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

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.
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PMID:Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition. 1554 57

A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.
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PMID:Novel interaction partners of the TPR/MET tyrosine kinase. 1554 61

Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. The fourth and fifth EGF-like domains of TM together form the minimal fragment with anticoagulant cofactor activity. A short linker connects the fourth and fifth EGF-like domains of TM, and Met 388 in the middle of the linker interacts with both domains. Several different structures of TMEGF45 variants are now available, and these show that mutation of Met 388 alters the structure of the fifth domain, as well as the connectivity of the two domains. To probe this phenomenon more thoroughly, NMR backbone dynamics experiments have been carried out on the individual fourth and fifth domains as well as on the wild type, the Met 388 Leu mutant, and the variant in which Met 388 is oxidized. The results presented here show that changes at Met 388 cause significant changes in backbone dynamics in both the fourth and fifth EGF-like domains of TM. Backbone dynamics within the small loop of the fourth domain Tyr 358 correlate with anticoagulant cofactor activity. Backbone dynamics of the thrombin-binding residues Tyr 413 and Ile 414 are inversely correlated with thrombin binding. The preordering of the backbone of Tyr 413 and Ile 414 only occurs in the two-domain fragments, revealing a role for the fourth domain in thrombin binding as well as in anticoagulant cofactor activity.
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PMID:Dynamics of the fragment of thrombomodulin containing the fourth and fifth epidermal growth factor-like domains correlate with function. 1566 16

Thrombomodulin (TM) is an endothelial cell surface glycoprotein that is responsible for switching the catalytic activity of thrombin away from fibrinogen cleavage (pro-coagulant) and towards protein C cleavage (anticoagulant). Although TM is a large protein, only the fourth and fifth epidermal growth factor-like (EGF-like) domains are required for anticoagulant function. These two domains must work together, and the linker between the two domains contains a single methionine residue, Met 388. Oxidation of Met 388 is deleterious for TM activity. Structural studies, both X-ray and NMR, of wild type and variants at position 388 show that Met 388 provides a key linkage between the two domains. Oxidation of the methionine has consequences for the structure of the fifth domain, which binds to thrombin. Oxidation also appears to disrupt the interdomain contacts resulting in structural and dynamic changes. The functional consequences of oxidation of Met 388 include decreased anticoagulant activity. Oxidative stress from several causes is reflected in lower serum levels of activated protein C and a higher thrombotic tendency, and this is thought to be linked to the oxidation of Met 388 in TM. Thus, TM structure and function are altered in a subtle but functionally critical way upon oxidation of Met 388.
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PMID:Structural and functional consequences of methionine oxidation in thrombomodulin. 1568 Feb 22

Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolic events. Aim of this study was to examine the relationship of hyperhomocysteinemia and thrombosis in IBD patients and to assess the role of this factor in addition to other known prothrombotic abnormalities. IBD patients with a history of thrombosis (n = 22) and sex-, age-, and diagnosis-matched IBD controls (n = 23) were studied. Homocysteine (tHcy) was assessed before and after methionine loading. Plasma levels of protein C, protein S, antithrombin III, and fibrinogen and the presence of anticardiolipin and antiphospholipid antibodies were determined and genetic testing for factor V Leiden and the prothrombin gene mutation was performed. Results showed that fasting homocysteine levels in IBD patients with a history of arterial or venous thrombosis tended to be higher than in IBD controls, although not significantly. The increase in homocysteine levels after methionine loading was significantly higher in IBD patients in the arterial thrombosis group than in IBD controls (40.9 +/- 17.7 vs. 27.2 +/- 9.9 microM; P < 0.05). Among the other prothrombotic factors, only factor V Leiden was significantly associated with a history of venous thrombosis (20 vs. 0%). At least one risk factor was found in 64% of the IBD patients with previous thromboembolic complications. We conclude that there is an association between hyperhomocysteinemia and a history of arterial thrombosis in IBD patients. We confirm the high prevalence of factor V Leiden in IBD patients with a history of venous thrombosis. In the majority of IBD patients with previous thromboembolic complications, at least one prothrombotic risk factor is detected.
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PMID:Risk factors for thromboembolic complications in inflammatory bowel disease: the role of hyperhomocysteinaemia. 1574 78

The temporal control of mitotic protein degradation remains incompletely understood. In particular, it is unclear why the mitotic checkpoint prevents the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of cyclin B and securin in early mitosis, but not cyclin A. Here, we show that another APC/C substrate, NIMA-related kinase 2A (Nek2A), is also destroyed in pro-metaphase in a checkpoint-independent manner and that this depends on an exposed carboxy-terminal methionine-arginine (MR) dipeptide tail. Truncation of the Nek2A C terminus delays its degradation until late mitosis, whereas Nek2A C-terminal peptides interfere with APC/C activity in an MR-dependent manner. Most importantly, we show that Nek2A binds directly to the APC/C, also in an MR-dependent manner, even in the absence of the adaptor protein Cdc20. As similar C-terminal dipeptide tails promote direct association of Cdc20, Cdh1 and Apc10-Doc1 with core APC/C subunits, we propose that this sequence also allows a substrate, Nek2A, to directly bind the APC/C. Thus, although Cdc20 is required for the degradation of Nek2A, it is not required for its recruitment and this renders its degradation insensitive to the mitotic checkpoint.
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PMID:Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C. 1664 45

