RESUMO
BACKGROUND: Maturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin. METHODS: In this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed. RESULTS: We found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6-1 that may contribute to development of MODY. Functional assessment of the NKX6-1 variants showed that they are functionally impaired. CONCLUSIONS: Our findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6-1, and these require further validation.
Assuntos
Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença/epidemiologia , Adolescente , Adulto , Estudos de Coortes , Exoma , Feminino , Biblioteca Gênica , Genômica , Hemoglobinas Glicadas/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Índia/epidemiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Análise de Sequência de DNA , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismo , Adulto JovemRESUMO
PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 µm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 µm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the ß-strand and a discontinuous short α-helix, and it engaged in the same ß-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.
Assuntos
Oligopeptídeos/farmacologia , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Células Hep G2 , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Biblioteca de Peptídeos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de LDL/química , Receptores de LDL/genética , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.
Assuntos
Colesterol/sangue , Furina/metabolismo , Pró-Proteína Convertases/metabolismo , Proteólise , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Animais , Anticorpos Monoclonais Murinos/química , Colesterol/genética , Furina/genética , Células Hep G2 , Humanos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , Estrutura Secundária de Proteína , Receptores de LDL/genética , Serina Endopeptidases/genéticaRESUMO
Hepatocyte growth factor (HGF) binds the extracellular domain and activates the Met receptor to induce mitogenesis, morphogenesis, and motility. The extracellular domain of Met is comprised of Sema, PSI, and four IPT subdomains. We investigated the contribution of these subdomains to Met receptor dimerization. Our observations indicate that the Sema domain is necessary for dimerization in addition to HGF binding. Treatment of Met-overexpressing tumor cells with recombinant Sema in the presence or absence of HGF results in decreased Met-mediated signal transduction, cell motility, and migration, behaving in a manner similar to an antagonistic anti-Met Fab. These data suggest that the Sema domain of Met may not only represent a novel anticancer therapeutic target but also acts as a biotherapeutic itself.
Assuntos
Movimento Celular , Matriz Extracelular/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Reagentes de Ligações Cruzadas , Dimerização , Feminino , Fator de Crescimento de Hepatócito/genética , Humanos , Camundongos , Camundongos Nus , Testes de Precipitina , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-met/imunologia , Deleção de Sequência , Células Tumorais CultivadasRESUMO
Proprotein convertase subtilisin/kexin 9 (PCSK9) has become an important therapeutic target for lipid lowering, since it regulates low-density lipoprotein cholesterol (LDL-c) levels by binding to liver LDL receptors (LDLR) and effecting their intracellular degradation. However, the development of small molecule inhibitors is hampered by the lack of attractive PCSK9 target sites. We recently discovered helical peptides that are able to bind to a cryptic groove site on PCSK9, which is situated in proximity to the main LDLR binding site. Here, we designed potent bipartite PCSK9 inhibitors by appending organic moieties to a helical groove-binding peptide to reach a hydrophobic pocket in the proximal LDLR binding region. The ultimately designed 1-amino-4-phenylcyclohexane-1-carbonyl extension improved the peptide affinity by >100-fold, yielding organo-peptide antagonists that potently inhibited PCSK9 binding to LDLR and preserved cellular LDLR. These new bipartite antagonists have reduced mass and improved potency compared to the first-generation peptide antagonists, further validating the PCSK9 groove as a viable therapeutic target site.
Assuntos
Inibidores de PCSK9 , Peptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Células Hep G2 , Humanos , Estrutura Molecular , Peptídeos/química , Peptídeos/metabolismo , Pró-Proteína Convertase 9/química , Pró-Proteína Convertase 9/metabolismo , Ligação Proteica , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismoRESUMO
Activating mutations in receptor tyrosine kinases play a critical role in oncogenesis. Despite evidence that Met kinase is deregulated in human cancer, the role of activating mutations in cancers other than renal papillary carcinoma has not been well defined. Here we report the identification of somatic intronic mutations of Met kinase that lead to an alternatively spliced transcript in lung cancer, which encodes a deletion of the juxtamembrane domain resulting in the loss of Cbl E3-ligase binding. The mutant receptor exhibits decreased ubiquitination and delayed down-regulation correlating with elevated, distinct Met expression in primary tumors harboring the deleted receptor. As a consequence, phospho-Met and downstream mitogen-activated protein kinase activation is sustained on ligand stimulation. Cells expressing the Met deletion reveal enhanced ligand-mediated proliferation and significant in vivo tumor growth. A hepatocyte growth factor competitive Met antagonist inhibits receptor activation and proliferation in tumor cells harboring the Met deletion, suggesting the important role played by ligand-dependent Met activation and the potential for anticancer therapy. These results support a critical role for Met in lung cancer and somatic mutation-driven splicing of an oncogene that leads to a different mechanism for tyrosine kinase activation through altered receptor down-regulation in human cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Deleção de Genes , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Fatores de Crescimento/genética , Processamento Alternativo , Animais , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Regulação para Baixo , Ativação Enzimática , Éxons , Feminino , Humanos , Íntrons , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-met , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células 3T3 , Animais , Quinases relacionadas a CDC2 e CDC28/biossíntese , Células COS , Divisão Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Ativação Enzimática , Repressão Enzimática , Fase G1 , Camundongos , Camundongos Knockout , Ligação Proteica/fisiologia , Fase S , Técnicas do Sistema de Duplo-HíbridoRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P' helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.
