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1.
Fam Cancer ; 4(2): 73-5, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15951955

RESUMO

A recent report based on 68 families, including 17 with mutations in BRCA1, suggested that there was an excess of female offspring born to BRCA1 mutation carriers. We have examined the gender ratio among offspring of 511 mutation carriers from 116 BRCA1 families, 77 and 39 from Australia and the United States, respectively. We found no evidence for a significant deviation from the expected proportion of female offspring in the Australian pedigrees, but there was an excess of female offspring in pedigrees from the USA. Ascertainment bias probably explains this bias, rather than a link with X-chromosome inactivation as previously suggested, because the families from the USA were ascertained for the purposes of linkage studies whereas those from Australia were ascertained through Familial Cancer Clinics to which they had been referred for clinical genetic counseling and mutation testing.


Assuntos
Genes BRCA1 , Genes BRCA2 , Heterozigoto , Razão de Masculinidade , Adulto , Austrália/epidemiologia , Neoplasias da Mama/genética , Cromossomos Humanos X , Estudos de Coortes , Feminino , Humanos , Recém-Nascido , Masculino , Neoplasias Ovarianas/genética , Linhagem , Estados Unidos/epidemiologia
2.
Breast Cancer Res ; 3(5): 346-9, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11597326

RESUMO

BACKGROUND: Mutations in BRCA1 and BRCA2 account for approximately 50% of breast cancer families with more than four affected cases, whereas exonic mutations in p53, PTEN, CHK2 and ATM may account for a very small proportion. It was recently reported that an intronic variant of p53--G13964C--occurred in three out of 42 (7.1%) 'hereditary' breast cancer patients, but not in any of 171 'sporadic' breast cancer control individuals (P = 0.0003). If this relatively frequent occurrence of G13964C in familial breast cancer and absence in control individuals were confirmed, then this would suggest that the G13964C variant plays a role in breast cancer susceptibility. METHOD: We genotyped 71 familial breast cancer patients and 143 control individuals for the G13964C variant using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. RESULTS: Three (4.2%; 95% confidence interval [CI] 0-8.9%) G13964C heterozygotes were identified. The variant was also identified in 5 out of 143 (3.5%; 95% CI 0.6-6.4%) control individuals without breast cancer or a family history of breast cancer, however, which is no different to the proportion found in familial cases (P = 0.9). CONCLUSION: The present study would have had 80% power to detect an odds ratio of 4.4, and we therefore conclude that the G13946C polymorphism is not a 'high-risk' mutation for familial breast cancer.


Assuntos
Neoplasias da Mama/genética , Genes p53/genética , Predisposição Genética para Doença/genética , Adulto , Estudos de Casos e Controles , Primers do DNA , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição
3.
J Biol Chem ; 276(10): 7643-53, 2001 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-11116155

RESUMO

The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, and INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation of INP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.


Assuntos
Retículo Endoplasmático/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Sequência de Aminoácidos , Membrana Celular/metabolismo , Clonagem Molecular , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Inositol Polifosfato 5-Fosfatases , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Água/metabolismo
5.
J Biol Chem ; 272(31): 19471-9, 1997 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-9235949

RESUMO

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly beta-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.


Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70/metabolismo , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , DNA/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo
6.
Curr Eye Res ; 14(6): 449-58, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7671626

RESUMO

The role of tissue-type plasminogen activator (tPA) in the 'spontaneous' as well as 'experimental' metastasis of ocular melanomas in mice was evaluated by transfecting the D5.1G4 murine melanoma cell line that possesses low metastatic activity and low tPA activity with a full length cDNA encoding human tPA. For comparison, a highly metastatic melanoma cell line (Queen's) that constitutively expresses high tPA production, was transfected with a cDNA coding for human plasminogen activator inhibitor type 1 (PAI-1). Unlike non-transfected controls, transfected D5.1G4 melanoma cells expressed high levels of tPA and produced extensive pulmonary metastases following intravenous injection. By contrast, PAI-1 transfected Queen's melanoma cells expressed low tPA activity and displayed significantly reduced metastatic potential compared with nontransfected controls. Moreover, PAI-1 transfected Queen's melanoma cells did not metastasize from the eye while nontransfected parental cells produced extensive spontaneous metastases. Expression of tPA activity in transfected and nontransfected cell lines was completely blocked by an anti-tPA antibody. This antibody significantly inhibited the organ localization and frequency of lung metastases of both Queen's and tPA-transfected D5.1G4 melanomas. This study demonstrates that tPA is involved in the metastasis of murine intraocular melanomas.


