RESUMO
Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signaling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. While inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterized a pair of nanobodies that are specific for mouse Plexin-B1 and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signaling and provides a potential innovative route for therapeutic modulation of Plexin-B1.
Assuntos
Moléculas de Adesão Celular , Semaforinas , Anticorpos de Domínio Único , Animais , Camundongos , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , Transdução de Sinais , Moléculas de Adesão Celular/metabolismoRESUMO
Osteoporosis and multiple sclerosis are highly prevalent diseases with limited treatment options. In light of these unmet medical needs, novel therapeutic approaches are urgently sought. Previously, the activation of the transmembrane receptor Plexin-B1 by its ligand semaphorin 4D (Sema4D) has been shown to suppress bone formation and promote neuroinflammation in mice. However, it is unclear whether inhibition of this receptor-ligand interaction by an anti-Plexin-B1 antibody could represent a viable strategy against diseases related to these processes. Here, we raised and systematically characterized a monoclonal antibody directed against the extracellular domain of human Plexin-B1, which specifically blocks the binding of Sema4D to Plexin-B1. In vitro, we show that this antibody inhibits the suppressive effects of Sema4D on human osteoblast differentiation and mineralization. To test the therapeutic potential of the antibody in vivo, we generated a humanized mouse line, which expresses transgenic human Plexin-B1 instead of endogenous murine Plexin-B1. Employing these mice, we demonstrate that the anti-Plexin-B1 antibody exhibits beneficial effects in mouse models of postmenopausal osteoporosis and multiple sclerosis in vivo. In summary, our data identify an anti-Plexin-B1 antibody as a potential therapeutic agent for the treatment of osteoporosis and multiple sclerosis.
Assuntos
Anticorpos Monoclonais , Antígenos CD , Esclerose Múltipla , Proteínas do Tecido Nervoso , Osteoporose Pós-Menopausa , Receptores de Superfície Celular , Semaforinas , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Ligantes , Camundongos , Esclerose Múltipla/terapia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Osteoporose Pós-Menopausa/terapia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Semaforinas/antagonistas & inibidores , Semaforinas/metabolismoRESUMO
Peptic ulcer disease is a frequent clinical problem with potentially serious complications such as bleeding or perforation. A decisive factor in the pathogenesis of peptic ulcers is gastric acid, the secretion of which is controlled by the hormone gastrin released from gastric G cells. However, the molecular mechanisms regulating gastrin plasma concentrations are poorly understood. Here, we identified a semaphorin-plexin signaling pathway that operates in gastric G cells to inhibit gastrin expression on a transcriptional level, thereby limiting food-stimulated gastrin release and gastric acid secretion. Using a systematic siRNA screening approach combined with biochemical, cell biology, and in vivo mouse experiments, we found that the RasGAP protein Rasal1 is a central mediator of plexin signal transduction, which suppresses gastrin expression through inactivation of the small GTPase R-Ras. Moreover, we show that Rasal1 is pathophysiologically relevant for the pathogenesis of peptic ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs), a main risk factor of peptic ulcers in humans. Last, we show that application of recombinant semaphorin 4D alleviates peptic ulcer disease in mice in vivo, demonstrating that this signaling pathway can be harnessed pharmacologically. This study unravels a mode of G cell regulation that is functionally important in gastric homeostasis and disease.
Assuntos
Úlcera Péptica , Semaforinas , Animais , Moléculas de Adesão Celular , Proteínas Ativadoras de GTPase , Gastrinas/efeitos adversos , Gastrinas/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso , Úlcera Péptica/induzido quimicamente , Transdução de SinaisRESUMO
The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.
Assuntos
Moléculas de Adesão Celular/metabolismo , Divisão Celular/fisiologia , Células Epidérmicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Células-Tronco/metabolismo , Animais , Carcinoma Basocelular/patologia , Proteínas de Transporte/metabolismo , Adesão Celular , Proliferação de Células , Desenvolvimento Embrionário/fisiologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Junções Intercelulares , Queratinócitos , Camundongos , Mitose , Morfogênese , OrganogêneseRESUMO
A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Bacteriófago M13/genética , GTP Fosfo-Hidrolases/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Guanosina Trifosfato/metabolismo , Biblioteca de Peptídeos , Mapeamento de Peptídeos/métodos , Peptídeos/genética , Conformação Proteica , Proteínas Recombinantes/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.
