Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0033036 (APC)
 10,214 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF) epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 gamma-carboxy glutamic acid residues in the vitamin K-dependent domain, a beta-hydroxylated aspartic acid in the first EGF-like domain and a beta-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical alpha-chains and one beta-chain. The alpha- and beta-chains are linked by disulphide bridges. The cDNA cloning of the beta-chain showed the alpha- and beta-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the beta-chain to contain the single protein S binding site on C4BP, whereas each of the alpha-chains contains a binding site for the complement protein, C4b. As C4BP lacking the beta-chain is unable to bind protein S, the beta-chain is required for protein S binding, but not for the assembly of the alpha-chains during biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Protein S and C4b-binding protein: components involved in the regulation of the protein C anticoagulant system. 183 51

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm2), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By 125I-EGF binding analysis and [35S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8-10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2-3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity.
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PMID:Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity. 810 61

The cDNA encoding a chimeric human protein C (PC), in which its epidermal growth factor-(EGF) like regions have been replaced with equivalent structures from human factor IX (fIX), was constructed and the gene product was expressed in human 293 cells. A molecular subpopulation of the recombinant chimeric protein (r-[PC/delta EGF-1,2/delta fIXEGF-1,2]) was purified that contained the full complement (9 residues/mol) of gamma-carboxyglutamic acid (Gla). After conversion by thrombin to its activated form (r-[APC/delta EGF-1,2/delta fIXEGF-1,2]), this latter enzyme was found to possess approximately 10% of the activity of wild-type recombinant APC (wtr-APC) in an APTT assay. In assay systems employing purified components, the activity of the mutant enzyme toward prothrombinase cofactor Va (fVa) and tenase cofactor VIII (fVIII) was approximately 30% and < 10%, respectively, of that of wtr-APC. The chimeric protein displayed full reactivity with a Ca(2+)-dependent monoclonal antibody to the Gla domain of PC, yielding a C50 for Ca2+ that was very similar to that obtained with wtr-PC (ca. 3.7 mM). Titrations of the dependency on Ca2+ of the intrinsic fluorescence of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] allowed calculation of a C50 value of 0.34 mM, again very similar to that of wtr-PC. As with wtr-PC, Ca2+ inhibited the thrombin-catalyzed activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] with aKi of 148 microM, as compared to a Ki of 125 microM for wtr-PC. At a saturating level of Ca2+, activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2/] by the thrombin/thrombomodulin (thrombin/TM) complex occurred at approximately 70% of the rate of that of wtr-PC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Construction, expression, and properties of a recombinant chimeric human protein C with replacement of its growth factor-like domains by those of human coagulation factor IX. 829 11

In this study we sought factors that determine the survival of human colonic epithelial cells. Normal colonic epithelial cells are dependent on cell-cell contacts and survival factors for the inhibition of apoptosis whereas, during colorectal tumorigenesis, cells develop mechanisms to evade these controls. The ability to survive loss of cell-cell contacts and/or growth factor deprivation is a marker of tumour progression. Many adenoma (premaligant) cultures survive only if cell-cell contacts are maintained in vitro and die by apoptosis if trypsinized to single cells. This also occurs in adenomas derived from familial adenomatous polyposis (FAP) patients, therefore APC mutations do not confer resistance to cell death in response to loss of cell-cell contacts. We show here that if cell-cell contacts are maintained such cells are capable of survival in suspension. Adenoma cells also undergo apoptosis in response to removal of serum and growth factors from the medium. After removal of serum and growth factors c-myc is down-regulated within 2 h. Therefore, the induction of apoptosis is not an inappropriate response of the cells due to a deregulated c-myc gene. The apoptotic response is also p53 independent. Such cultures have been used to determine specific survival factors for colonic epithelial cells. Insulin, the insulin-like growth factors I and II, hydrocortisone and epidermal growth factor (EGF) protect cells from the induction of apoptosis in the absence of serum over a short-term period of 24 h. This approach may give insight into the factors governing growth and survival of colonic epithelial cells in vivo. This is the first report of specific growth factors protecting against apoptosis in human colonic epithelial cells.
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PMID:Cell-cell contact and specific cytokines inhibit apoptosis of colonic epithelial cells: growth factors protect against c-myc-independent apoptosis. 908 30

