RESUMO
The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.
Assuntos
Proteína de Ligação a Androgênios , Proteínas de Transporte/metabolismo , Membranas Artificiais , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Bovinos , Dicroísmo Circular , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Transferência de Fosfolipídeos , Espectrometria de FluorescênciaRESUMO
In rat and human cells, RKIP (previously known as PEBP) was characterized as an inhibitor of the MEK phosphorylation by Raf-1. In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found. The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well. We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E. coli, respectively, after which we determined their crystallographic structures. These structures verify that YBHB and YBCL belong to the same structural family as mammalian RKIP/PEBP proteins. The general fold and the anion binding site of these proteins are extremely well conserved between mammals and bacteria and suggest functional similarities. However, the bacterial proteins also exhibit some specific structural features, like a substrate binding pocket formed by the dimerization interface and the absence of cis peptide bonds. This structural variety should correspond to the recognition of multiple cellular partners.
Assuntos
Proteína de Ligação a Androgênios , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Escherichia coli , Escherichia coli/química , Sequência de Aminoácidos , Ânions/metabolismo , Proteínas Arqueais/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Transporte/genética , Clonagem Molecular , Biologia Computacional , Sequência Conservada/genética , Cristalografia por Raios X , Citoplasma/química , Dimerização , Escherichia coli/citologia , Escherichia coli/genética , Genes Bacterianos/genética , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Periplasma/química , Proteína de Ligação a Fosfatidiletanolamina , Proteínas de Transferência de Fosfolipídeos , Prostateína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Secretoglobinas , Alinhamento de Sequência , Homologia de Sequência , Especificidade por Substrato , UteroglobinaRESUMO
Transgenic mice expressing the Simian virus 40 large T antigen under the control of the liver-specific human antithrombin-III promoter all develop well-differentiated hepatocellular carcinoma. During tumour development serum ceruloplasmin (Cp) increases gradually until it reaches 30 times control levels in all transgenic mice at 6 months of age. The accumulation of Cp in the serum is due to the increased transcription of the Cp gene as well as to the increase in Cp mRNA stability in the livers of the transgenic mice. One-half of the overproduced Cp is charged with copper and Cp-associated serum oxidase activity increases in parallel with the holo-Cp concentration. Through its ferroxidase activity Cp is involved prominently in iron metabolism. Analysis of copper and iron in serum and liver revealed increased copper levels in the serum of tumour-bearing animals and which increased in parallel with Cp concentration; the amounts of copper in the liver were unchanged. In contrast, serum iron remained constant during tumour development whereas the iron concentration in the livers of the transgenic mice decreased.
Assuntos
Carcinoma Hepatocelular/sangue , Ceruloplasmina/metabolismo , Neoplasias Hepáticas/sangue , Animais , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Antitrombina III/genética , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Ceruloplasmina/química , Ceruloplasmina/genética , Ceruloplasmina/isolamento & purificação , Cromatografia por Troca Iônica , Cobre/sangue , Cobre/metabolismo , Eletroforese em Gel Bidimensional , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Ferro/sangue , Ferro/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Transgênicos , Peso Molecular , Regiões Promotoras Genéticas/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vírus 40 dos Símios/genética , Transgenes/genéticaRESUMO
The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.