The ability of peptides to form stable complexes with MHC class II molecules expressed in the host determines their ability to recruit CD4 T cells during an immune response. In this study, we sought to define the features of the antigenic peptides that control their kinetic stability with I-A(d) because of the diversity of peptides that this molecule is known to present. Peptide dissociation assays indicated that each pocket of I-A(d) displays exquisite sensitivity to side chain structure, size, and charge. Most surprising were results related to the P1 pocket, which has been difficult to define by conventional competition assays. Our studies revealed a considerable degree of specificity in the P1 pocket but also an unexpected degree of structural flexibility. Amino acids with neutral side chains such as Met and the alternatively negatively charged Glu are both highly favored at P1. Interestingly, these two options at the P1 pocket in I-A(d) display dramatically different pH-dependent interactions with the class II molecule. These findings are discussed in the context of a structural model to explain these data and in light of the immunological implications of pH-dependent behavior of class II-peptide complexes in acidic endosomal compartments, where DM-catalyzed loading of class II molecules takes place, and at the neutral pH of the APC cell surface, where class II-peptide complexes promote activation of CD4 T cells.
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PMID:Peptide-binding motifs for the I-Ad MHC class II molecule: alternate pH-dependent binding behavior. 1670 May 53

Hyperhomocysteinemia is a risk factor for thrombosis, but the mechanisms are not well defined. We tested the hypothesis that hyperhomocysteinemia accelerates arterial thrombosis in mice. Mice heterozygous for a targeted disruption of the cystathionine beta-synthase gene (Cbs+/-) and wild-type littermates (Cbs+/+) were fed either a control diet or a high methionine/low folate (HM/LF) diet for 6 to 8 months to produce graded hyperhomocysteinemia. The time to occlusion of the carotid artery after photochemical injury was shortened by more than 50% in Cbs+/+ or Cbs+/- mice fed the HM/LF diet (P < .001 versus control diet). Carotid artery thrombosis was not accelerated in mice deficient in endothelial nitric oxide synthase (Nos3), which suggests that decreased endothelium-derived nitric oxide is not a sufficient mechanism for enhancement of thrombosis. Cbs+/+ and Cbs+/- mice fed the HM/LF diet had elevated levels of reactive oxygen species in the carotid artery, increased aortic expression of the NADPH oxidase catalytic subunit, Nox4, and decreased activation of anticoagulant protein C in the aorta (P < .05 versus control diet). We conclude that hyperhomocysteinemia enhances susceptibility to arterial thrombosis through a mechanism that is not caused by loss of endothelium-derived nitric oxide but may involve oxidative stress and impairment of the protein C anticoagulant pathway.
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PMID:Enhanced susceptibility to arterial thrombosis in a murine model of hyperhomocysteinemia. 1680 15

The conversion of the reactive center bond of the serpin alpha1-proteinase inhibitor (alpha1-PI, also known as alpha1-antitrypsin) from Met-Ser to Arg-Ser decreases the rate at which it inhibits neutrophil elastase and endows it with the ability to inhibit thrombin and activated protein C (APC). Another serpin, heparin cofactor II (HCII), contains a unique N-terminal extension that binds thrombin exosite 1. We fused residues 1-75 of HCII to the N-terminus of alpha1-PI M358R, forming an HCII-alpha1-PI chimera (HAPI M358R). It inhibited alpha-thrombin 21-fold faster than alpha1-PI M358R, with second-order rate constants of 2.3 x 10(8) M(-1) min(-1) versus 1.1 x 10(7) M(-1) min(-1), respectively. When gammaT-thrombin, which lacks an intact exosite 1, was substituted for alpha-thrombin, the kinetic advantage of HAPI M358R over alpha1-PI M358R was reduced to 9-fold, whereas APC and trypsin, proteases lacking exosite 1-like regions, were inhibited only 1.3- and 2-fold more rapidly by HAPI M358R than by alpha1-PI M358R, respectively. Maximal enhancement of alpha1-PI M358R activity required the acidic residues found between HCII residues 55 and 75, because no enhancement was observed either by fusion of residues 1-54 alone or by fusion of a mutated HCII acidic extension in which all Glu and Asp residues between positions 55 and 75 were neutralized by mutation. Fusing residues 55-75 to alpha1-PI M358R yielded a relative rate enhancement of only 6-fold, suggesting a need for the full tail region to achieve maximal enhancement. Our results suggest that transfer of the N-terminal acidic extension of HCII to alpha1-PI M358R enhanced its inhibition of thrombin by conferring the ability to bind exosite 1 on HAPI M358R. This enhancement may aid in efforts to tailor this inhibitor for therapeutic use.
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PMID:The transferable tail: fusion of the N-terminal acidic extension of heparin cofactor II to alpha1-proteinase inhibitor M358R specifically increases the rate of thrombin inhibition. 1698 4

The leading cause of premature death in smokers is cardiovascular disease. Diabetics also suffer from increased cardiovascular disease. This results, in part, from the hypercoagulable state associated with these conditions. However, the molecular cause(s) of the elevated risk of cardiovascular disease and the prothrombotic state of smokers and diabetics remain unknown. It is well known that oxidative stress is increased in both conditions. In smokers, it is established that oxidation of methionine residues takes place in alpha(1)-antitrypsin in lungs and that this leads to emphysema. Thrombomodulin is a key regulator of blood clotting and is found on the endothelium. Oxidation of methionine 388 in thrombomodulin is known to slow the rate at which the thrombomodulin-thrombin complex activates protein C, a protein which, in turn, degrades the factors which activate thrombin and lead to clot formation. In analogy to the cause of emphysema, it is hypothesized that oxidation of this methionine is elevated in smokers relative to non-smokers and, perhaps, in conditions such as diabetes that impose oxidative stress on the body. Evidence for the hypothesis that such an oxidation and concomitant reduction in activated protein C levels would lead to elevated cardiovascular risk is presented.
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PMID:Does the oxidation of methionine in thrombomodulin contribute to the hypercoaguable state of smokers and diabetics? 1706 53


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