RESUMO
The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fatores de Troca do Nucleotídeo Guanina , Proteínas rac1 de Ligação ao GTP , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Ligação Proteica , Conformação Proteica , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/químicaRESUMO
Matrix metalloproteinase-9 (MMP-9) is a secreted zinc-dependent endopeptidase that degrades the extracellular matrix and basement membrane of neurons, and then contributes to synaptic plasticity by remodeling the extracellular matrix. Inhibition of MMP-9 activity has therapeutic potential for neurodegenerative diseases such as fragile X syndrome. This paper reports the molecular design, synthesis, and in vitro studies of novel indole derivatives as inhibitors of proMMP-9 activation. High-throughput screening (HTS) of our internal compound library and subsequent merging of hit compounds 1 and 2 provided compound 4 as a bona-fide lead. X-ray structure-based design and subsequent lead optimization led to the discovery of compound 33, a highly potent and selective inhibitor of proMMP-9 activation.
Assuntos
Precursores Enzimáticos , Metaloproteinase 9 da Matriz , Metaloproteinase 9 da Matriz/metabolismo , Precursores Enzimáticos/metabolismo , Matriz Extracelular/metabolismo , Indóis/farmacologia , Indóis/metabolismo , Metaloendopeptidases/metabolismo , Inibidores de Metaloproteinases de MatrizRESUMO
Dedicator of cytokinesis 10 (DOCK10), an evolutionarily conserved guanine nucleotide exchange factor (GEF) for Rho GTPases, has the unique specificity within the DOCK-D subfamily to activate both Cdc42 and Rac, but the structural bases for these activities remained unknown. Here we present the crystal structures of the catalytic DHR2 domain of mouse DOCK10, complexed with either Cdc42 or Rac1. The structures revealed that DOCK10DHR2 binds to Cdc42 or Rac1 by slightly changing the arrangement of its two catalytic lobes. DOCK10 also has a flexible binding pocket for the 56th GTPase residue, allowing a novel interaction with Trp56Rac1. The conserved residues in switch 1 of Cdc42 and Rac1 showed common interactions with the unique Lys-His sequence in the ß5/ß6 loop of DOCK10DHR2. However, the interaction of switch 1 in Rac1 was less stable than that of switch 1 in Cdc42, due to amino acid differences at positions 27 and 30. Structure-based mutagenesis identified the DOCK10 residues that determine the Cdc42/Rac1 dual specificity.
Assuntos
Fatores de Troca do Nucleotídeo Guanina , Proteínas rac1 de Ligação ao GTP , Animais , Camundongos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Citocinese , Mutagênese , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.
Assuntos
Receptores de Ativinas Tipo I/antagonistas & inibidores , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Miosite Ossificante/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Camundongos , Estrutura Molecular , Mutação , Miosite Ossificante/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.
Assuntos
Receptores de Ativinas Tipo I/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Administração Oral , Animais , Área Sob a Curva , Disponibilidade Biológica , Simulação por Computador , Cristalografia por Raios X , Meia-Vida , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Estrutura Molecular , Miosite Ossificante/enzimologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Espectroscopia de Prótons por Ressonância Magnética , Pirazóis/administração & dosagem , Pirazóis/química , Pirazóis/farmacocinética , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokinesis 2-engulfment and cell motility protein 1 (DOCK2â¢ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Å resolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2â¢ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2â¢ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2â¢ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismo , Domínios de Homologia de srcRESUMO
Biologics have become an important component of treatment strategies for a variety of diseases, but the immunogenicity of large immune complexes (ICs) and aggregates of biologics may increase risk of adverse events is a concern for biologics and it remains unclear whether large ICs consisting of intrinsic antigen and therapeutic antibodies are actually involved in acute local inflammation such as injection site reaction (ISR). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. Treatment with ozoralizumab has been shown to provide beneficial effects in the treatment of rheumatoid arthritis (RA) comparable to those obtained with other TNFα inhibitors. Very few ISRs (2%) have been reported after ozoralizumab administration, and the drug has been shown to have acceptable safety and tolerability. In this study, in order to elucidate the mechanism underlying the reduced incidence of ISRs associated with ozoralizumab administration, we investigated the stoichiometry of two TNFα inhibitors (ozoralizumab and adalimumab, an anti-TNFα IgG) ICs and the induction by these drugs of Fcγ receptor (FcγR)-mediated immune responses on neutrophils. Ozoralizumab-TNFα ICs are smaller than adalimumab-TNFα ICs and lack an Fc portion, thus mitigating FcγR-mediated immune responses on neutrophils. We also developed a model of anti-TNFα antibody-TNFα IC-induced subcutaneous inflammation and found that ozoralizumab-TNFα ICs do not induce any significant inflammation at injection sites. The results of our studies suggest that ozoralizumab is a promising candidate for the treatment of RA that entails a lower risk of the IC-mediated immune cell activation that leads to unwanted immune responses.