Assuntos
Neoplasias Oculares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/secundário , Ativador de Plasminogênio Tecidual/fisiologia , Animais , Câmara Anterior/patologia , Feminino , Expressão Gênica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/imunologia , Transfecção , Células Tumorais Cultivadas
7.
J Biol Chem ; 269(48): 30470-8, 1994 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-7982963

RESUMO

We have studied the binding of synthetic peptides to three hsp70 molecular chaperones, DnaK, BiP, and hsc70, as a model for the interaction of hsp70 proteins with unfolded regions of target polypeptides. We measured the ability of 53 peptides to inhibit the formation of complexes between the hsp70 proteins and denatured lactalbumin. Peptides that bound with highest affinity to all three hsp70 proteins contained stretches of at least 7 residues that included large hydrophobic and basic amino acids, but few or no acidic residues. Amino acid substitutions within one heptameric peptide showed that an important feature for its binding to all three chaperones was a large hydrophobic residue in position 4, while specificity differences between the chaperones were revealed by substitutions at positions 2 and 6. Such specificity differences were frequently observed with other peptides, the most extreme example being a peptide rich in basic residues that bound with high affinity to DnaK, intermediate affinity to hsc70, and negligible affinity to BiP. Substitution of a lysine residue at position 2 in this peptide by tyrosine abolished the specificity difference by increasing the affinities of the DnaK and hsc70 proteins 5- and 20-fold, respectively, and that of BiP by greater than 2 orders of magnitude. Thus, hsp70 proteins can exhibit common or exclusive binding specificities, depending on the peptide sequence.


Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70/metabolismo , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Bovinos , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSC70 , Cinética , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Peptídeos/síntese química , Peptídeos/química , Especificidade por Substrato
8.
J Biol Chem ; 269(43): 26933-43, 1994 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-7523415

RESUMO

Human small intestinal lactase-phlorizin hydrolase (LPH) is synthesized as a single-chain polypeptide precursor, prepro-LPH, that undergoes two sequential cleavage steps: the first in the endoplasmic reticulum to pro-LPH (215-kDa) and the second, following terminal glycosylation in the Golgi apparatus, to mature 160-kDa LPH (denoted LPH beta). The LPH beta molecule is subsequently targetted to the brush-border membrane. Characterization of the N-terminal profragment (denoted LPH alpha) of pro-LPH using an epitope-specific, anti-peptide polyclonal antibody reveals that LPH alpha (i) has an apparent molecular weight of approximately 100,000, (ii) is not associated with LPH beta after cleavage of pro-LPH has occurred, and (iii) is not transported to the cell surface or secreted into the extracellular medium. In biosynthetic labeling experiments, a clear precursor/product relationship could be demonstrated between pro-LPH and the LPH alpha and LPH beta polypeptides. Further, LPH alpha has a significantly shorter half-life than LPH beta. LPH alpha is neither N- nor O-glycosylated, despite the presence of 5 potential N-glycosylation sites. LPH alpha, which is rich in cysteine and hydrophobic amino acid residues, may fold rapidly into a tight and rigid globular domain in which carbohydrate attachment sites are no longer accessible to glycosyltransferases. When expressed independently in COS-1 cells, the LPH beta polypeptide forms a misfolded, transport-incompetent molecule. We propose a role for the LPH alpha domain within the pro-LPH molecule as an intramolecular chaperone during folding in the ER.


Assuntos
Precursores Enzimáticos/metabolismo , Intestino Delgado/enzimologia , Lactase-Florizina Hidrolase/metabolismo , Fragmentos de Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Biópsia , Compartimento Celular , Células Cultivadas , Precursores Enzimáticos/genética , Precursores Enzimáticos/imunologia , Epitopos , Imunofluorescência , Glicosilação , Meia-Vida , Humanos , Lactase-Florizina Hidrolase/genética , Lactase-Florizina Hidrolase/imunologia , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
9.
Am J Pathol ; 145(3): 661-70, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8080048

RESUMO

By administering physiological doses of interleukin-1 (IL-1) concurrently with multiple low doses of the beta cell toxin streptozotocin (MSZ), we observed an augmentation of diabetes by IL-1 in four different strains of mice. Augmentation of hyperglycemia by IL-1 was most prominent in the two MSZ-resistant mouse strains Balb/cJ and A/J. Furthermore, concurrent treatment with IL-1 and MSZ rendered these MSZ-resistant mice susceptible to the development of significant insulitis when compared to mice treated with MSZ alone. Development of insulitis was dependent upon the dose of IL-1; it was only observed at an IL-1 dose of 250 ng/kg body weight. Analysis of the leukocytic infiltrate in the islets of mice after treatment with 250 ng/kg IL-1 plus MSZ revealed the presence of L3T4+ and Lyt-2+ T lymphocytes. Administration of MSZ alone or IL-1 alone did not produce diabetes in the MSZ-resistant mice, indicating that neither of these agents was toxic to the beta cells by itself. We conclude that IL-1 synergizes with MSZ to augment diabetes in mice that are normally resistant to the diabetogenic effects of MSZ.


