RESUMO
Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes of cno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3-independent mechanism critical to the biogenesis of lysosome-related organelles. (Blood. 2000;96:4227-4235)
Assuntos
Modelos Animais de Doenças , Síndrome de Hermanski-Pudlak/genética , Proteínas de Membrana/genética , Camundongos Mutantes/genética , Proteínas Monoméricas de Montagem de Clatrina , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Difosfato de Adenosina/sangue , Animais , Plaquetas/química , Plaquetas/patologia , Mapeamento Cromossômico , Olho/patologia , Genes , Genes Recessivos , Heterogeneidade Genética , Cor de Cabelo/genética , Síndrome de Hermanski-Pudlak/epidemiologia , Síndrome de Hermanski-Pudlak/patologia , Humanos , Rim/enzimologia , Rim/ultraestrutura , Lipofuscina/metabolismo , Fígado/enzimologia , Fígado/ultraestrutura , Lisossomos/enzimologia , Melanossomas/patologia , Camundongos , Camundongos Endogâmicos C3H , Modelos Animais , Fenótipo , Porto Rico/epidemiologia , Serotonina/sangue , Pele/patologia , Especificidade da EspécieRESUMO
The complete amino acid sequence of a basic liver fatty acid-binding protein (L-FABP) from catfish (Rhamdia sapo) was determined. Alignment of sequences shows that it has more similarity to chicken basic L-FABP than to mammalian L-FABP. The phylogenetic analysis suggests that basic L-FABP from catfish, chicken and iguana diverged from the mammalian protein before the fish-tetrapod divergence, thus implying that the two types are encoded by different genes. Supporting this conclusion, a 14-kDa protein, structurally closely related to mammalian L-FABP, was isolated from catfish intestine, indicating the presence of the two genes in the same species. The catfish basic L-FABP binds only one fatty acid/molecule, while mammalian L-FABP bind two. The former has more affinity for trans-parinaric acid than for cis-parinaric acid, in constrast to the latter proteins.
Assuntos
Proteínas de Transporte/química , Peixes-Gato/genética , Evolução Molecular , Ácidos Graxos/metabolismo , Fígado/metabolismo , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Filogenia , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Galinhas , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Iguanas , Mucosa Intestinal/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Fragmentos de Peptídeos/química , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TubarõesRESUMO
A low-molecular-mass fatty acid-binding protein was isolated from the cytosol of the yeast Yarrowia lipolytica. Purification was achieved by a two-step procedure involving size-exclusion and cation-exchange chromatography. The isolated protein exists as a monomer of 15 kDa, is basic and has a blocked N-terminus. Internal amino acid sequencing suggests that this protein may belong to a novel class of fatty acid-binding proteins.
Assuntos
Proteínas de Transporte/química , Proteínas Fúngicas/química , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Saccharomycetales/química , Sequência de Aminoácidos , Proteínas de Transporte/isolamento & purificação , Cromatografia em Gel , Cromatografia por Troca Iônica , Brometo de Cianogênio/metabolismo , Citoplasma/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação a Ácido Graxo , Proteínas Fúngicas/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Proteína P2 de Mielina/isolamento & purificação , Ácido Palmítico/metabolismo , Fragmentos de Peptídeos/química , Análise de Sequência , Tripsina/metabolismoRESUMO
We report here the isolation of a fatty acid-binding protein (FABP) from the liver of the catfish Rhamdia sapo. The purification procedure involves gel filtration, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The purified protein is basic (pI > 8.7) and migrates on sodium dodecyl sulfate-gel electrophoresis as a single entity of about 15 kDa. Its amino acid composition resembles those of FABPs isolated from other animals. Unlike mammalian liver FABPs, catfish liver FABP contains at least one tryptophan residue per molecule. No significant cross-reactivity was observed between the purified protein and polyclonal antibodies against either rat liver FABP or rat heart FABP. Amino acid sequencing of peptides obtained by digestion with Lys-C revealed that the catfish protein is structurally more similar to chicken liver FABP (69% identity in a 67-residue overlap) than to human liver FABPs (36%), nurse shark (Ginglymostoma cirratum) liver FABP (30%) and human heart FABP (31%). Taken together, these results suggest that catfish liver FABP is far more closely related to chicken liver FABP than to the FABPs isolated from the liver of mammals or elasmobranchs.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Peixes-Gato/metabolismo , Fígado/metabolismo , Proteína P2 de Mielina/química , Proteína P2 de Mielina/isolamento & purificação , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Ácidos Palmíticos/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Galinhas , Citosol/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Dados de Sequência Molecular , Proteína P2 de Mielina/metabolismo , Ácido Palmítico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Calgranulin A (CAGA) and calgranulin B (CAGB) are two S100-like calcium-binding proteins that in human, bovine and mouse granulocytes are associated into a heterocomplex. We have previously identified in pig granulocytes the porcine homologue of CAGA and a novel S100-like protein which was named calgranulin C (CAGC). As pig CAGA is not associated with CAGC, we herein investigate its possible association with other proteins. CAGA was purified from pig granulocytes by gel filtration followed by Mono Q chromatography. The purified fractions were analysed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, mass spectrometry, chemical cross-linking and hydrophobic interaction chromatography. The CAGA-associated protein was further characterized by amino acid sequencing. Two CAGA-containing fractions were isolated. One of them was identified as a CAGA homodimer. The other fraction consists of a heterocomplex containing CAGA and a pI 7.0 calcium-binding protein; this protein has a molecular mass of 15,877.9 +/- 3.8 Da (mean +/- SD) whereas it migrates on 10 and 16% polyacrylamide gels as a 24- and 20-kDa protein, respectively. The pI 7.0 protein was identified by internal amino acid sequencing as the porcine homologue of CAGB. The stoichiometry of the heterocomplex was estimated to be 1:1. Both the CAGA homodimer and CAGA/CAGB were found to be non-covalently associated. Unlike the homodimer, CAGA/CAGB was bound to a Phenyl Superose column in a calcium-dependent manner. Our results suggest that pig granulocytes contain, in addition to CAGC, a CAGA homodimer and a CAGA/CAGB heterodimer. It is proposed that CAGB/CAGB and the CAGA homodimer may play different roles in vivo.
