RESUMEN

Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT(4) receptor. Until now, the AT(4) receptor has eluded molecular characterization. Here we identify the AT(4) receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT(4) receptor binding characteristics; the AT(4) receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [(125)I]Nle(1)-angiotensin IV with IC(50) values of 32 and 140 nm, respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT(4) receptor determined by radioligand binding. We also show that AT(4) receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT(4) receptor is IRAP and propose that AT(4) receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates.

Asunto(s)

Aminopeptidasas/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Receptores de Angiotensina/metabolismo , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/genética , Aminopeptidasas/aislamiento & purificación , Angiotensina II/química , Antagonistas de Receptores de Angiotensina , Animales , Autorradiografía , Encéfalo/citología , Encéfalo/enzimología , Encéfalo/metabolismo , Línea Celular , Cistinil Aminopeptidasa , Hemoglobinas/metabolismo , Humanos , Inmunohistoquímica , Hibridación in Situ , Radioisótopos de Yodo/química , Radioisótopos de Yodo/metabolismo , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Ensayo de Unión Radioligante , Receptores de Angiotensina/genética , Receptores de Angiotensina/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transfección

RESUMEN

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Asunto(s)

Neoplasias del Colon/química , Electroforesis en Gel Bidimensional/métodos , Proteínas de Neoplasias/aislamiento & purificación , Proteoma/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Neoplasias del Colon/genética , Citosol/química , Humanos , Focalización Isoeléctrica/métodos , Espectrometría de Masas/métodos , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Proteoma/genética , Tripsina , Células Tumorales Cultivadas

RESUMEN

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Asunto(s)

Proteínas Portadoras/metabolismo , Melanoma Experimental/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Melanoma Experimental/patología , Proteínas de la Membrana/inmunología , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas R-SNARE , Células Tumorales Cultivadas

RESUMEN

gp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys(279) and Cys(469)), and one (Cys(397)) is modified by S-cysteinylation. Of the 11 potential N-glycosylation sites, Asn(21), Asn(61), Asn(109), Asn(135), Asn(205), Asn(357), Asn(361), Asn(531), and Asn(542) are glycosylated but not Asn(224) and Asn(368). The disulfide bonds, Cys(112)-Cys(122) and Cys(150)-Cys(160), are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys(6)-Cys(32) and Cys(26)-Cys(81)) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys(436)-Cys(444). We have created a model predicting that this loop maintains Cys(469) in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130.

Asunto(s)

Antígenos CD/química , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Antígenos CD/aislamiento & purificación , Receptor gp130 de Citocinas , Disulfuros/química , Glicosilación , Humanos , Glicoproteínas de Membrana/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Mapeo Peptídico

RESUMEN

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.

Asunto(s)

Adhesión Celular , Neoplasias del Colon/patología , Receptores ErbB/metabolismo , Integrinas/fisiología , Laminina/fisiología , Secuencia de Aminoácidos , Comunicación Autocrina , Neoplasias del Colon/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/aislamiento & purificación , Proteínas de la Matriz Extracelular/fisiología , Humanos , Laminina/genética , Laminina/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/fisiología , Células Tumorales Cultivadas

RESUMEN

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.

Asunto(s)

Apoptosis , Proteínas Portadoras/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Proteínas Mitocondriales , Nucleopoliedrovirus , Proteínas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Línea Celular Transformada , Clonación Molecular , Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , Péptidos y Proteínas de Señalización Intracelular , Mamíferos , Ratones , Datos de Secuencia Molecular , Proteínas/genética , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Proteínas Virales/genética , Proteína Inhibidora de la Apoptosis Ligada a X