Assuntos
Diabetes Mellitus Experimental/induzido quimicamente , Hiperglicemia/etiologia , Interleucina-1/administração & dosagem , Pancreatite/etiologia , Animais , Diabetes Mellitus Experimental/imunologia , Suscetibilidade a Doenças , Sinergismo Farmacológico , Imunidade Inata , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pancreatite/patologia , Especificidade da Espécie , Estreptozocina
10.
Biochem J ; 302 ( Pt 3): 929-35, 1994 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-7945222

RESUMO

Lactase-phlorizin hydrolase (LPH) is expressed on the intestinal brush border and is responsible for the hydrolysis of lactose, the chief sugar in mammalian milk. The enzyme activity of LPH peaks soon after birth in most mammals and declines to much lower levels before adolescence. The molecular basis of this pattern of expression has not been clearly established. We have measured relative amounts of LPH mRNA in intestine from sheep with ages across a developmental spectrum, including third trimester fetal lambs, newborn lambs and adult sheep. LPH mRNA levels in the jejunum decline approximately 50-fold between infancy and adulthood, in parallel with the reduction in both lactase specific activity and immunologically reactive lactase protein expression in sheep jejunum. LPH mRNA is present in high concentration in the duodenum of newborn lambs, but steadily declines by day 34 and is dramatically reduced in adults. Because the changes in LPH mRNA, protein, and enzymic activity are generally parallel, we conclude that the developmental regulation of LPH in sheep is probably mediated primarily at the mRNA level.


Assuntos
Regulação Enzimológica da Expressão Gênica , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Lactase-Florizina Hidrolase/genética , RNA Mensageiro/genética , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Western Blotting , Duodeno/enzimologia , Humanos , Hidrólise , Jejuno/enzimologia , Lactase-Florizina Hidrolase/biossíntese , Microvilosidades/enzimologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ovinos
11.
Invest Ophthalmol Vis Sci ; 35(9): 3533-9, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8056529

RESUMO

PURPOSE: To characterize intraocular tumors that arise by in situ transformation in the choroid-retinal pigment epithelium (RPE) in transgenic mice bearing the SV40 oncogene under the control of the mouse tyrosinase promoter. METHODS: Tumors from TySV40 transgenic mice were characterized in vivo and in vitro by immunohistology, compound microscopy, and electron microscopy. Tumor cell lines were established and characterized for growth and metastatic potential in the eyes of nude mice. RESULTS: On light microscopy, ocular tumors were predominantly epithelioid, although occasional clusters of spindle cells were also present. Transmission electron microscopy revealed the presence of numerous basal infoldings and abundant multilaminated basement membranes on the ocular tumors. Tumors stained with antibodies to melanoma-associated antigens, gangliosides GD2 and GD3, and the SV40 T antigen. Radiolabeled transgenic tumor cells preferentially localized in the liver after intravenous injection in normal mice. Intracamerally transplanted transgenic tumors metastasized from the eyes to the livers of nude mice. CONCLUSIONS: In TySV40 transgenic mice, intraocular tumors develop that arise at the choroid-RPE interface, and they display morphologic and ultrastructural features consistent with RPE carcinomas. However, the transgenic tumors express melanoma-associated antigens and a propensity to metastasize to the liver, two features characteristic of uveal melanomas. The TySV40 transgenic murine tumors represent potentially useful tools for investigations into the biology and metastasis of intraocular neoplasms.