Assuntos
Cálcio/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Músculo Liso/fisiologia , Proteína rhoA de Ligação ao GTP/química , Western Blotting , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Modelos Moleculares , Conformação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Mutations in the Lis1 gene result in lissencephaly (smooth brain), a debilitating developmental syndrome caused by the impaired ability of postmitotic neurons to migrate to their correct destination in the cerebral cortex. Sequence similarities suggest that the LIS1 protein contains a C-terminal seven-blade beta-propeller domain, while the structure of the N-terminal fragment includes the LisH (Lis-homology) motif, a pattern found in over 100 eukaryotic proteins with a hitherto unknown function. We present the 1.75 A resolution crystal structure of the N-terminal domain of mouse LIS1, and we show that the LisH motif is a novel, thermodynamically very stable dimerization domain. The structure explains the molecular basis of a low severity form of lissencephaly.
Assuntos
Proteínas Associadas aos Microtúbulos/química , 1-Alquil-2-acetilglicerofosfocolina Esterase , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Dimerização , Relação Dose-Resposta a Droga , Guanidina/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/química , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Regulação Alostérica , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.
Assuntos
Antineoplásicos/química , Engenharia de Proteínas/métodos , Receptor EphA2/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Biblioteca de Peptídeos , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PDZ domains are ubiquitous protein interaction modules that play a key role in cellular signaling. Their binding specificity involves recognition of the carboxyl-terminus of various proteins, often belonging to receptor and ion channel families. PDZ domains also mediate more complicated molecular networks through PDZ-PDZ interactions, recognition of internal protein sequences or phosphatidylinositol moieties. The domains often form a tandem of multiple copies, but equally often such tandems or single PDZ domain occur in combination with other signaling domains (for example SH3, DH/PH, GUK, LIM, CaMK). Common occurrence of PDZ domains in Metazoans strongly suggests that their evolutionary appearance results from the complication of signaling mechanisms in multicellular organisms. Here, we focus on their structure, specificity and role in signaling pathways.
Assuntos
Proteínas de Transporte/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas de Transporte/classificação , Guanilato Quinases , Humanos , Ligantes , Núcleosídeo-Fosfato Quinase/fisiologia , Fosforilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Estrutura Terciária de Proteína/genéticaRESUMO
Vps34 is the ancestral phosphatidylinositol 3-kinase (PtdIns3K) isoform and is essential for endosomal trafficking of proteins to the vacuole/lysosome, autophagy and phagocytosis. Vps34-containing complexes associate with specific cellular compartments to produce PtdIns(3)P. Understanding the roles of Vps34 has been hampered by the lack of potent, specific inhibitors. To boost development of Vps34 inhibitors, we determined the crystal structures of Vps34 alone and in complexes with multitargeted PtdIns3K inhibitors. These structures provided a first glimpse into the uniquely constricted ATP-binding site of Vps34 and enabled us to model Vps34 regulation. We showed that the substrate-binding "activation" loop and the flexibly attached amphipathic C-terminal helix are crucial for catalysis on membranes. The C-terminal helix also suppresses ATP hydrolysis in the absence of membranes. We propose that membrane binding shifts the C-terminal helix to orient the enzyme for catalysis, and the Vps15 regulatory subunit, which binds to this and the preceding helix, may facilitate this process. This C-terminal region may also represent a target for specific, non-ATP-competitive PtdIns3K inhibitors.
Assuntos
Autofagia/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fosfatidilinositol 3-Quinase/químicaRESUMO
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases with diverse roles in health and disease. The primordial PI3K, Vps34, is present in all eukaryotes and has essential roles in autophagy, membrane trafficking, and cell signaling. We solved the crystal structure of Vps34 at 2.9 angstrom resolution, which revealed a constricted adenine-binding pocket, suggesting the reason that specific inhibitors of this class of PI3K have proven elusive. Both the phosphoinositide-binding loop and the carboxyl-terminal helix of Vps34 mediate catalysis on membranes and suppress futile adenosine triphosphatase cycles. Vps34 appears to alternate between a closed cytosolic form and an open form on the membrane. Structures of Vps34 complexes with a series of inhibitors reveal the reason that an autophagy inhibitor preferentially inhibits Vps34 and underpin the development of new potent and specific Vps34 inhibitors.