Assuntos
Artrite Reumatoide , Produtos Biológicos , Humanos , Complexo Antígeno-Anticorpo , Adalimumab/uso terapêutico , Receptores de IgG , Artrite Reumatoide/tratamento farmacológico , Anticorpos Monoclonais/efeitos adversos , Inflamação/tratamento farmacológico , Produtos Biológicos/uso terapêuticoRESUMO
The control of cell movement through manipulation of cytoskeletal structure has therapeutic prospects notably in the development of novel anti-metastatic drugs. In this study, we determine the structure of Ras-binding domain (RBD) of ELMO1, a protein involved in cytoskeletal regulation, both alone and in complex with the activator RhoG and verify its targetability through computational nanobody design. Using our dock-and-design approach optimized with native-like initial pose selection, we obtain Nb01, a detectable binder from scratch in the first-round design. An affinity maturation step guided by structure-activity relationship at the interface generates 23 Nb01 sequence variants and 17 of them show enhanced binding to ELMO1-RBD and are modeled to form major spatial overlaps with RhoG. The best binder, Nb29, inhibited ELMO1-RBD/RhoG interaction. Molecular dynamics simulation of the flexibility of CDR2 and CDR3 of Nb29 reveal the design of stabilizing mutations at the CDR-framework junctions potentially confers the affinity enhancement.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Simulação de Dinâmica Molecular , Proteínas rho de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).
Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/química , Complexos Multienzimáticos/química , Regulação Alostérica/fisiologia , Animais , Cristalografia por Raios X , Ativação Enzimática/fisiologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutação , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Fatores de Troca de Nucleotídeo Guanina Rho , Relação Estrutura-AtividadeRESUMO
Alzheimer's disease (AD) is one of the most common, progressive neurodegenerative disorders affecting the aged populations. Though various disease pathologies have been suggested for AD, the impairment of the cholinergic system is one of the critical factors for the disease progression. Restoration of the cholinergic transmission through acetylcholinesterase (AChE) inhibitors is a promising disease modifying therapy. Being the first marketed drug for AD, tacrine reversibly inhibits AChE and thereby slows the breakdown of the chemical messenger acetylcholine (ACh) in the brain. However, the atomic level of interactions of tacrine towards human AChE (hAChE) is unknown for years. Hence, in the current study, we report the X-ray structure of hAChE-tacrine complex at 2.85 Å resolution. The conformational heterogeneity of tacrine within the electron density was addressed with the help of molecular mechanics assisted methods and the low-energy ligand configuration is reported, which provides a mechanistic explanation for the high binding affinity of tacrine towards AChE. Additionally, structural comparison of reported hAChE structures sheds light on the conformational selection and induced fit effects of various active site residues upon binding to different ligands and provides insight for future drug design campaigns against AD where AChE is a drug target.
Assuntos
Doença de Alzheimer , Tacrina , Acetilcolinesterase/metabolismo , Idoso , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Inibidores da Colinesterase/química , Descoberta de Drogas , Humanos , Ligantes , Estrutura Molecular , Tacrina/química , Tacrina/farmacologia , Tacrina/uso terapêuticoRESUMO
The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) promotes cell motility, phagocytosis, and cancer metastasis through activation of Rho guanosine triphosphatases. Engulfment and cell motility (ELMO) proteins are binding partners of DOCK and regulate Rac activation. Here, we report the cryo-electron microscopy structure of the active ELMO1-DOCK5 complex bound to Rac1 at 3.8-Å resolution. The C-terminal region of ELMO1, including the pleckstrin homology (PH) domain, aids in the binding of the catalytic DOCK homology region 2 (DHR-2) domain of DOCK5 to Rac1 in its nucleotide-free state. A complex α-helical scaffold between ELMO1 and DOCK5 stabilizes the binding of Rac1. Mutagenesis studies revealed that the PH domain of ELMO1 enhances the GEF activity of DOCK5 through specific interactions with Rac1. The structure provides insights into how ELMO modulates the biochemical activity of DOCK and how Rac selectivity is achieved by ELMO.
