RESUMEN
Heparin-binding neurite-promoting factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins. It exhibits a neurite-outgrowth promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development. A high interspecies sequence conservation suggests important, presently unknown, biological functions. HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine). To elucidate biological roles of these proteins, recombinant forms of the proteins were produced. Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein. Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea. Refolding of the reduced and denatured protein occurred upon dialysis at pH 7.4. Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography. Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides. Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation. The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Portadoras/biosíntesis , Citocinas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/farmacología , Bovinos , Línea Celular , Cricetinae , Cisteína/metabolismo , Cistina/biosíntesis , Citocinas/química , Citocinas/aislamiento & purificación , Citocinas/farmacología , ADN Complementario/metabolismo , Ditiotreitol/farmacología , Escherichia coli , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Mesocricetus , Midkina , Datos de Secuencia Molecular , Pliegue de Proteína , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Homología de Secuencia de AminoácidoRESUMEN
The organization of the human heparin-binding neurite outgrowth promoting factor (HBNF) gene is presented. Based on Southern analysis and the isolation of genomic DNA clones from a lambda phage library, the minimum size of the gene is 42 kb. Sequences comprising the HBNF mRNA are contained in five exons which account for the 1650 nt mRNA size observed by northern analysis. From the structure of the gene it is predicted that a variant human HBNF cDNA with a three basepair deletion is a result of alternative splicing at the acceptor site of exon 5. Evidence is presented that indicates the existence of a variant HBNF protein, des-Ala119-HBNF, in bovine brain which has a corresponding amino acid deletion. This alternate form comprises approximately 20% of the total HBNF protein present in bovine brain.
Asunto(s)
Empalme Alternativo , Proteínas Portadoras/genética , Citocinas/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/aislamiento & purificación , Bovinos , Clonación Molecular , Citocinas/aislamiento & purificación , ADN , Variación Genética , Humanos , Datos de Secuencia Molecular , Eliminación de SecuenciaRESUMEN
The retinoic acid-inducible MK gene shows a distinct developmental pattern of expression, which implies that it has potential growth regulation and differentiation functions, particularly in the brain. We report here the cloning of the human MK gene from a phage library constructed from placental tissue. The structure of this gene has been determined using Southern hybridization and DNA sequence analysis. An isolated fragment was cloned and found to contain sequences identical to those of a previously isolated human MK cDNA clone, MKHC4. The gene contains three introns within the MK coding region as well as additional sequence, which indicates the presence of an intron prior to the putative protein start site. As judged by sequence analysis of cDNA clones, primer extension studies, and Northern analysis, the most abundant human MK message corresponds to the major mRNA of the previously described mouse gene. Primer extension studies and cDNA sequence data suggest that minor messages may be transcribed from the human gene, but no evidence of additional messages has been found by Northern analysis. This is in contrast to the mouse MK gene, from which three mRNAs are transcribed. Nevertheless, the similarity in the overall genomic structure of the human and mouse genes is striking.
Asunto(s)
Factores de Crecimiento Nervioso , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Exones , Regulación de la Expresión Génica/efectos de los fármacos , Biblioteca Genómica , Humanos , Intrones , Midkina , Datos de Secuencia Molecular , Células Madre Neoplásicas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
Human metastasis-suppressor genes nm23-1 (NME1) and nm23-2 (NME2) are implicated in control of the metastatic potential of malignant cells. Using somatic cell hybrid analysis and fluorescence in situ hybridization we co-localized both genes to 17q21.3. The 17q21 region carries the locus responsible for early-onset familial breast-ovarian cancer and several other genes that are involved in tumorigenesis and differentiation and undergo frequent rearrangements during neoplastic development. Thus, our mapping places the NME genes in a region that may be subjected to multiple selection pressures. NME1 and NME2 genes were expressed as soluble proteins in a T7 bacterial expression system. Both proteins are independently active nucleotide diphosphate kinases and readily form intra- and intermolecular disulfide bonds. The biochemical properties of these proteins may explain the diversity of mature eucaryotic nucleoside diphosphate kinases.
Asunto(s)
Mapeo Cromosómico , Genes Supresores de Tumor , Metástasis de la Neoplasia , Nucleósido-Difosfato Quinasa/análisis , Animales , Secuencia de Bases , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/análisisRESUMEN
Recombinant HIV-1 Nef proteins with either thr-15 or ala-15 have been constructed and expressed in the T7 bacterial system. From the soluble portion of bacterial lysates both Nef(thr-15) and Nef(ala-15) have been purified to near homogeneity through 6 nondenaturing chromatographic steps in the presence of MgCl2. Neither purified proteins display the previously reported GTP binding activity. Additionally Nef(thr-15) does not have autophosphorylating activity with either [gamma-32P]GTP or [gamma-32P]ATP. Although GTPase activity is present in the preparations of Nef proteins, it does not increase during purification and is attributed to bacterial contaminations.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Productos del Gen nef/metabolismo , Guanosina Trifosfato/metabolismo , VIH-1 , Proteínas de Unión al GTP/metabolismo , Productos del Gen nef/aislamiento & purificación , Genes nef , VIH-1/genética , Cinética , Fosforilación , Proteínas Recombinantes/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
This report describes the cloning, expression and characterization of two members of a novel human gene family of proteins, HBNF and MK, which exhibit neurite outgrowth-promoting activity. The HBNF cDNA gene codes for a 168-residue protein which is a precursor for a previously described brain-derived heparin-binding protein of 136 amino acids. The second human gene identified in this study, called MK, codes for a 143-residue protein (including a 22-amino acid signal sequence) which is 46% homologous with HBNF. Complementary DNA constructs coding for the mature HBNF and MK proteins were expressed in bacteria and purified by heparin affinity chromatography. These recombinant proteins exhibited neurite-outgrowth promoting activity, but lacked mitogenic activity. The HBNF gene is expressed in the brain of adult mice and rats, but only minimal expression of MK was observed in this tissue. Different patterns of developmental expression were observed in the embryonic mouse, with MK expression peaking in the brain between days E12 and E14 and diminishing to minimal levels in the adult, while expression of HBNF mRNA was observed to gradually increase during embryogenesis, reaching a maximal level at birth and maintaining this level into adulthood. Expression of these genes was also observed in the human embryonal carcinoma cell line, NT2/D1. Retinoic acid induced the expression of HBNF and MK 6- and 11-fold, respectively, in this cell line. Our studies indicate that HBNF and MK are members of a new family of highly conserved, developmentally regulated genes that may play a role in nervous tissue development and/or maintenance.