Assuntos
Carcinoma/secundário , Neoplasias da Coroide/patologia , Epitélio Pigmentado Ocular/ultraestrutura , Doenças Retinianas/patologia , Animais , Antígenos de Neoplasias/análise , Transformação Celular Neoplásica , Feminino , Imunofluorescência , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Oncogenes/genética , Vírus 40 dos Símios/genética
12.
Curr Eye Res ; 13(5): 361-9, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-7914482

RESUMO

The present study examined the role of tumor infiltrating lymphocytes (TIL) in the rejection of intraocular tumors from SV40 transgenic mice. Tumor cells from an intraocular tumor arising in an SV40 transgenic FVB/N mouse were transplanted into the eyes of syngeneic FVB/N mice and the TIL isolated. TIL were assessed for direct cytolytic activity in vitro. TIL were also transferred passively to immunosuppressed FVB/N mice to determine if they could mediate intraocular tumor rejection. The role of CD4+ and CD8+ T cells in intraocular tumor rejection was evaluated by depleting the respective cell populations in FVB/N hosts prior to intraocular tumor challenge. The results showed that intraocular tumors undergoing rejection in immunocompetent syngeneic hosts became infiltrated with T cells, with the CD8+ subset predominating at the time of rejection. By contrast, athymic nude mice did not reject the intraocular tumors nor did the tumors become infiltrated with TIL. TIL displayed direct, tumor-specific cytolytic activity immediately after isolation from the tumor-containing eyes. FVB/N hosts depleted of CD4+ T cells were unable to reject their intraocular tumors. In vivo depletion of CD8+ T cells delayed, but did not prevent tumor rejection. Adoptively transferred TIL mediated swift rejection of intraocular tumors in immunoincompetent recipients. Recipients of TIL, but not recipients of normal spleen cells, acquired significant tumor-specific CTL activity that was demonstrable in vitro. The results strongly suggest, but do not prove, that TIL mediate rejection of intraocular tumors from transgenic mice by direct cytolysis. Although CD4+ T cells are necessary for tumor rejection and are capable of direct cytolysis, the predominant effector cells are CD8+ CTL.


Assuntos
Neoplasias Oculares/imunologia , Rejeição de Enxerto/imunologia , Linfócitos do Interstício Tumoral/imunologia , Animais , Câmara Anterior/imunologia , Linfócitos T CD4-Positivos/imunologia , Neoplasias Oculares/patologia , Feminino , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/patologia , Linfócitos do Interstício Tumoral/transplante , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Células Tumorais Cultivadas
13.
Thromb Haemost ; 71(1): 124-8, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8165630

RESUMO

Platelet-rich clots are inefficiently lysed by current fibrinolytic agents. Platelets contain a great deal of plasminogen activator inhibitor 1 (PAI-1), the principal endogenous inhibitor of tissue-type plasminogen activator (t-PA). We have tested whether PAI-1 resistant t-PAs would be more effective thrombolytic agents in an in vitro model of platelet-rich clots. Clots were formed with recalcified human plasma without or with the addition of platelets. The lysis of these clots was followed by the release of incorporated 125I-fibrinogen. Mutant and wild-type t-PA were almost equally effective against clots lacking platelets but the mutant was twice as effective at lysing platelet-rich clots. A mechanism for this effect is suggested by the demonstration that a complex between wild-type t-PA and extruded platelet contents resembles that between purified t-PA and PAI-1 and that the PAI-1 resistant t-PA does not interefer with formation of this adduct. Because of its enhanced ability to lyse platelet-rich clots in vitro, further in vivo work may find that PAI-1 resistant t-PA is a more efficacious therapeutic agent than wild-type t-PA in situations where platelets contribute to the failure of thrombolysis.


Assuntos
Fibrinolíticos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Fibrinólise/efeitos dos fármacos , Humanos , Engenharia de Proteínas , Ativador de Plasminogênio Tecidual/genética
14.
Cell ; 75(4): 717-28, 1993 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-7902213

RESUMO

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.


Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico/metabolismo , Oligopeptídeos/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Chaperoninas , Sequência Consenso , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Biblioteca Gênica , Técnicas In Vitro , Inovirus/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Ligação Proteica , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
15.
Science ; 262(5132): 419-21, 1993 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-8211162

RESUMO

Unlike most serine proteases of the chymotrypsin family, tissue-type plasminogen activator (tPA) is secreted from cells as an active, single-chain enzyme with a catalytic efficiency only slightly lower than that of the proteolytically cleaved form. A zymogenic mutant of tPA has been engineered that displays a reduction in catalytic efficiency by a factor of 141 in the single-chain form while retaining full activity in the cleaved form. The residues introduced in the mutant, serine 292 and histidine 305, are proposed to form a hydrogen-bonded network with aspartate 477, similar to the aspartate 194-histidine 40-serine 32 network found to stabilize the zymogen chymotrypsinogen.


