RESUMO
Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.
Assuntos
Encefalinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica , Cálcio/metabolismo , Embrião de Mamíferos/fisiologia , Encefalinas/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Hibridização In Situ , Ligantes , Dados de Sequência Molecular , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Peptídeos Opioides/metabolismo , Filogenia , Ligação Proteica , Precursores de Proteínas/genética , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Alinhamento de Sequência , Distribuição TecidualRESUMO
Major vault protein (MVP) is the main constituent of vaults, large ribonucleoprotein particles implicated in resistance to cancer therapy and correlated with poor survival prognosis. Here, we report the structure of the main repeat element in human MVP. The approximately 55 amino acid residue MVP domain has a unique, novel fold that consists of a three-stranded antiparallel beta-sheet. The solution NMR structure of a two-domain fragment reveals the interdomain contacts and relative orientations of the two MVP domains. We use these results to model the assembly of 672 MVP domains from 96 MVP molecules into the ribs of the 13MDa vault structure. The unique features include a thin, skin-like structure with polar residues on both the cytoplasmic and internal surface, and a pole-to-pole arrangement of MVP molecules. These studies provide a starting point for understanding the self-assembly of MVP into vaults and their interactions with other proteins. Chemical shift perturbation studies identified the binding site of vault poly(ADP-ribose) polymerase, another component of vault particles, indicating that MVP domains form a new class of interaction-mediating modules.
Assuntos
Sequências Repetitivas de Ácido Nucleico , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Poli(ADP-Ribose) Polimerases/metabolismo , Conformação Proteica , Alinhamento de Sequência , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
The solution structure of the second PDZ domain from human phosphatase hPTP1E in complex with a C-terminal peptide from the guanine nucleotide exchange factor RA-GEF-2 has been determined using 2D and 3D heteronuclear NMR experiments. Compared to previously solved structures, the hPTP1E complex shows an enlarged interaction surface with the C terminus of the bound peptide. Novel contacts were found between the long structured beta2/beta3 loop of the PDZ domain and the sixth amino acid residue from the C terminus of the peptide. This work underlines the importance of the beta2/beta3 loop for ligand selection by PDZ domains.
Assuntos
Proteínas de Transporte/química , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas Tirosina Fosfatases/química , Sequência de Aminoácidos , Citosol , Humanos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 13 , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
By germline insertion, a long terminal repeat (LTR) of an intracisternal A-particle type IAP retrovirus has overtaken the transcriptional control of the rat oncomodulin (OM) gene, which codes for a high affinity Ca2+-binding protein with modulatory capacity. In order to get insights into regulatory mechanisms of LTR directed OM gene expression we tested promoter activity of this LTR by transient transfection of transformed rat fibroblasts with this sequence placed 5' of the human growth hormone hGH reporter gene. The OM LTR is a strong promoter but does not follow an expression pattern similar to the one of the OM gene. Genomic sequencing showed a good correlation between CpG hypomethylation in the OM LTR and OM transcription among various cell lines and tissues. DNase I mapping of a 18 kb fragment containing the OM gene and 5' flanking sequences revealed cell-line specific hypersensitivity sites located within the U3 region of the LTR element. Several cis-elements in the OM LTR promoter exhibiting cell-line specific occupancy were identified by in vivo DMS-footprinting. Detailed analysis of protein interactions with two such sequence elements in vitro revealed binding of ubiquitously expressed nuclear factors within an AP-1 (activator protein 1) and a intracisternal A-particle upstream enhancer recognition sequence. Protein occupancy to the latter sequence is significantly reduced by CpG methylation. These results indicate that cell-line specificity of OM expression is dictated by factor accessibility to the LTR promoter.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Sequências Repetidas Terminais/genética , Animais , Células Cultivadas , RatosRESUMO
Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys(49) that forms an intramolecular disulfide bond with the catalytic Cys(104) even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity.
