RESUMEN
Development proceeds through successive activation of different sets of genes by specific transcription factors as a consequence of cell interactions and signaling. It is thus of primary interest to identify new putative transcriptional regulators. We report here the isolation of chicken clones bearing sequences coding for a chicken zinc finger protein (chZFp) which contains four pairs of zinc fingers of mixed type C2-H-C/C2-H2. At least five chZFp isoforms are produced through differential splicing of four small exons. The amino acid domains encoded by these four exons are highly conserved across species. Northern blot analysis and RNase-protection assays showed that chZFp transcripts are present in brain, heart, skin and liver during chick development. Reverse transcription mediated polymerase chain reaction (RT-PCR) experiments suggested that the relative amount of some chZFp isoforms increases at critical stages of development and skin morphogenesis. Finally, the main chZFp isoforms are able to directly interact in vitro with the scaffold attachment factor-A (SAF-A, also known as heterogenous nuclear ribonucleoprotein U) through both their aminoterminal and carboxyterminal domains.
Asunto(s)
ADN Complementario/genética , Isoformas de Proteínas/biosíntesis , Elementos Reguladores de la Transcripción/genética , Dedos de Zinc/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/embriología , Núcleo Celular/metabolismo , Embrión de Pollo , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Ribonucleoproteína Heterogénea-Nuclear Grupo U/metabolismo , Hígado/embriología , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piel/embriologíaRESUMEN
The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.
Asunto(s)
Lisosomas/metabolismo , Monocitos/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Manosafosfatos/metabolismo , Datos de Secuencia Molecular , Monocitos/ultraestructura , Proteínas/aislamiento & purificación , Células U937RESUMEN
The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP.
Asunto(s)
Proteínas Portadoras/metabolismo , Dictyostelium/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Dictyostelium/genética , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Filogenia , Plásmidos , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic [2Fe-2S](Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one [2Fe-2S] cluster per subunit. This protein is undamaged by heating to 100 degrees C for at least three hours. The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A. aeolicus protein relate it to well characterized [2Fe-2S] proteins from Clostridium pasteurianum and Azotobacter vinelandii. These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. The A. aeolicus [2Fe-2S] protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds.
Asunto(s)
Proteínas Bacterianas , Bacilos y Cocos Aerobios Gramnegativos/química , Proteínas Hierro-Azufre/química , Secuencia de Aminoácidos , Azotobacter vinelandii/química , Secuencia de Bases , Clostridium/química , Dimerización , Estabilidad de Medicamentos , Escherichia coli/genética , Expresión Génica , Bacilos y Cocos Aerobios Gramnegativos/genética , Calor , Proteínas Hierro-Azufre/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Recombinantes , Alineación de Secuencia , EspectrofotometríaRESUMEN
Capillary zone electrophoresis (CZE) in the presence of ethanolamine was used in a micropreparative mode. Sample volumes up to 1 microL could be loaded onto a 100 microns diameter capillary without loss in resolution. Coupled to narrow-bore reversed-phase high-performance liquid chromatography, ethanolamine-CZE allowed the collection of sufficient amounts of pure peptidic material to perform amino acid sequence analysis.
Asunto(s)
Electroforesis Capilar/métodos , Etanolamina , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Indicadores y Reactivos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Análisis de Secuencia , Tripsina/metabolismoRESUMEN
Owing to the complexity of higher eukaryotic cells, characterization of a complete proteome is likely to be difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and "physiology" of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting, but other methods were also used (N-terminal microsequencing, blotting). The optimization steps in two-dimensional (2-D) electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Mitocondrias/química , Proteínas Gestacionales/química , Electroforesis en Gel Bidimensional/normas , Humanos , Proteínas Gestacionales/aislamiento & purificación , Valores de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normasRESUMEN
When St. Joseph's Hospital in St. Paul, MN, owned and operated by the Sisters of St. Joseph of Carondelet, joined the ecumenical HealthEast healthcare system in 1987, many observers were skeptical of the venture's success. But an emphasis on their shared Judeo-Christian values has enabled the Catholic, Lutheran, and Baptist facilities to build a strong system. The beginnings of the merger were difficult, as facilities closed, others expanded their services, and staff shifted between them. An open communications policy between HealthEast leaders and staff members and dedication to the mission of healthcare that all the system facilities shared helped blend denominational identity and traditions at each member hospital and establish a corporate identity. The HealthEast system has adopted some of St. Joseph's policies and practices, particularly in the areas of mission, ethics, and spiritual care. HealthEast St. Joseph's has also benefited from being part of the HealthEast system, gaining a more diverse staff respectful of each others' beliefs, expanded spiritual care, and the means to continue serving its community. HealthEast plans to discontinue inpatient services at HealthEast St. Joseph's in downtown St. Paul and build a suburban facility, but the Sisters of St. Joseph of Carondelet are working with HealthEast to assess the downtown community's healthcare needs, especially among the homeless and immigrant populations, and ensure those needs will continue to be met.