Asunto(s)
Proteínas Portadoras/genética , Citocinas/genética , Factores de Crecimiento Nervioso , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Expresión Génica/efectos de los fármacos , Humanos , Midkina , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas del Tejido Nervioso/genética , Homología de Secuencia de Ácido Nucleico , Tretinoina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
A partial rat cDNA clone coding for a novel neurotrophic factor HBNF was isolated. Nucleotide sequence determination, in combination with the known N-terminal sequence of rat HBNF, allowed deduction of the amino acid sequence of the first 102 residues of mature rat HBNF. HBNF shares high structural homology (55%) with the MK protein (Tomomura et al., J. Biol. Chem. 265, 10765, 1990). Complete alignment of 9 cysteine residues suggests further that the two proteins have similar 3-dimensional structures. HBNF was reported to stimulate neurite outgrowth in neurons and to be expressed in a developmentally regulated manner in the rat brain. MK mRNA was found in retinoid acid-induced teratocarcinoma cells and during early development of the mouse embryo, but no biological activity for MK is yet known. These data suggest that HBNF and MK are members of a novel family of structurally and probably functionally related proteins.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/genética , Citocinas/genética , Familia de Multigenes , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ratones , Midkina , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN/genética , ARN/aislamiento & purificación , Ratas , Ratas Endogámicas , Homología de Secuencia de Ácido NucleicoRESUMEN
Recent evidence supports a role for transient c-fos expression as one step in signalling pathways by which membrane receptor-ligand interactions are transduced into appropriate intracellular responses. The activity of adrenergic receptors is mediated by second messenger systems which include ion fluxes, changes in cAMP concentration and enhanced phosphoinositide turnover. In order to determine if C-fos induction was also a step in adrenergic signal transduction in the brain, we performed in vivo studies with drugs specific for different adrenergic receptor types. Unexpectedly, we found that the stress associated with a single intraperitoneal (i.p.) injection of drug vehicle produced a transient increase (averaging 4.0-fold) in c-fos mRNA levels in rat brain. Injection of the alpha 2-adrenoreceptor antagonist, yohimbine, produced a transient increase which was larger in magnitude (averaging 9.6-fold) and longer in duration than that produced by injection of the drug vehicle alone. In experiments designed to ask whether either of these inductions was mediated by specific types of adrenergic receptors, we found that the alpha 2- and beta-adrenoreceptors were involved in both responses, while the alpha 1-receptor played a role in mediating the yohimbine induction, but no detectable role in the solvent induction. One hypothesis consistent with our results is that the norepinephrine (NE) released due to the stress associated with an i.p. injection interacts with postsynaptic beta-adrenergic receptors, resulting in the observed c-fos mRNA induction. When the negative feedback effect of NE, mediated by presynaptic alpha 2-receptors, is blocked by yohimbine, the postsynaptic response is enhanced and prolonged.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Receptores Adrenérgicos/fisiología , Estrés Fisiológico/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Animales , Encéfalo/fisiopatología , Masculino , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Receptores Adrenérgicos/metabolismo , Sistemas de Mensajero Secundario , Yohimbina/farmacologíaRESUMEN
Rapid, transient expression of the c-fos proto-oncogene is induced by the beta-adrenergic agonist, isoproterenol, in vivo and by numerous factors promoting growth and differentiation in cultured cells. We wanted to determine whether cellular stressors, which are known to induce expression of the gene encoding the major heat shock protein, hsp 70, also induced expression of the c-fos gene. Our findings demonstrate that c-fos mRNA levels increase transiently under conditions of heat stress or sodium arsenite treatment which induce expression of hsp70 mRNA in cultured cell lines. When 3T3 cells are heat shocked in the presence of amiloride, an inhibitor of Na+/H+ exchange, the induction of c-fos mRNA is partially inhibited, whereas that of hsp70 is somewhat enhanced. However, neither response requires ongoing protein synthesis. In fact, dramatic superinduction of c-fos mRNA is observed in cells which are heat shocked in the presence of the protein synthesis inhibitor, anisomycin. A comparison of relative rates of protein synthesis and c-fos mRNA levels during either heat shock or sodium arsenite treatment suggests that the transient suppression of protein synthesis accompanying these treatments may be one factor responsible for the observed c-fos mRNA induction.