RESUMEN

The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. However, due to the charge heterogeneity and poor solubility of membrane-associated proteins this subsection of the cell's proteome is often refractory to two-dimensional electrophoresis (2-DE), the current paradigm technology for studying protein expression profiles. Here, we describe a non-2-DE method for identifying membrane proteins. Proteins from an enriched membrane preparation of the human colorectal carcinoma cell line LIM1215 were initially fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4-20%). The unstained gel was cut into 16 x 3 mm slices, and peptide mixtures resulting from in-gel tryptic digestion of each slice were individually subjected to capillary-column reversed phase-high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS). Interrogation of genomic databases with the resulting collision-induced dissociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane proteins) were identified, including many integral membrane proteins not previously identified by 2-DE, many proteins seen at the genomic level only, as well as several proteins identified by expressed sequence tags (ESTs) only. Additionally, a number of peptides, identified by de novo MS sequence analysis, have not been described in the databases. Further, a "targeted" ion approach was used to unambiguously identify known low-abundance plasma membrane proteins, using the membrane-associated A33 antigen, a gastrointestinal-specific epithelial cell protein, as an example. Following localization of the A33 antigen in the gel by immunoblotting, ions corresponding to the theoretical A33 antigen tryptic peptide masses were selected using an "inclusion" mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the standard automated "nontargeted" approach, were identified. The membrane protein database may be accessed via the World Wide Web (WWW) at http://www.ludwig. edu.au/jpsl/jpslhome.html.

Asunto(s)

Bases de Datos Factuales , Proteínas de la Membrana , Proteínas de Neoplasias/análisis , Proteoma , Neoplasias del Colon , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Espectrometría de Masas/métodos , Células Tumorales Cultivadas

RESUMEN

Insulin regulates glucose metabolism in adipocytes via a phosphatidylinositide 3-kinase (PI3K)-dependent pathway that appears to involve protein phosphorylation. However, the generation of phosphoinositides is not sufficient for insulin action, and it has been suggested that insulin regulation of glucose metabolism may involve both PI3K-dependent and -independent pathways, the latter being insulin specific. To test this hypothesis, we have designed a phosphoprotein screen to study insulin-specific phosphoproteins that may be either downstream or in parallel to PI3K. Nineteen insulin-regulated phosphospots were detected in the cytosol and high speed pellet fractions, only six of which were significantly regulated by platelet-derived growth factor. Importantly, almost all (92%) of the insulin-specific phosphoproteins identified using this approach were sensitive to the PI3K inhibitor wortmannin. Thus, we obtained no evidence for an insulin-specific, PI3K-independent signaling pathway. A large proportion (62%) of the insulin-specific phosphoproteins were enriched in the same high speed pellet fraction to which PI3K was recruited in response to insulin. Thus, our data suggest that insulin specifically stimulates the phosphorylation of a novel subset of downstream targets and this may in part be because of the unique localization of PI3K in response to insulin in adipocytes.

Asunto(s)

Adipocitos/metabolismo , Insulina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal , Células 3T3 , ATP Citrato (pro-S)-Liasa/química , ATP Citrato (pro-S)-Liasa/metabolismo , Adipocitos/efectos de los fármacos , Secuencia de Aminoácidos , Androstadienos/farmacología , Animales , Citosol/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Factor 2 de Elongación Peptídica/química , Factor 2 de Elongación Peptídica/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Fosfoproteínas/química , Fosfoproteínas/aislamiento & purificación , Fosforilación , Transducción de Señal/efectos de los fármacos , Wortmanina

RESUMEN

PIP: After years of achieving only disappointing participation rates in a prenatal program for pregnant adolescents, public health nurses in Brantford, Ontario, evaluated the program and reviewed other prenatal programs to determine how to improve attendance. The results of the evaluation indicated that the adolescents thought the prenatal program was not important or relevant. Using adolescent development theory, a new prenatal program was designed with the help of adolescents. This new program utilizes three teaching approaches: provision of relevant information, offering of information on a "need to know" basis, and provision of problem-solving activities. Topics such as how to deal with the pain of labor; how to care for an infant; and the health hazards of drugs, alcohol, and tobacco are presented using small group exercises and games. Problem-solving skills are developed through large and small group discussions, games, puzzles, and activities incorporating movement. The new 10-week program has been in effect since 1993 and has proved to be so popular that additional series have been necessary. Program participants are pregnant teenagers, the fathers of their babies, and/or their labor coaches. Because the classes are scheduled at supper time, they include a hands-on nutritional component in the form of meal preparation. Ongoing evaluations echo the good results revealed by the improved attendance figures and allow the participants to recommend new methods and ideas for the course. One such idea developed into a weekly postnatal drop-in program.^ieng

Asunto(s)

Servicios de Salud del Adolescente/organización & administración , Padres/educación , Embarazo en Adolescencia , Atención Prenatal/organización & administración , Adolescente , Femenino , Humanos , Recién Nacido , Masculino , Ontario , Embarazo , Enfermería en Salud Pública