Assuntos
Proteínas de Ligação ao Cálcio/química , Granulócitos/química , Proteínas S100/química , Sequência de Aminoácidos , Animais , Calgranulina A , Calgranulina B , Bovinos , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , SuínosRESUMO
The intracellular lipid-binding proteins are a group of homologous proteins which bind and facilitate the transport of fatty acids, bile acids and retinoids. The evolutionary relationship of 51 family members from vertebrates and invertebrates was analyzed. The inferred phylogeny implies the occurrence of at least 14 gene duplications and contains five regions where the branching order is statistically non-significant--this uncertainty explaining most inconsistencies between previous phylogenetic analyses. The phylogeny also suggests that the intestinal fatty acid-binding protein and the liver fatty acid-binding protein/ileal lipid-binding protein subfamilies diverged from the other subfamilies before the vertebrate-invertebrate split. Finally, results presented herein indicate that the putative fatty acid-binding domain of NMDA receptor 1 is unlikely to be a member of this family.
Assuntos
Proteínas de Transporte , Evolução Molecular , Metabolismo dos Lipídeos , Família Multigênica , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Intestinos/química , Invertebrados , Fígado/química , Filogenia , Receptores de N-Metil-D-Aspartato/química , Alinhamento de Sequência , VertebradosRESUMO
In this paper we report the biochemical characterization of calgranulin C, a new member of the S100 protein family. The protein is highly abundant in the cytosol of pig granulocytes, with relatively small amounts in lymphocytes. A simple protocol for the rapid purification of calgranulin C is described. The purified protein migrates as a single entity on SDS-polyacrylamide gel electrophoresis while it has two isoforms focusing at pH 5.8 and 5.5. Gel filtration and cross-linking experiments indicate that calgranulin C is capable of dimerization. The complete amino acid sequence was determined by Edman degradation of peptides generated by trypsin and V8 protease digestion. Calgranulin C consists of 91 residues and has a calculated molecular mass of 10,614 daltons. This value is virtually identical to that obtained by electrospray mass spectrometry. Sequence analysis predicts two EF-hand calcium-binding motifs, the first having an extended loop that is distinctive of the S100 protein family. The metal-binding properties were studied by means of a direct 45Ca(2+)-binding assay and by tyrosine fluorescence titration. Calgranulin C binds not only calcium but also zinc ions. A single high affinity Zn(2+)-binding site per monomer was evidenced by fluorimetric titration. Zinc binding to calgranulin C induces a remarkable increase in the protein affinity for calcium; in the absence of zinc, the protein binds 1 Ca2+/monomer with a binding constant of about 2 x 10(4) M-1, whereas the Zn(2+)-loaded form binds 2 Ca2+/monomer with Ka values of approximately 3 x 10(7) and 6 x 10(4) M-1. Circular dichroism analysis showed that the binding of calcium to calgranulin C induces a 15% decrease in the apparent alpha-helix content. This result and the calcium-dependent binding of the protein to a phenyl-Superose column strongly suggest that calgranulin C undergoes a gross conformational change upon calcium binding, thus supporting the idea that this protein may be involved in Ca(2+)-dependent signal transduction events.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Granulócitos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/isolamento & purificação , Humanos , Dados de Sequência Molecular , Conformação Proteica , Proteínas S100/química , Proteínas S100/metabolismo , Proteína S100A12 , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Suínos , Tirosina/químicaRESUMO
1. Two small, abundant calcium-binding proteins were isolated from pig granulocytes. They were named p7A and p7B. Relative molecular masses were approx. 32,000 for p7A and 13,000 for p7B, when obtained by Sephadex G-75 gel filtration, while it was 7000 for both proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 2. N-terminal sequence analysis suggests that p7A is homologous to human and mouse MRP-8 and that p7B may be related to human and mouse MRP-14, though some properties of the latter--such as mobility on SDS-PAGE--were found to be different. In addition, p7A and p7B could be resolved under native conditions, contrasting with the fact that human and mouse MRP-8/MRP-14 form noncovalent complexes.
Assuntos
Proteínas de Ligação ao Cálcio/isolamento & purificação , Granulócitos/química , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/sangue , Proteínas de Ligação ao Cálcio/química , Citosol/química , Granulócitos/ultraestrutura , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , SuínosRESUMO
High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.
Assuntos
Ácidos Palmíticos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Catalase/metabolismo , Meios de Cultura , Ácidos Graxos/metabolismo , Microcorpos/efeitos dos fármacos , Microcorpos/metabolismo , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.