Assuntos
Proteínas Imediatamente Precoces/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias , Ressonância Magnética Nuclear Biomolecular , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Relação Estrutura-AtividadeRESUMO
The phosphoinositide 3-kinase (PI 3-kinase) signaling pathway has been shown to play a pivotal role in intracellular signal transduction pathways involved in cell growth, cellular transformation and tumorigenesis. Analysis of several colon adenocarcinoma cell lines indicates that the PI 3-kinase signaling pathway is up-regulated in colon cancers. In particular, the protein levels and phosphorylation status of Akt and p70 S6 kinase are up-regulated in colon adenocarcinoma cell lines. More significantly, we have demonstrated for the first time that the phosphorylation of FKHR, a downstream target of Akt, is increased in these cell lines. Intriguingly, phosphorylation of three components of the PI 3-kinase signaling pathway, namely Akt, p70 S6 kinase and FKHR, are in direct correlation with the degree of tumorigenic potential of the colon cell lines tested. No differences in the protein levels of the two subunits of PI 3-kinase, p85 and p110alpha, and PTEN were noted. Real-time quantitative PCR indicated an increase in levels of Akt message only, and not of the other signaling pathway components. Inhibition of the PI 3-kinase with wortmannin decreased the anchorage-independent growth of colon cells in a soft agar assay. Hence, the components of the PI 3-kinase signaling pathway could serve as potential candidates for drug development in treatment of colon cancer.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias do Colo/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Androstadienos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Genes Supressores de Tumor , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , WortmaninaRESUMO
A mammalian two-hybrid system was developed for high-throughput screening of compounds that disrupt specific protein-protein interactions. The existing mammalian systems are unsatisfactory for drug screening due to nonregulated expression of interacting proteins. To construct a tightly regulated system, the tetracycline repressor was fused with the inhibitory KRAB domain as a suppressor. The binding of the suppressor to the tet operator entirely blocked expression of two interacting proteins. When both the inducer doxycycline and drugs were added to the culture, the reporter gene was either activated by interaction of the paired proteins with ineffective drugs or remained silent due to disruption of the protein interactions by the effective drugs. We demonstrate that interactions of the type I receptor for TGFbeta with FKBP12 and the epidermal growth factor receptor (EGFR) with p85 are effectively disrupted by FK506 and EGFR kinase inhibitor AG1478, respectively. The power of this system for drug screening was further demonstrated by rapid identification of inhibitors from a druglike library for the receptor kinases.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas/metabolismo , Tetraciclina/farmacologia , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Doxiciclina/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes Reporter , Engenharia Genética , Humanos , Proteínas/química , Proteínas/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
Endoglin is a component of the transforming growth factor-beta receptor complex abundantly expressed at the surface of endothelial cells and plays an important role in cardiovascular development and vascular remodeling. By using the cytoplasmic domain of endoglin as a bait for screening protein interactors, we have identified ZRP-1 (zyxin-related protein 1), a 476-amino acid member that belongs to a family of LIM containing proteins that includes zyxin and lipoma-preferred partner. The endoglin interacting region was mapped within the three double zinc finger LIM domains of the ZRP-1 C terminus. Analysis of the subcellular distribution of ZRP-1 demonstrated that in the absence of endoglin, ZRP-1 mainly localizes to focal adhesion sites, whereas in the presence of endoglin ZRP-1 is found along actin stress fibers. Because the LIM family of proteins has been shown to associate with the actin cytoskeleton, we investigated the possibility of a regulatory role for endoglin with regard to this structure. Expression of endoglin resulted in a dramatic reorganization of the actin cytoskeleton. In the absence of endoglin, F-actin was localized to dense aggregates of bundles, whereas in the presence of endoglin, expressed in endothelial cells, F-actin was in stress fibers and colocalized with ZRP-1. Furthermore, small interfering RNA-mediated suppression of endoglin or ZRP-1, or clustering of endoglin in endothelial cells, led to mislocalization of F-actin fibers. These results suggest a regulatory role for endoglin, via its interaction with ZRP-1, in the actin cytoskeletal organization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Fatores de Transcrição/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Actinas/química , Animais , Antígenos CD , Western Blotting , Células COS , Linhagem Celular , Células Cultivadas , Citoplasma/metabolismo , DNA Complementar/metabolismo , Endoglina , Endotélio Vascular/citologia , Humanos , Proteínas com Domínio LIM , Microscopia de Fluorescência , Plasmídeos/metabolismo , Testes de Precipitina , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de Superfície Celular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
PTEN is a tumor suppressor that primarily dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to down-regulate the phosphoinositide 3-kinase/Akt signaling pathway. Although the cellular functions of PTEN as a tumor suppressor have been well characterized, the mechanism by which PTEN activity is modulated by other signal molecules in vivo remains poorly understood. In searching for potential PTEN modulators through protein-protein interaction, we identified the major vault protein (MVP) as a dominant PTEN-binding protein in a yeast two-hybrid screen. MVP is the major structural component of vault, the largest intracellular ribonucleoprotein particle. Co-immunoprecipitation confirmed the interaction between PTEN and MVP in transfected mammalian cells. More importantly, we found that a significant portion of endogenous PTEN associates with vault particles in human HeLa cells. Deletion mutation analysis demonstrated that MVP binds to the C2 domain of PTEN and that PTEN interacts with MVP through its EF hand-like motif. Furthermore, the in vitro binding experiments revealed that the interaction of PTEN with MVP is Ca(2+)-dependent.