RESUMO
Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.
Assuntos
Plaquetas/química , Eletroforese das Proteínas Sanguíneas/métodos , Proteínas Sanguíneas/isolamento & purificação , Eletroforese em Gel Bidimensional/métodos , Fosfoproteínas/sangue , Fosfotirosina/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adulto , Algoritmos , Western Blotting , Citosol/química , Bases de Dados Factuais , Humanos , Focalização Isoelétrica , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/síntese química , Mapeamento de Peptídeos , Fosforilação , Processamento de Proteína Pós-Traducional , SoftwareRESUMO
AIMS/HYPOTHESIS: To identify a GTPase of 24,000 M(r) which we recently found to co-localize with GLUT4 in cardiac muscle. METHODS: A 24,000 M(r)-GTP-binding fraction was purified from pig heart by a three-step chromatographic procedure, followed by two-dimensional electrophoresis and electrospray ionization-mass spectrometry. Subcellular distribution of the GTPase was assessed by western blotting. Co-localization with GLUT4 was assessed by continuous sucrose density gradient fractionation and immunoadsorption of GLUT4-containing vesicles. RESULTS: The Rab11 protein was identified as a major component of the GTP-binding fraction and its expression in rat cardiac muscle was confirmed. In vivo insulin treatment resulted in the recruitment of Rab11 from the microsomal fraction to the plasma membrane. Subcellular fractionation indicated two immunoreactive GLUT4 pools. Most of the intracellular pool of Rab11 overlapped with the high-density GLUT4 pool and most of the transferrin receptor pool. The Rab11 protein also co-sedimented with the low-density, non-endosomal GLUT4 pool and substantially increased in this fraction after insulin treatment. It was specifically present in GLUT4-containing vesicles and insulin increased its abundance in these vesicles 2.2-fold relative to the amount of GLUT4. These vesicles also containend Rab4 and Akt-2, the latter being only associated after insulin stimulation. Insulin was unable to alter the cellular localization of Rab11 in insulin-resistant obese Zucker rats. CONCLUSION/INTERPRETATION: These results support the hypothesis that at least two GTPases of the Rab family participate in GLUT4-vesicle trafficking. We suggest that Rab11 is involved in the endosomal recycling, sorting and exocytotic movement of the glucose transporter.
Assuntos
Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Miocárdio/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Fracionamento Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Eletroforese em Gel Bidimensional , Transportador de Glucose Tipo 4 , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/metabolismo , Ventrículos do Coração , Membranas Intracelulares/metabolismo , Masculino , Microssomos/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Ratos Zucker , Espectrometria de Massas por Ionização por Electrospray , Suínos , Proteínas rab de Ligação ao GTP/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/isolamento & purificaçãoRESUMO
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate complex protein mixtures including whole cell lysates. In combination with immunoblotting techniques or radioactive labeling techniques it is a fast and convenient way to demonstrate the presence of certain proteins or protein modifications. With the development of extremely sensitive analytical techniques such as matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization (ESI)-MS, it has become possible to use 2-D gels not only as an analytical but also as a preparative tool. Starting with a number of spots excised from 2-D gels, a protein can be identified using different strategies involving enzymatic cleavage of the protein in the gel matrix, elution of the resulting peptides and analysis of these peptides by mass spectrometry. The obtained peptide mass fingerprint or fragment ion spectra from peptides can be used to screen protein or nucleic acid databases in order to identify the protein. We have used the techniques described above to identify proteins from human platelets which change their phosphorylation state following activation of platelets by thrombin. Platelets were radioactively labeled with [32P]orthophosphate and stimulated. Several protein spots in the observed range of 10-80 kDa and an isoelectric point of 3-10 showed a significant increase or decrease in phosphorylation. We present the results from the investigation of a spot group representing different isoforms and phosphorylation states of myosin light chain.
Assuntos
Plaquetas/química , Fosfoproteínas/sangue , Ativação Plaquetária , Trombina/farmacologia , Sequência de Aminoácidos , Plaquetas/fisiologia , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel Bidimensional/métodos , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Fosfoproteínas/isolamento & purificação , Fosforilação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massa de Íon Secundário/métodosRESUMO
Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell-free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl-calmodulin synthetase (ubiquitin-calmodulin ligase, EC 6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+-binding loop of calmodulin and has the octapeptide structure -L-F-D-K21-D-G-D-G- with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl-calmodulin synthetase. Removal of the 41 C-terminal amino acids (fourth Ca2+-binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site-specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site -V-F-D-K94-D-G-N-G- in the third Ca2+-binding loop is apparently not ubiquitylated by the synthetase.
Assuntos
Calmodulina/química , Calmodulina/metabolismo , Peptídeo Sintases/metabolismo , Complexo de Endopeptidases do Proteassoma , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Brometo de Cianogênio , Ativação Enzimática , Cinética , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/metabolismo , Mapeamento de Peptídeos , Testículo/metabolismo , Enzimas Ativadoras de Ubiquitina , Ubiquitina-Proteína Ligases , Ubiquitinas/química , VertebradosRESUMO
HIV-1 can be neutralized by soluble factors produced and secreted by activated CD8+ T cells. Production of such anti-viral CD8 factors (including chemokines) can be induced with IL-2 or phytohaemagglutinin (PHA). In addition to PHA or IL-2, we have co-stimulated CD8+ T cells with PHA/IL-2 and a mixture of thymic peptides (TP) of molecular weights below 10 kD. For the activation, CD8+ T cells were purified from peripheral blood mononuclear cells of HIV-1- individuals and any resultant anti-viral activity was monitored using an HIV-1 neutralization assay. Using HIV-1 isolates highly resistant to chemokine inhibition we detected significantly higher levels of HIV-1 neutralizing activity in CD8+ T cell culture supernatants which had been co-activated with TP. When the TP-induced anti-viral activity was monitored, neutralization of both non-syncytia-inducing (NSI) and syncytia-inducing (SI) patient isolates was enhanced by 38% (NSI, PHA +/- TP), 66% (SI, PHA +/- TP), 28% (NSI, IL-2 +/- TP), and 57% (SI, IL-2 +/- TP) compared with the anti-viral activity present in supernatants from CD8+ T cell cultures stimulated only with PHA or IL-2. Peptide sequence analysis of purified TP showed that the TP mixture predominantly contains peptides with homology to human histone and collagen sequences. Our data demonstrate that CD8+ T cells are additionally activated by a mixture of TP. In this way, the production of HIV-1 neutralizing CD8 factors can be enhanced.