ZIKV protease might be a suitable target for the development of antiviral agents.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/fisiologia , Proteínas Virais/metabolismo , Zika virus/enzimologia , Zika virus/patogenicidade , Sítios de Ligação , Caspases/metabolismo , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Biológicos , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a Fosfato/química , Proteólise , Especificidade por Substrato , Infecção por Zika virus/etiologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologiaRESUMO
Urease is a nickel-containing enzyme that is essential for the survival of several and often deadly pathogenic bacterial strains, including Helicobacter pylori. Notwithstanding several attempts, the development of direct urease inhibitors without side effects for the human host remains, to date, elusive. The recently solved X-ray structure of the HpUreDFG accessory complex involved in the activation of urease opens new perspectives for structure-based drug discovery. In particular, the quaternary assembly and the presence of internal tunnels for nickel translocation offer an intriguing possibility to target the HpUreDFG complex in the search of indirect urease inhibitors. In this work, we adopted a theoretical framework to investigate such a hypothesis. Specifically, we searched for putative binding sites located at the protein-protein interfaces on the HpUreDFG complex, and we challenged their druggability through structure-based virtual screening. We show that, by virtue of the presence of tunnels, some protein-protein interfaces on the HpUreDFG complex are intrinsically well suited for hosting small molecules, and, as such, they possess good potential for future drug design endeavors.
Assuntos
Inibidores Enzimáticos/farmacologia , Helicobacter pylori/metabolismo , Complexos Multiproteicos/metabolismo , Urease/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Urease/química , Urease/metabolismoRESUMO
UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions.
Assuntos
Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Helicobacter pylori/metabolismo , Níquel/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Infecções por Helicobacter/microbiologia , Helicobacter pylori/química , Humanos , Modelos Moleculares , Proteínas de Ligação a Fosfato/química , Conformação ProteicaRESUMO
Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)-retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (â¼18%), dimer (â¼35%), trimer (â¼24%), and tetramer (â¼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.
Assuntos
Bicamadas Lipídicas/química , Complexos Multiproteicos/química , Proteínas de Ligação a Fosfato/química , Nexinas de Classificação/química , Proteínas rab de Ligação ao GTP/química , Humanos , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Nexinas de Classificação/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1ß (pro-IL18/pro-IL1ß). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1ß cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.
Assuntos
Caspases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Piroptose , Animais , Catálise , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.