Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The thrombin mutant W215A/E217A features a drastically impaired catalytic activity toward chromogenic and natural substrates but efficiently activates the anticoagulant protein C in the presence of thrombomodulin. As the remarkable anticoagulant properties of this mutant continue to be unraveled in preclinical studies, we solved the x-ray crystal structures of its free form and its complex with the active site inhibitor H-d-Phe-Pro-Arg-CH(2)Cl (PPACK). The PPACK-bound structure of W215A/E217A is identical to the structure of the PPACK-bound slow form of thrombin. On the other hand, the structure of the free form reveals a collapse of the 215-217 strand that crushes the primary specificity pocket. The collapse results from abrogation of the stacking interaction between Phe-227 and Trp-215 and the polar interactions of Glu-217 with Thr-172 and Lys-224. Other notable changes are a rotation of the carboxylate group of Asp-189, breakage of the H-bond between the catalytic residues Ser-195 and His-57, breakage of the ion pair between Asp-222 and Arg-187, and significant disorder in the 186- and 220-loops that define the Na(+) site. These findings explain the impaired catalytic activity of W215A/E217A and demonstrate that the analysis of the molecular basis of substrate recognition by thrombin and other proteases requires crystallization of both the free and bound forms of the enzyme.
 ...
PMID:The anticoagulant thrombin mutant W215A/E217A has a collapsed primary specificity pocket. 1525 33

We previously demonstrated expression of full-length transcripts for sublingual mucin apoprotein, Muc19, of approximately 24 kb (Fallon MA, Latchney LR, Hand AR, Johar A, Denny PA, Georgel PT, Denny PC, and Culp DJ. Physiol Genomics 14: 95-106, 2003). We now describe the complete sequence and genomic organization of the apomucin encoded by 43 exons. Southern analyses indicate a central exon of approximately 18 kb containing 36 tandem repeats, each encoding 163 residues rich in serine and threonine. Full-length transcripts are an estimated 22,795 bp in length that span 106 kb of genomic DNA. The transcriptional start site is 24 bp downstream of a TATA box and 42 bp upstream of the conceptual translational start codon. The putative apoprotein has an estimated mass of 693.4 kDa and contains 7,524 amino acids (80% serine, threonine, glycine, alanine, and proline). We present a model for rat Muc19 transcripts and compare the conceptually translated Muc19 proteins for mouse, rat, pig, and the 3' end of human Muc19. Conserved among these apoproteins are a signal peptide, a large tandem repeat region, von Willebrand factor type C and D domains, a trypsin inhibitor-like Cys-rich domain, and a COOH-terminal cystine knot-like domain. Southern blot analyses indicate transcripts for Muc19 and Smgc (submandibular gland protein C) are splice variants of a larger gene, Muc19/Smgc. Comparative Northern analyses between the major salivary glands demonstrate highly selective Muc19 expression in neonatal and adult sublingual glands, whereas Smgc is expressed in neonatal submandibular and sublingual glands. Regulation of Muc19/Smgc gene expression is discussed with respect to alternative splicing and mucous cell cytodifferentiation.
 ...
PMID:The gene encoding mouse Muc19: cDNA, genomic organization and relationship to Smgc. 1534 Jan 21

Cdh1 contributes to proper exit from mitosis and maintenance of G(1) phase in eukaryotic cells by activating a large ubiquitin ligase called the anaphase-promoting complex, or cyclosome (APC/C). At the end of G(1), APC/C(Cdh1) is inhibited by cyclin-dependent kinase (CDK) phosphorylation of Cdh1. The specific Cdh1 phosphorylation sites used to regulate APC/C(Cdh1) activity have not been directly identified. Here, we used a mass spectrometric approach to identify the in vivo phosphorylation sites on yeast Cdh1. Surprisingly, in addition to several expected CDK phosphorylation sites, we discovered numerous nonCDK phosphorylation sites. In total, at least 19 serine and threonine residues on Cdh1 are phosphorylated in vivo. Seventeen of these sites are located in the N-terminal half of Cdh1, outside the highly conserved WD40 repeats. The pattern of phosphorylation was the same when Cdh1 was purified from yeast cultures arrested in S, early M and late M. Mutation of CDK consensus sequences eliminated detectable phosphorylation at many of the nonCDK sites. In contrast, mutation of nonCDK sites had no significant effect on CDK phosphorylation. We conclude that phosphorylation of CDK sites promotes the subsequent recognition of Cdh1 by at least one additional kinase. The function of nonCDK phosphorylation may differ from CDK phosphorylation because mutation of nonCDK sites did not result in constitutive activation of APC and consequent cell cycle arrest. These results suggest that phosphoregulation of APC/C(Cdh1) activity is much more complex than previously thought.
 ...
PMID:Multi-kinase phosphorylation of the APC/C activator Cdh1 revealed by mass spectrometry. 1546 59

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na(+) site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.
 ...
PMID:Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C. 1558 90

