Assuntos
Evolução Molecular , Genoma , Animais , Células Eucarióticas , Genômica , Humanos , Íntrons , Modelos Genéticos , FilogeniaAssuntos
Ácido Aspártico Endopeptidases/fisiologia , Ciclo Celular/fisiologia , Proteínas Fúngicas/fisiologia , Retroviridae/enzimologia , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Células Eucarióticas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de AminoácidosRESUMO
A key challenge in studying protein/protein interactions is to accurately identify contact surfaces, i.e. regions of two proteins that are in direct physical contact. Aside from x-ray crystallography and NMR spectroscopy few methods are available that address this problem. Although x-ray crystallography often provides detailed information about contact surfaces, it is limited to situations when a co-crystal of proteins is available. NMR circumvents this requirement but is limited to small protein complexes. Other methods, for instance protection from proteolysis, are less direct and therefore less informative. Here we describe a new method that identifies candidate contact surfaces in protein complexes. The complexes are first stabilized by cross-linking. They are then digested with a protease, and the cross-linked fragments are analyzed by mass spectrometry. We applied this method, referred to as COSUMAS (contact surfaces by mass spectrometry), to two proteins, retinal guanylyl cyclase 1 (RetGC1) and guanylyl cyclase-activating protein-1 (GCAP-1), that regulate cGMP synthesis in photoreceptors. Two regions in GCAP-1 and three in RetGC1 were identified as possible contact sites. The two regions of RetGC1 that are in the vicinities of Cys(741) and Cys(780) map to a kinase homology domain in RetGC1. Their identities as contact sites were independently evaluated by peptide inhibition analysis. Peptides with sequences from these regions block GCAP-1-mediated regulation of guanylyl cyclase at both high and low Ca2+ concentrations. The two regions of GCAP-1 cross-linked to these peptides were in the vicinities of Cys(17) and Cys(105) of GCAP-1. Peptides with sequences derived from these regions inhibit guanylyl cyclase activity directly. These results support a model in which GCAP-1 binds constitutively to RetGC1 and regulates cyclase activity by structural changes caused by the binding or dissociation of Ca2+.
Assuntos
Proteínas de Ligação ao Cálcio/química , Guanilato Ciclase/química , Receptores de Superfície Celular , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Bovinos , Proteínas Ativadoras de Guanilato Ciclase , Espectrometria de Massas , Dados de Sequência MolecularRESUMO
Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.
Assuntos
Proteínas de Ligação ao Cálcio/química , Guanilato Ciclase/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Bovinos , Linhagem Celular , Proteínas Ativadoras de Guanilato Ciclase , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A composite morphological examination of chondromatous pulmonary hamartoma showed this tumor always to contain epithelial structures determining the peculiar cytological picture in transthoracal puncture biopsy. Therefore, the diagnostic signs in cytological examinations of chondromatous hamartoma should include cluster-like accumulations of cubic epithelial cells, typical complexes of epithelial and stromal cells, while fragments of immature cartilage and mesenchyma make the picture distinctly trustworthy. The source of epithelial and cartilage elements in the punctate consists of the less compact peripheral parts of the tumor where the main portion of epithelial structures, immature cartilage tissue and mesenchyma are located. Some epithelial elements in their ultrastructural organization resemble the lining of embryonal lungs. otworthy. The source of epithelial and cartilage elements in the punctate consists of the less compact peripheral parts of the tumor where the main portion of epithelial structures, immature cartilage tissue and mesenchyma are located. Some epithelial elecments in their ultrastructural organization resemble the lining of embryonal lungs. The histogenesis of chondromatous hamartoma should apparently be associated with mesodermal and ectodermal components of the bronchial germ which explains the regular presence of the epithelium in this tumor.