RESUMO
The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.
Assuntos
Inflamassomos , Sistemas de Secreção Tipo III , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Flagelina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Proteína Inibidora de Apoptose Neuronal/metabolismoRESUMO
Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through a highly conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity.
Assuntos
Imunoglobulina A Secretora/química , Cadeias J de Imunoglobulina/metabolismo , Imunoglobulina M/química , Componente Secretório/metabolismo , Linhagem Celular , Humanos , Imunoglobulina A Secretora/metabolismo , Imunoglobulina M/metabolismo , Modelos Moleculares , Conformação Proteica , Homologia Estrutural de Proteína , TranscitoseRESUMO
The phosphoinositide PI(3,5)P2, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P2 levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P2 from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P2 production by PIKfyve in vivo.
Assuntos
Membrana Celular/metabolismo , Flavoproteínas/metabolismo , Homeostase , Lisossomos/metabolismo , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Flavoproteínas/química , Flavoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Transporte ProteicoRESUMO
Secretory immunoglobulin A (sIgA) represents the immune system's first line of defense against mucosal pathogens. IgAs are transported across the epithelium, as dimers and higher-order polymers, by the polymeric immunoglobulin receptor (pIgR). Upon reaching the luminal side, sIgAs mediate host protection and pathogen neutralization. In recent years, an increasing amount of attention has been given to IgA as a novel therapeutic antibody. However, despite extensive studies, sIgA structures have remained elusive. Here, we determine the atomic resolution structures of dimeric, tetrameric, and pentameric IgA-Fc linked by the joining chain (JC) and in complex with the secretory component of the pIgR. We suggest a mechanism in which the JC templates IgA oligomerization and imparts asymmetry for pIgR binding and transcytosis. This framework will inform the design of future IgA-based therapeutics.
Assuntos
Imunoglobulina A Secretora/química , Fragmentos Fc das Imunoglobulinas/química , Multimerização Proteica , Humanos , Cadeias J de Imunoglobulina/química , Receptores de Imunoglobulina Polimérica/química , TranscitoseRESUMO
Kindlins are essential FERM-domain-containing focal adhesion (FA) proteins required for proper integrin activation and signaling. Despite the widely accepted importance of each of the three mammalian kindlins in cell adhesion, the molecular basis for their function has yet to be fully elucidated, and the functional differences between isoforms have generally not been examined. Here, we report functional differences between kindlin-2 and -3 (also known as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not kindlin-3, can rescue α5ß1 integrin activation defects in kindlin-2-knockdown fibroblasts. Using chimeric kindlins, we show that the relatively uncharacterized kindlin-2 F2 subdomain drives FA targeting and integrin activation. We find that the integrin-linked kinase (ILK)-PINCH-parvin complex binds strongly to the kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated kindlin-2, we establish that efficient kindlin-2-mediated integrin activation and FA targeting require binding to the ILK complex. Thus, ILK-complex binding is crucial for normal kindlin-2 function and differential ILK binding contributes to kindlin isoform specificity.
Assuntos
Adesões Focais/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Isoformas de Proteínas , Transdução de SinaisRESUMO
Diabetic patients are known to be more susceptible to atherosclerosis and its associated cardiovascular complications. However, the effects of hyperglycemia on atherosclerosis regression remain unclear. We hypothesized that hyperglycemia impairs atherosclerosis regression by modulating the biological function of lesional macrophages. HypoE (Apoe(h/h)Mx1-Cre) mice express low levels of apolipoprotein E (apoE) and develop atherosclerosis when fed a high-fat diet. Atherosclerosis regression occurs in these mice upon plasma lipid lowering induced by a change in diet and the restoration of apoE expression. We examined the morphological characteristics of regressed lesions and assessed the biological function of lesional macrophages isolated with laser-capture microdissection in euglycemic and hyperglycemic HypoE mice. Hyperglycemia induced by streptozotocin treatment impaired lesion size reduction (36% versus 14%) and lipid loss (38% versus 26%) after the reversal of hyperlipidemia. However, decreases in lesional macrophage content and remodeling in both groups of mice were similar. Gene expression analysis revealed that hyperglycemia impaired cholesterol transport by modulating ATP-binding cassette A1, ATP-binding cassette G1, scavenger receptor class B family member (CD36), scavenger receptor class B1, and wound healing pathways in lesional macrophages during atherosclerosis regression. Hyperglycemia impairs both reduction in size and loss of lipids from atherosclerotic lesions upon plasma lipid lowering without significantly affecting the remodeling of the vascular wall.