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.
 ...
PMID:Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. 1571 Jun 22

Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.
 ...
PMID:Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1. 1571 55

Dysfunction of the protein C anticoagulant system is associated with venous thromboembolism (VTE) and thrombomodulin (TM) is a critical cofactor within the protein C system. The aim of this study was to test the hypotheses that polymorphisms or haplotypes within the TM gene are common risk factors for VTE. We screened the TM putative promoter, exon and 3'-untranslated region for sequence variations in a random sample (n = 266) of consecutive idiopathic, objectively confirmed non-Olmsted County VTE patients referred to the Mayo Clinic. We then genotyped a sample of Olmsted County, MN residents with a first lifetime, objectively confirmed VTE in the 25-year period, 1966-90 (n = 223), and a sample of Olmsted County residents without VTE (n = 237) for polymorphisms either discovered in the screening population or previously published, and tested for an association of VTE with TM genotype or haplotypes using unconditional logistic regression and generalized linear models, respectively. We also genotyped these Olmsted County cases and controls at 20 'null' genetic maker loci and tested for population admixture. Nine novel and three previously described mutations were identified in the screening population. Mutations within the TM promoter, EGF(1-5), serine/threonine-rich, transmembrane, and cytoplasm regions were absent or uncommon. TM845G-->A (Ala25Thr; lectin region), TM2136T-->C (Ala455Val; EGF(6) region), TM2470C deletion (3'-untranslated region), and 4363A-->G (3'-flanking region) were more common, but were not associated with VTE by genotype or haplotype. Null genetic marker allele frequencies did not differ significantly among cases and controls. We conclude that polymorphisms or haplotypes within the TM gene are not common risk factors for incident VTE.
 ...
PMID:Thrombomodulin gene polymorphisms or haplotypes as potential risk factors for venous thromboembolism: a population-based case-control study. 1635 29

Thrombomodulin (TM) is a cell membrane-bound glycoprotein that functions as a thrombin cofactor in the activation of protein C. Its protein structure includes a N-terminal lectin-like domain (D1), 6 epidermal growth factor repeats (D2), a serine-threonine-rich region (D3), a transmembrane domain (D4) and a short cytoplasmic tail (D5). Recent studies have demonstrated the direct effect of TM on cellular proliferation, adhesion and inflammation. In the study, we investigated the role of TM in vascular remodeling and neointima formation in a mouse carotid ligation model. TM expressions on the endothelium, neointima and media were examined in the ligated carotid artery by immunohistochemistry and quantitative real-time reverse transcription PCR. Endothelial TM expression decreased after ligation and appeared later in the media and neointima, which is quite similar to the appearance of TM in the human atherosclerotic process. Recombinant TMD123 was prepared. It was effective for thrombin-dependent protein C activation and the inhibition of leukocyte adhesion to the vessel wall after carotid ligation. Recombinant TMD123 and saline was administered immediately before and after carotid ligation. The TM-treated arteries demonstrated significantly less arterial dilatation (30279 +/- 12605 vs 73789 +/- 15073 microm(2), p < 0.05) in response to less neointima formation (14179 +/- 6538 vs 42227 +/- 8754 microm(2), p < 0.05) at 4 weeks after ligation. Our data indicated that there was a compensatory increase in TM expression in the media and neointima in relation to the reduced endothelial TM after carotid ligation. Early recombinant TM treatment in mice undergoing carotid ligation altered vascular remodeling and decreased the severity of neointima formation.
 ...
PMID:Thrombomodulin plays an important role in arterial remodeling and neointima formation in mouse carotid ligation model. 1654 71

Members of the protein kinase C (PKC) family of serine/threonine kinases play key regulatory roles in numerous cellular processes, including differentiation and proliferation. Of the 11 mammalian PKC isoforms known, several have been implicated in tumor development and progression. However, in most cases, isotype specificity is poorly defined, and even contrary functions for a single PKC have been reported mostly because appropriate molecular and genetic tools were missing to specifically assess the contribution of single PKC isoforms in vivo. In this report, we therefore used PKC genetic targeting to study the role of PKCalpha and PKCzeta in colorectal cancer. Both isoforms were found to be strongly down-regulated in intestinal tumors of ApcMin/+ mice. A deletion of PKCzeta did not affect tumorigenesis in this animal model. In contrast, PKCalpha-deficient ApcMin/+ mice developed more aggressive tumors and died significantly earlier than their PKCalpha-proficient littermates. Even without an additional Apc mutation, PKCalpha knockout mice showed an elevated tendency to develop spontaneous intestinal tumors. Transcriptional profiling revealed a role for this kinase in regulating epidermal growth factor receptor (EGFR) signaling and proposed a synergistic mechanism for EGFR/activator protein and WNT/APC pathways in mediating intestinal tumor development.
 ...
PMID:Protein kinase C alpha but not PKCzeta suppresses intestinal tumor formation in ApcMin/+ mice. 1684 39

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.
 ...
PMID:Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters. 1734 20


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>