Variant proteins containing charge-to-alanine mutations of single amino acid residues and clusters of such groups contained in the epidermal growth factor 1 (EGF1) homology unit of human protein C (PC) have been accomplished, resulting in the following recombinant (r) mutant proteins: r-[E56A/H57A]PC; r-[H66A]PC; r-[D71A]PC; r-[D79A/R81A]PC; r-[E85A/R87A]PC; and r-[R91A/E92A]PC. Studies of the mutant proteins with a variety of Ca2+-dependent and Ca2+-independent monoclonal antibodies not only led to identification of the epitopes of these antibodies, but also confirmed the importance of D/beta-hydroxyaspartic acid (Hya)71 as one probable coordination site for Ca2+. Employing these antibodies, it was also revealed that Ca2+ binding to its site in the EGF1 region of PC did not influence Ca2+ binding or adoption of the Ca2+-dependent conformation of the gamma-carboxyglutamic acid domain of this same protein. In addition, the Ca2+-induced inhibition of PC activation by thrombin, and the kinetic constants for activation of PC by the thrombin/thrombomodulin complex, were only modestly affected by any of the mutations. The mutants r-[E56A/H57A]APC and r-[H66A]APC displayed at least 70% of wild type r-APC activity in a fVIII inactivation assay, while r-[D79A/R81A]APC, r-[E85A/R87A]APC and r-[R91A/E92A]APC possessed only approximately 40% activity in that same assay. The special role of D/Hya71 in this process was confirmed by showing that r-[D71A]APC was inactive in the fVIII-inactivation assay. These findings demonstrate that some of the charged residues of EGF1, most notably those in the carboxy-terminal region of this domain, participate as partial determinants of the anticoagulant activity of APC. Overall, with the exceptions noted, the data generally suggest that the charged residues of the EGF1 domain of PC, and the Ca2+ binding site contained within this module, are likely more involved with maintenance of the overall structural integrity of this module rather than with its direct functional interactions with effectors, activators, or substrates of PC and APC. Lastly, functional Ca2+ binding to the Gla domain of PC is not significantly influenced by the binding of Ca2+ to the EGF1 module.
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PMID:The functions of the first epidermal growth factor homology region of human protein C as revealed by a charge-to-alanine scanning mutagenesis investigation. 946 48

The soluble thrombomodulin (TM) subspecies in human urine detected by polyclonal anti-human TM IgG were isolated and characterized. 105, 85, 80, 56, 33, 31 and 28 kDa subspecies under reducing conditions was comparable to 78, 66, 56, 200, 52, 30 and 25 kDa under non-reducing conditions, respectively, in the two-dimensional electrophoresis. Each subspecies under non-reducing conditions, except the 200 and 52 kDa molecules, was constituted of single subspecies, whereas the 200 and 52 kDa molecules were constituted of the tetramer of the 56 kDa subspecies of reducing conditions and a dimer of the 33 kDa subspecies, respectively. NH2-terminal amino acid sequences of the 105, 85 and 80 kDa subspecies maintained Ala1-Pro2-Ala3- of intact human TM, however, 56, 33, 31 and 28 kDa subspecies started from Glu137-Gln138-, Gln214-Gly215-, Ser228-Val229- and Ala240-Ile241-, respectively. All subspecies obtained under non-reducing conditions exhibited cofactor activity for thrombin-dependent protein C activation ranging from 58 to 162 pmol APC/min/nmol TM at 0.4 mM Ca2+ indicating that all of the subspecies maintained the fourth to sixth repeat of epidermal growth factor-like structure of intact TM. 85, 80, 56, 33, 31 and 28 kDa subspecies were suggested to lack both chondroitin sulfate glycosaminoglycan (CSGAG), transmembrane and cytoplasmic domains of intact TM, while 105 kDa subspecies lack only CSGAG from the results of kinetic properties and the interaction with phospholipid vesicles composed from phosphatidylcholine and phosphatidylethanolamine.
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PMID:Characterization of soluble thrombomodulin fragments in human urine. 949 86

Despite the localization of several human diseases to 11q13, the majority of the genes responsible for these disorders have not yet been cloned. Exon amplification and EST mapping were performed using clones derived from an approximately 1.65-Mb P1 artificial chromosome contig encompassing the region that reportedly harbors the gene mutated in the dominantly inherited eye disorder, Best disease. Fifty-eight exons isolated from the region were sequenced, resulting in 41.3% showing weak or no similarity to database sequences. Four exons had exact matches with human ESTs and 2 exons were highly similar to mouse ESTs. The sequence of 1 of these human ESTs was highly similar to that of the rat Rabin3 and mouse Pat-12 genes, which potentially encode Ras-like GTPase binding proteins. Three exon sequences were similar to those of the inner centromere proteins of Gallus gallus and Xenopus laevis, which are mitotic phosphoproteins, and 1 exon sequence had similarity to the epidermal growth factor-like repeat from several proteins. High-resolution mapping of 34 ESTs binned to the 11q12-q13 region by the Human Transcript Mapping Project identified 5 present in the PAC contig, with 1 of these ESTs identifying a human homologue of the rat synaptotagmin VII gene. Database searches identified two overlapping cDNA clones representing almost the entire open reading frame of this human gene and a sequenced cosmid indicating its partial genomic structure. Further database analyses identified another sequenced cosmid from this region that contained both exon-trap and mapped EST sequences. PowerBLAST and GRAIL analysis of this cosmid sequence identified matches with several other ESTs, the previously described FEN1 gene, and a novel evolutionarily conserved gene. These experiments identify candidate genes for disorders that map to this region and indicate that this is a gene-rich region of the human genome.
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PMID:Transcript mapping of the human chromosome 11q12-q13.1 gene-rich region identifies several newly described conserved genes. 961 27

Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.
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PMID:Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling. 1022 94

Due to its increasing incidence, esophageal adenocarcinoma and its precursor lesions have received increasing attention in recent years. The histopathologic steps in the process of malignant progression in Barrett's esophagus are well described and include the following: (a) metaplasia of the normal esophageal squamous epithelium to a specialized intestinal glandular epithelium, (b) development of dysplasia (classified histologically as low and high grade), and (c) development of adenocarcinoma characterized by invasive and metastatic potential. Intestinal metaplasia can be identified by the presence of goblet cells, the detection of which can be aided by finding mucin stained by Alcian blue at low pH. Despite this well-characterized sequence, the timing of the development of dysplasia and the subsequent transition to carcinoma and the risk of development of carcinoma in low- and high-grade dysplasia are not precisely known. In addition, there are problems in the identification of dysplasia, including sampling error and interobserver discrepancies among pathologists. A better understanding of the mechanisms of these events would allow early identification and elimination of high-risk lesions before adenocarcinoma with its attendant poor prognosis were able to develop. In order to better understand this process and to potentially identify early markers of malignant transformation, a variety of molecular studies have been carried out in recent years on adenocarcinoma and its precursor lesions in Barrett's esophagus. On the phenotypic level, increased expression and changes in pattern of expression of proliferation marker (Mib-1) Ki-67 antigen, overexpression of p53 protein, overexpression of growth factors such as epidermal growth factor (EGF), c-erbB2, and transforming growth factor (TGF)-a, decreased and abnormal expression of the cell adhesion molecule E-cadherin, and, in carcinomas, increased expression of serine proteases have all been described. A new area of interest is the family of rab proteins, which play an important role in maintaining cell polarity in the gastrointestinal tract. Increased expression of one of these proteins, rab11, has been described in low-grade, but not high-grade dysplasia. In cytogenetic studies, an increased S-phase fraction, followed by an increased tetraploid (4N) fraction and then aneuploidy, has been described. So far, the specific genes which have been most thoroughly investigated have been p53, APC, p16, and the sites of probable tumor suppressor genes, including 3p (FHIT), 13q, and 18q. With only a few exceptions (i.e., rab11 expression, and possibly mutations of FHIT), the numerous molecular abnormalities which have been described occur late in malignant progression, which means that the best marker which presently exists to identify high-risk lesions in Barrett's esophagus is the histologic identification of dysplasia in endoscopic biopsies, especially high-grade dysplasia. We are presently beginning studies using laser microdissection and competitive genomic hybridization (CGH), which could help to identify new chromosomal areas that might contain genes that are crucial in the early phases of malignant progression in Barrett's esophagus. In the future, identification of such early molecular events which predispose to carcinoma development will allow more precise and earlier risk assessment for individual patients, therefore, enabling more effective therapy.
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PMID:Malignant progression in Barrett's esophagus: pathology and molecular biology. 1069 36

The prevalence of Barrett's oesophagus has risen over a short time interval implying environmental in addition to genetic aetiological factors. Bile salt effects from duodenogastro-reflux are assuming increasing importance with deoxycholic and taurodeoxycholic acid being particularly associated with Barrett's oesophagus. The cellular biology changes appear to follow a progression from initial inflammation and oesophagitis to metaplasia and dysplasia through to adenocarcinoma. Mechanisms of restitution include epidermal growth factor mediated increases in epithelial thickness. This results in basal stem cells becoming superficially placed and exposed further to luminal refluxed bile salts. Immature stem cells result which undergo mutation to a metaplastic glandular phenotype with intestinal metaplasia. P53 mutation increasingly occurs in progression to dysplasia and carcinoma and may confer a survival advantage of these cell clones by delaying apoptosis. Cell cycling gene mutations occur with accumulation of cells in G2 phase. Disruption of cellular checkpoint mechanisms in the mitotic process result in loss of heterozygosity and aneuploidy including loss of the Y chromosome. Identical mutations between adjacent areas of dysplasia and adenocarcinoma supports clonal expansion as the mechanism of carcinogenesis. APC tumour suppressor gene mutations are conserved in synchronous carcinomas in Barrett's dysplasia and are associated with beta-catenin accumulation in the nucleus and cellular migration with invasion. Cumulative genetic errors result in abnormal clones with metastatic or angiogenic potential. When a clone with malignant potential occurs adenocarcinoma can result completing the progression from inflammation to metaplasia and dysplasia through to adenocarcinoma.
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PMID:Genetic versus environmental interactions in the oesophagitis-metaplasia-dysplasia-adenocarcinoma sequence (MCS) of Barrett's oesophagus. 1090 14


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