Assuntos
Proteína de Ligação a Androgênios , Química Encefálica , Proteínas de Transporte/química , Proteínas do Tecido Nervoso/química , Conformação Proteica , Ar , Animais , Varredura Diferencial de Calorimetria , Bovinos , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , Análise de Fourier , Temperatura Alta , Concentração de Íons de Hidrogênio , Modelos Moleculares , Oligodendroglia/química , Proteínas de Transferência de Fosfolipídeos , Desnaturação Proteica , Dobramento de Proteína , Espectrofotometria Infravermelho , Propriedades de Superfície , ÁguaRESUMO
BACKGROUND: Phosphatidylethanolamine-binding protein (PEBP) is a basic protein found in numerous tissues from a wide range of species. The screening of gene and protein data banks defines a family of PEBP-related proteins that are present in a variety of organisms, including Drosophila and inferior eukaryotes. PEBP binds to phosphatidylethanolamine and nucleotides in vitro, but its biological function in vivo is not yet known. The expression of PEBP and related proteins seems to be correlated with development and cell morphogenesis, however. To obtain new insights into the PEBP family and its potential functions, we initiated a crystallographic study of bovine brain PEPB. RESULTS: The X-ray crystal structure of bovine brain PEBP has been solved using multiple isomorphous replacement methods, and refined to 1.84 A resolution. The structure displays a beta fold and exhibits one nonprolyl cis peptide bond. Analysis of cavities within the structure and sequence alignments were used to identify a putative ligand-binding site. This binding site is defined by residues of the C-terminal helix and the residues His85, Asp69, Gly109 and Tyr119. This site also corresponds to the binding site of phosphorylethanolamine, the polar head group of phosphatidylethanolamine. CONCLUSIONS: This study shows that PEBP is not related to the G-protein family nor to known lipid-binding proteins, and therefore defines a novel structural family of phospholipid-binding proteins. The PEBP structure contains no internal hydrophobic pocket, as described for lipocalins or small phospholipid-transfer proteins. Nevertheless, in PEBP, a small cavity close to the protein surface has a high affinity for anions, such as phosphate and acetate, and also phosphorylethanolamine. We suggest that this cavity corresponds to the binding site of the polar head group of phosphatidylethanolamine.
Assuntos
Proteína de Ligação a Androgênios , Encéfalo/metabolismo , Proteínas de Transporte/química , Conformação Proteica , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Bovinos , Cristalografia por Raios X/métodos , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidiletanolaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Liver cancer is one of the most frequent and lethal malignancies worldwide. Early detection is hampered by the absence of reliable markers. Mice transgenic for the SV40 large T antigen under the control of a liver-specific promoter spontaneously develop well-differentiated hepatocellular carcinomas between 8 to 10 weeks of age. They are excellent models to investigate the alterations of protein expression in the early stages of tumor development and to follow these changes during tumor progression. In the present study, we analyzed the glycosylation changes occurring during tumor development in transgenic mice expressing the SV40 T antigen under the control of the antithrombin III promoter. The analysis of serum and liver glycoproteins by an ELISA type assay, using the lectin from Sambucus nigra (SNA) as a probe, revealed the presence of increased levels of Neu5Ac alpha2,6Gal beta1,4GlcNAc on N-glycans in the tumor-bearing transgenic mice as compared to controls. On serum glycoproteins the increase in alpha2,6 sialylation followed tumor progression, reaching up to 10 times control levels. However, significantly higher SNA binding (2-fold) could already be observed on serum glycoproteins from mice exhibiting only microscopically small neoplastic foci. On liver membrane glycoproteins, the increase in alpha2,6 sialylation was less pronounced, reaching two to three times control values in 6-month-old mice. Western blotting of serum and liver proteins with radiolabeled SNA showed that all glycoproteins that bind the lectin in controls exhibit larger amounts of Neu5Ac alpha2,6Gal beta1,4GlcNAc on N-glycans in the tumor-bearing mice. This general increase in alpha2,6 sialylation on all glycoproteins is due to the increased activity of the galactoside:alpha2,6 sialyltransferase (ST6Gal I), which specifically transfers Neu5Ac residues in alpha2,6 linkage to Gal beta1,4GlcNAc units on N-glycans. As for the structures synthesized by the enzyme, the increase of ST6Gal I activity in the serum as well as in liver microsomes of the transgenic mice followed tumor progression. Interestingly, the activity of the galactoside:alpha2,3 sialyltransferase (ST3Gal III), which uses the same acceptor substrate (Gal beta1,4GlcNAc), was unchanged in the earlier stages of tumor development but decreased in the serum and in liver microsomes from later stages. Using a rat ST6Gal I cDNA as a probe, Northern blots of total RNA extracted from the livers of control and transgenic mice revealed an increased (4-fold) expression of the ST6Gal I gene. The single transcripts detected in both normal and cancerous liver showed identical size.