Assuntos
Precursores Enzimáticos/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/química , Sequência de Bases , Catálise , Quimotripsina/química , Quimotripsina/metabolismo , Precursores Enzimáticos/química , Histidina/química , Ligação de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Serina/química , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/genética
16.
J Biol Chem ; 268(17): 12730-5, 1993 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8509407

RESUMO

The molecular chaperone BiP purified from bovine liver (bBiP) exhibits a low basal level of ATPase activity that can be stimulated 3-6-fold by synthetic peptides (Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390). By contrast, recombinant murine BiP (rBiP) purified to homogeneity following expression in Escherichia coli exhibits a higher basal level of ATPase activity and is much less stimulated by synthetic peptides. Nondenaturing gel electrophoresis showed that rBiP is predominantly monomeric, while bBiP exists in multiple forms probably corresponding to differentially modified monomeric, dimeric, and higher oligomeric species. Some, but not all, synthetic peptides cause conversion of the oligomeric and modified species of bBiP to a monomeric form. We propose that the peptide-dependent ATPase stimulation observed for BiP reflects the conversion of inactive oligomeric and/or modified species into active monomers.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Clonagem Molecular , DNA Recombinante/isolamento & purificação , DNA Recombinante/metabolismo , Eletroforese em Gel de Poliacrilamida , Chaperona BiP do Retículo Endoplasmático , Escherichia coli/genética , Cinética , Substâncias Macromoleculares , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
17.
J Virol ; 66(12): 7136-45, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1331514

RESUMO

The contribution of each of the seven asparagine-linked oligosaccharide side chains on the hemagglutinin of the A/Aichi/68 (X31) strain of influenza virus was assessed with respect to its effect on the folding, intracellular transport, and biological activities of the molecule. Twenty mutant influenza virus hemagglutinins were constructed and expressed, each of which had one or more of the seven glycosylation sites removed. Investigations of these mutant hemagglutinins indicated that (i) no individual oligosaccharide side chain is necessary or sufficient for the folding, intracellular transport, or function of the molecule, (ii) at least five oligosaccharide side chains are required for the X31 hemagglutinin molecule to move along the exocytic pathway to the plasma membrane, and (iii) mutant hemagglutinins having less than five oligosaccharide side chains form intracellular aggregates and are retained in the endoplasmic reticulum.


Assuntos
Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Vírus da Influenza A/genética , Dobramento de Proteína , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Linhagem Celular , DNA Viral/genética , DNA Viral/metabolismo , Eritrócitos/metabolismo , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Vírus da Influenza A/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus 40 dos Símios/genética , Proteínas do Envelope Viral/genética
18.
Biochem J ; 286 ( Pt 1): 275-80, 1992 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-1520279

RESUMO

Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis.


Assuntos
Ativador de Plasminogênio Tecidual/biossíntese , Sequência de Aminoácidos , Animais , Sistema Livre de Células , Cães , Glicosilação , Humanos , Cinética , Microssomos/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pâncreas/metabolismo , Biossíntese de Proteínas , Conformação Proteica , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/isolamento & purificação , Transcrição Gênica
19.
Proc Natl Acad Sci U S A ; 89(16): 7422-6, 1992 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-1502153

RESUMO

Tissue-type plasminogen activator and urokinase are serine proteases secreted by many cell types that participate in biological processes, such as tissue restructuring, cell migration, and tumor metastasis. Clinically, these proteases are used to dissolve coronary fibrin clots that are the proximal causes of acute myocardial infarction. In vivo, the activity of these enzymes is controlled by plasminogen-activator inhibitors, members of the serpin family of protease inhibitors. This study shows that tissue-type plasminogen activator-inhibitor complexes bind in solution to low density lipoprotein receptor-related protein (LRP), a large heterodimeric ubiquitous membrane receptor. In cultured cells, endocytosis and degradation of these complexes is reduced by polyclonal antibodies directed against LRP and inhibited by a M(r) 39,000 protein that binds to LRP and inhibits its interaction with previously known ligands, including apolipoprotein E and alpha 2-macroglobulin. We propose a role for LRP in the clearance of plasminogen activator-inhibitor complexes that is analogous to its function in the endocytosis of alpha 2-macroglobulin-protease complexes.


Assuntos
Endocitose , Inativadores de Plasminogênio/metabolismo , Receptores Imunológicos/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Anticorpos Monoclonais , Linhagem Celular , Immunoblotting , Cinética , Neoplasias Hepáticas Experimentais , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
20.
J Cell Biol ; 118(2): 227-44, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1321156

RESUMO

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.


Assuntos
Hemaglutininas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Linhagem Celular , Clonagem Molecular , Dissulfetos/metabolismo , Escherichia coli/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Conformação Proteica , Vírus 40 dos Símios/genética , Transfecção , Proteínas do Envelope Viral/metabolismo
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