Assuntos
Apolipoproteínas E , Aterosclerose , Regulação da Expressão Gênica/genética , Hiperglicemia , Lipídeos/sangue , Macrófagos , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/sangue , Aterosclerose/complicações , Aterosclerose/genética , Aterosclerose/patologia , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Feminino , Hiperglicemia/sangue , Hiperglicemia/complicações , Hiperglicemia/genética , Hiperglicemia/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos TransgênicosRESUMO
OBJECTIVE: To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)-lipoprotein levels. APPROACH AND RESULTS: Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoe(h/h)Ldlr(-/-)Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non-high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6C(high) monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. CONCLUSIONS: Restoring apoE expression in Apoe(h/h)Ldlr(-/-)Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non-high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6C(high) monocytes levels and genetic reprogramming of lesional macrophages.
Assuntos
Apolipoproteínas E/genética , Terapia Genética/métodos , Placa Aterosclerótica/genética , Placa Aterosclerótica/terapia , Receptores de LDL/genética , Animais , Apolipoproteína B-100 , Apolipoproteínas B/sangue , Apolipoproteínas B/genética , Apolipoproteínas E/sangue , Apolipoproteínas E/deficiência , Apoptose/fisiologia , Colesterol/sangue , HDL-Colesterol/sangue , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica/fisiologia , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Hiperlipidemias/terapia , Macrófagos/citologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Placa Aterosclerótica/metabolismo , Receptores de LDL/deficiência , Triglicerídeos/sangueRESUMO
BACKGROUND: Apolipoprotein (apo) E is best known for its ability to lower plasma cholesterol and protect against atherosclerosis. Although the liver is the major source of plasma apoE, extra-hepatic sources of apoE, including from macrophages, account for up to 10% of plasma apoE levels. This study examined the contribution of macrophage-derived apoE expression levels in diet-induced hyperlipidemia and atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Hypomorphic apoE (Apoe(h/h)) mice expressing wildtype mouse apoE at â¼2-5% of physiological levels in all tissues were derived by gene targeting in embryonic stem cells. Cre-mediated gene repair of the Apoe(h/h) allele in Apoe(h/h)LysM-Cre mice raised apoE expression levels by 26 fold in freshly isolated peritoneal macrophages, restoring it to 37% of levels seen in wildtype mice. Chow-fed Apoe(h/h)LysM-Cre and Apoe(h/h) mice displayed similar plasma apoE and cholesterol levels (55.53±2.90 mg/dl versus 62.70±2.77 mg/dl, nâ=â12). When fed a high-cholesterol diet (HCD) for 16 weeks, Apoe(h/h)LysM-Cre mice displayed a 3-fold increase in plasma apoE and a concomitant 32% decrease in plasma cholesterol when compared to Apoe(h/h) mice (602.20±22.30 mg/dl versus 888.80±24.99 mg/dl, nâ=â7). On HCD, Apoe(h/h)LysM-Cre mice showed increased apoE immunoreactivity in lesional macrophages and liver-associated Kupffer cells but not hepatocytes. In addition, Apoe(h/h)LysM-Cre mice developed 35% less atherosclerotic lesions in the aortic root than Apoe(h/h) mice (167×10(3)±16×10(3) µm(2) versus 259×10(3)±56×10(3) µm(2), nâ=â7). This difference in atherosclerosis lesions size was proportional to the observed reduction in plasma cholesterol. CONCLUSIONS/SIGNIFICANCE: Macrophage-derived apoE raises plasma apoE levels in response to diet-induced hyperlipidemia and by such reduces atherosclerosis proportionally to the extent to which it lowers plasma cholesterol levels.