Assuntos
Neoplasias Hepáticas Experimentais/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Lectinas de Plantas , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sialiltransferases/metabolismo , Animais , Antígenos Virais de Tumores/fisiologia , Proteínas Sanguíneas/metabolismo , Sequência de Carboidratos , Progressão da Doença , Indução Enzimática , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/metabolismo , Glicosilação , Lectinas/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Neoplásico/análise , RNA Neoplásico/genética , Ratos , Proteínas Inativadoras de Ribossomos , Sialiltransferases/genética , Vírus 40 dos Símios/genéticaRESUMO
We created fully and stable xenogeneic hematopoietic chimeras in "poorly concordant" rat-mouse strain combinations defined by their high histocompatibility-antigen disparity and by the high titer of mouse-serum natural cytotoxic antibodies (NcAb) to rat donor bone marrow cells (BMC). Recipients were adult male (C57BL/6 x DBA/2)F1 (BDF1) mice, and donors of untreated BMC were adult male WAG strain rats. We tried several approaches to improve the quality and the stability of the rat-cell engraftment and to avoid the risk of graft-vs.-host reaction (GVHR). Best results were obtained when: 1) BDF1 recipients were previously thymectomized and then heavily irradiated to lower their immunocompetence; 2) irradiated recipients were implanted with a newborn BDF1 thymus, which allows maturing rat T lymphoid cells to be made tolerant to mouse antigens in vivo, which lowered the risk of GVHR; and 3) recipients were given a high number of untreated rat BMC (4-5 injections of 1.6 x 10(7) cells) to reduce the risk of rejection of rat BMC by mouse NcAb. We found that rat BMC engraftment was highly effective (75 to 100% rat hemoglobin and 100% rat IgG) and long-lasting (more than 10 months). The grafted rat cells were very tolerant toward host histocompatibility antigens but maintained all their immunological potentialities. Moreover, using the "poorly concordant" Wistar Furth (WF)-BDF1 combination, we showed that a genetically controlled characteristic of the hematopoietic system of the WF rat donor was maintained and functionally expressed in the xenogeneic environment of BDF1 mice.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Quimera , Citotoxicidade Imunológica , Transplante de Células-Tronco Hematopoéticas , Transplante Heterólogo/imunologia , Animais , Testes de Hemaglutinação , Hemoglobinas/análise , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Fenótipo , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos , Especificidade da Espécie , Fatores de Tempo , Transplante Heterólogo/métodosRESUMO
Two months after adult-thymectomy, male B10.D2 mice were irradiated (6.5 Gy), injected with B10.D2 bone marrow cells and grafted with untreated neonate (DBA/2 X B10.D2)F1 (H-2d/H-2d) thymus [D2.F1]. Control (D2.D2) mice were implanted with B10.D2 thymus. T cells from [D2.F1] mice revealed to be fully immunocompetent while being tolerant to the minor histocompatibility antigens (MiHA) differing between DBA/2 and B10.D2 strains by in vitro and in vivo assays. Their transplantation into F1 irradiated (8.5 Gy) recipients did not induce any clinical sign of graft-versus-host reaction (GVHR) whereas this reaction is lethal when the transplantation is realized with normal B10.D2 or [D2.D2] hemopoietic cells. (D2.F1) transplanted cells were not stimulated to divide in non-lymphoid organs of the F1 hosts while this stimulation is a characteristic feature of the early period of the GVHR. Tolerance was due neither to detectable chimerism of the thymus-grafted host nor to active suppression. It is concluded that the thymus plays a sufficient and essential role for the proper acquisition of T cell immunocompetence and tolerance to all the minor histocompatibility antigens of the thymus-donor strain. Since we have previously reported that MiHA are numerous and highly organ-specific, the expression or the presence of all these MiHA in the non-lymphoid components of the thymus is questioned.