Assuntos
Adenina/análogos & derivados , Autofagia/efeitos dos fármacos , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Inibidores Enzimáticos/metabolismo , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase , Adenina/metabolismo , Adenina/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Catálise , Domínio Catalítico , Membrana Celular/metabolismo , Cristalografia por Raios X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Furanos/química , Furanos/metabolismo , Furanos/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologiaRESUMO
Rab11-FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient cause loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific because expression of the equivalent Rab11-binding deficient mutant of Rab-coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.
Assuntos
Proteínas de Transporte/fisiologia , Endossomos/fisiologia , Linhagem Celular Tumoral , Centrossomo/fisiologia , Endossomos/química , HumanosRESUMO
The Dbl homology nucleotide exchange factors (GEFs) activate Rho family cytosolic GTPases in a variety of physiological and pathophysiological events. These signaling molecules typically act downstream of tyrosine kinase receptors and often facilitate nucleotide exchange on more than one member of the Rho GTPase superfamily. Three unique GEFs, i.e. p115, PDZ-RhoGEF, and LARG, are activated by the G-protein coupled receptors via the Galpha(12/13), and exhibit very selective activation of RhoA, although the mechanism by which this is accomplished is not fully understood. Based on the recently solved crystal structure of the DH-PH tandem of PDZ-RhoGEF in complex with RhoA (Derewenda, U., Oleksy, A., Stevenson, A. S., Korczynska, J., Dauter, Z., Somlyo, A. P., Otlewski, J., Somlyo, A. V., and Derewenda, Z. S. (2004) Structure (Lond.) 12, 1955-1965), we conducted extensive mutational and functional studies of the molecular basis of the RhoA selectivity in PDZ-RhoGEF. We show that while Trp(58) of RhoA is intimately involved in the interaction with the DH domain, it is not a selectivity determinant, and its interaction with PDZ-RhoGEF is unfavorable. The key selectivity determinants are dominated by polar contacts involving residues unique to RhoA. We find that selectivity for RhoA versus Cdc42 is defined by a small number of interactions.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Ativação Enzimática , Epitopos , Fatores de Troca do Nucleotídeo Guanina/genética , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fatores de Troca de Nucleotídeo Guanina Rho , Sensibilidade e Especificidade , Homologia de Sequência de Aminoácidos , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Proteases and their natural protein inhibitors are among the most intensively studied protein-protein complexes. There are about 30 structurally distinct inhibitor families that are able to block serine, cysteine, metallo- and aspartyl proteases. The mechanisms of inhibition can be related to the catalytic mechanism of protease action or include a mechanism-unrelated steric blockage of the active site or its neighborhood. The structural elements that are responsible for the inhibition most often include the N- or the C-terminus or exposed loop(s) either separately or in combination of several such elements. During complex formation, no major conformational changes are usually observed, but sometimes structural transitions of the inhibitor and enzyme occur. In many cases, convergent evolution, with respect to the inhibitors' parts that are responsible for the inhibition, can be inferred from comparisons of their structures or sequences, strongly suggesting that there are only limited ways to inhibit proteases by proteins.
Assuntos
Evolução Molecular , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Sítios de Ligação/genética , Ligação Competitiva , Catálise , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Conformação ProteicaRESUMO
PDZ-containing RhoGEF (PDZ-RhoGEF) is a multidomain protein composed of 1522 amino acids that belongs to the guanine nucleotide exchange factors family (GEF) active on Rho GTPases. It is highly specific for RhoA and is thought to transduce signals from Galpha(12/13)-coupled receptors to the RhoA-dependent regulatory cascades. The protein shows high sequence homology to LARG, p115-RhoGEF and Drosophila DRhoGEF2. The exchange reaction is catalyzed by a DH domain, which is directly downstream of a PH domain in all known Rho-specific GEFs. The DH/PH tandem of PDZ-RhoGEF and C-terminally truncated RhoA were overexpressed in Escherichia coli as TEV protease-cleavable fusion proteins containing GST and a hexahistidine tag at the N-termini, respectively. The nucleotide-free DH/PH-RhoA complex was purified by gel filtration and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 88.6, b = 119.0, c = 91.5 A, beta = 114.7 degrees.