RESUMO
The inside of a cell is highly crowded with proteins and other biomolecules. How proteins express their specific functions together with many off-target proteins in crowded cellular environments is largely unknown. Here, we investigate an inhibitor binding with c-Src kinase using atomistic molecular dynamics (MD) simulations in dilute as well as crowded protein solution. The populations of the inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), in bulk solution and on the surface of c-Src kinase are reduced as the concentration of crowder bovine serum albumins (BSAs) increases. This observation is consistent with the reduced PP1 inhibitor efficacy in experimental c-Src kinase assays in addition with BSAs. The crowded environment changes the major binding pathway of PP1 toward c-Src kinase compared to that in dilute solution. This change is explained based on the population shift mechanism of local conformations near the inhibitor binding site in c-Src kinase.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Quinases da Família src/efeitos dos fármacos , Quinases da Família src/metabolismo , Animais , Sítios de Ligação , Proteína Tirosina Quinase CSK/efeitos dos fármacos , Proteína Tirosina Quinase CSK/metabolismo , Biologia Computacional , Modelos Moleculares , Proteínas/química , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinases da Família src/químicaRESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare but severe genetic disorder in which acute inflammation elicits progressive heterotopic ossification in the muscles, tendons, and ligaments. Classic FOP is caused by the R206H mutation in ALK2/ACVR1. While several activin receptor-like kinase 2 (ALK2) inhibitors were found to be efficacious in animal models of FOP, most of the ALK2 (R206H) inhibitors lacked sufficient oral bioavailability for efficacy. Previously, the synthesis of a series of novel bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitors that achieved both substantial potency and an improved ADMET profile was reported. In the present study, the detailed procedure of the in silico approach employed to identify the initial bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitor RK-59638 and the analysis of the ALK2 (R206H) RK-59638 complex structure to guide the synthetic optimization of the chemical series, obtaining RK-71807 showing improved potency and metabolic stability, were described. According to the initial in silico screening, the screening efficiencies and chemical diversity of the hit compounds of both ligand-based and structure-based methods were evaluated. Then, X-ray structures of ALK2 (R206H) and the inhibitors were analyzed to assess the structure-activity relationships of the synthesized compounds. The 3D-RISM analysis indicated the existence of the additional hydrogen bond via water molecules restricting the attachment point in the pyrazole scaffold. The quantum mechanics calculation of the newly determined ALK2 (R206H) RK-71807 complex structure using a fragment molecular orbital method and pair interaction energy decomposition analysis was employed to evaluate the interaction energies between the inhibitor and each of the amino acid residues and decompose them to electrostatic, exchange-repulsion, and charge transfer energies. The pattern of decomposed interaction energies was then compared to that formed by RK-59638 and LDN-193189 to investigate the structural basis of ALK2 (R206H) inhibition.
RESUMO
The Dedicator Of CytoKinesis (DOCK) family of atypical guanine nucleotide exchange factors activates the Rho family GTPases Rac and/or Cdc42 through DOCK homology region 2 (DHR-2). Previous structural analyses of the DHR-2 domains of DOCK2 and DOCK9 have shown that they preferentially bind Rac1 and Cdc42, respectively; however, the molecular mechanism by which DHR-2 distinguishes between these GTPases is unclear. Here we report the crystal structure of the Cdc42-bound form of the DOCK7 DHR-2 domain showing dual specificity for Rac1 and Cdc42. The structure revealed increased substrate tolerance of DOCK7 at the interfaces with switch 1 and residue 56 of Cdc42. Furthermore, molecular dynamics simulations showed a closed-to-open conformational change in the DOCK7 DHR-2 domain between the Cdc42- and Rac1-bound states by lobe B displacement. Our results suggest that lobe B acts as a sensor for identifying different switch 1 conformations and explain how DOCK7 recognizes both Rac1 and Cdc42.