Assuntos
Inibidores Enzimáticos/metabolismo , Inibidores de PCSK9 , Peptídeos/metabolismo , Pró-Proteína Convertase 9/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/isolamento & purificação , Humanos , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Peptídeos/isolamento & purificaçãoRESUMO
LDL (low-density lipoprotein) receptor (LDLR) binds to its negative regulator proprotein convertase subtilisin/kexin type 9 (PCSK9) through the first EGF (epidermal growth factor-like) domain [EGF(A)]. The isolated EGF(A) domain is a poor antagonist due to its low affinity for PCSK9. To improve binding affinity, we used a phage display approach by randomizing seven PCSK9 contact residues of EGF(A), including the Ca(2+)-coordinating Asp310. The library was panned in Ca(2+)-free solution, and 26 unique clones that bind to PCSK9 were identified. Four selected variants demonstrated improved inhibitory activities in a PCSK9-LDLR competition binding ELISA. The Fc fusion protein of variant EGF66 bound to PCSK9 with a K(d) value of 71 nM versus 935 nM of wild type [EGF(A)-Fc] and showed significantly improved potency in inhibiting LDLR degradation in vitro and in vivo. The five mutations in EGF66 could be modeled in the EGF(A) structure without perturbation of the EGF domain fold, and their contribution to affinity improvement could be rationalized. The most intriguing change was the substitution of the Ca(2+)-coordinating Asp310 by a Lys residue, whose side-chain amine may have functionally replaced Ca(2+). EGF66-Fc and other EGF variants having the Asp310Lys change bound to PCSK9 in a Ca(2+)-independent fashion. The findings indicate that randomization of an important Ca(2+)-chelating residue in conjunction with "selection pressure" applied by Ca(2+)-free phage selection conditions can yield variants with an alternatively stabilized Ca(2+) loop and with increased binding affinities. This approach may provide a new paradigm for the use of diversity libraries to improve affinities of members of the Ca(2+)-binding EGF domain subfamily.
Assuntos
Cálcio/metabolismo , Pró-Proteína Convertases/antagonistas & inibidores , Receptores de LDL/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Biblioteca de Peptídeos , Pró-Proteína Convertase 9 , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de LDL/genética , Serina EndopeptidasesRESUMO
Tuberous sclerosis is a multi-organ disorder characterized by the formation of benign tumors, called hamartomas, which affects more than 1 million people worldwide. The syndrome is initiated by a mutation in one of two tumor suppressor genes, TSC1 or TSC2, that encode for the proteins hamartin and tuberin, respectively. Herein, we demonstrate that tuberin binds and regulates the G 2/M cyclin, cyclin B1. We have determined that this binding region encompasses a mutational hotspot within tuberin that is implicated in some of the most severe cases of TS. Mimicking a mutation found in a subset of patients with tuberous sclerosis, we found a significant reduction in the binding between tuberin and cyclin B1. Functionally, our data supports that tuberin plays a role in regulating the cellular localization of cyclin B1. These results demonstrate a novel and clinically relevant mechanism, where tuberin functions in mitotic onset.
Assuntos
Ciclina B1/metabolismo , Mitose , Proteínas Supressoras de Tumor/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Ciclina B1/genética , Regulação da Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ativação Transcricional , Transfecção , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Ligand dependent activity of receptor tyrosine kinases is critical for modulating downstream signaling and cell proliferation. In normal cellular context, hepatocyte growth factor (HGF) regulates MET kinase activation and mediates cell proliferation, migration and motility. Recent elucidation of the MET extracellular domain suggests that the Sema domain, which bears structural similarity to other Semaphorins and Plexin family members, plays a critical role in ligand mediated receptor activation. Overexpression of MET which is observed in many cancers leads to ligand independent receptor dimerization and activation. Evidence to support a role for the Sema domain in cancer and therapeutic implications of targeting the Met Sema domain are discussed in this review.