Assuntos
Proteínas Ativadoras de GTPase/química , Fatores de Troca do Nucleotídeo Guanina/química , Especificidade por Substrato , Proteína cdc42 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/química , Cristalização , Cristalografia por Raios X , Humanos , Conformação Molecular , Simulação de Dinâmica Molecular , MutagêneseRESUMO
Granzyme A (GzmA), together with perforin, are well-known for their cytotoxic activity against tumor or virus-infected cells. In addition to this cytotoxic function, GzmA stimulates several immune cell types and induces inflammation in the absence of perforin, however, its effect on the dendritic cell (DC) is unknown. In the current study, we showed that recombinant GzmA induced the phenotypic maturation of plasmacytoid DCs (pDCs) and conventional DCs (cDCs), but not their apoptosis. Particularly, GzmA made pDCs more functional, thus leading to production of type I interferon (IFN) via the TLR9-MyD88 pathway. We also demonstrated that GzmA binds TLR9 and co-localizes with it in endosomes. When co-administered with antigen, GzmA acted as a powerful adjuvant for eliciting antigen-specific cytotoxic CD8+ T lymphocytes (CTLs) that protected mice from tumor challenge. The induction of CTL was completely abolished in XCR1+ DC-depleted mice, whereas it was reduced to less than half in pDC-depleted or IFN-α/ß receptor knockout mice. Thus, CTL cross-priming was dependent on XCR1+cDC and also type I IFN, which was produced by GzmA-activated pDCs. These results indicate that GzmA -stimulated pDCs enhance the cross-priming activity of cDCs in situ. We also showed that the adjuvant effect of GzmA is superior to CpG-ODN and LPS. Our findings highlight the ability of GzmA to bridge innate and adaptive immune responses via pDC help and suggest that GzmA may be useful as a vaccine adjuvant.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Granzimas/farmacologia , Imunidade Celular/efeitos dos fármacos , Plasmócitos/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Células Dendríticas/citologia , Granzimas/genética , Granzimas/imunologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Plasmócitos/citologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologiaRESUMO
Although immune responses are essential to protect the body from infection, they can also harm tissues. Certain tissues and organs, including the eye, constitute specialized microenvironments that locally inhibit immune reactivity. Dedicator of cytokinesis protein 2 (DOCK2) is a Rac-specific guanine nucleotide exchange factor (GEF) that is predominantly found in hematopoietic cells. DOCK2 plays a key role in immune surveillance because it is essential for the activation and migration of leukocytes. DOCK2 mutations cause severe immunodeficiency in humans. We found that DOCK2-mediated Rac activation and leukocyte migration were effectively inhibited by cholesterol sulfate (CS), but not by cholesterol or other sulfated steroids. CS bound to the catalytic domain of DOCK2 and suppressed its GEF activity. Mass spectrometric quantification revealed that CS was most abundantly produced in the Harderian gland, which provides the lipids that form the oily layer of the tear film. Sulfation of cholesterol is mediated by the sulfotransferases SULT2B1b and, to a lesser extent, SULT2B1a, which are produced from the same gene through alternative splicing. By genetically inactivating Sult2b1, we showed that the lack of CS in mice augmented ultraviolet- and antigen-induced ocular surface inflammation, which was suppressed by administration of eye drops containing CS. Thus, CS is a naturally occurring DOCK2 inhibitor and contributes to the generation of the immunosuppressive microenvironment in the eye.
Assuntos
Ésteres do Colesterol/metabolismo , Olho/imunologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Evasão da Resposta Imune , Ceratite/prevenção & controle , Transtornos de Fotossensibilidade/prevenção & controle , Animais , Modelos Animais de Doenças , Olho/efeitos dos fármacos , Olho/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Ceratite/etiologia , Ceratite/imunologia , Ceratite/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos de Fotossensibilidade/etiologia , Transtornos de Fotossensibilidade/imunologia , Transtornos de Fotossensibilidade/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sulfotransferases/fisiologiaRESUMO
A rabbit monoclonal antibody (Abcam ab124797), with high affinity for a synthetic peptide corresponding to the C-terminal region of the receptor activator of nuclear factor (NF)-κB ligand (RANKL), specifically recognizes a 37 kDa protein by immunoblotting, in good agreement with the molecular mass of RANKL. However, our mass spectroscopy analysis revealed that the protein recognized by the antibody is the α-subunit of NAD(+)-dependent isocitrate dehydrogenase (ICDH), a key Krebs cycle enzyme in mitochondria. Consistently, immunocytochemical staining with the antibody revealed a network organization characteristic of mitochondria, which overlapped with staining by MitoTracker and was lost after the siRNA-mediated downregulation of ICDH. The C-terminal peptide of ICDH contains similar chemical characteristics to that of the RANKL peptide and interacts with the antibody, although the affinity is a hundred times weaker. The present study provides an example of the preferential recognition of a surrogate protein by a rabbit monoclonal antibody.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Isocitrato Desidrogenase/imunologia , Ligante RANK/imunologia , Animais , Reações Cruzadas , Isocitrato Desidrogenase/genética , Camundongos , Estrutura Terciária de Proteína , Ligante RANK/genética , CoelhosRESUMO
The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.