α-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012–37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that α-actinin expressed identical to the cytoskeletal/non-muscle isoform. A construct this isoform containing a His6 tag at amino terminus was generated. Robust tyrosine phosphorylation recombinant protein detected cells treated...

Productive infections of oncogenic human papillomaviruses (HPVs) are closely linked to the differentiation host epithelial cells, a process that virus manipulates create conditions favorable produce virion progeny. This viral interference involves altering expression numerous genes. Among these, proprotein convertases (PCs) have emerged as potential oncogenes due their central role in cellular functions. Using RT-qPCR, aberrant PC gene was detected across progression from early HPV infection...

Proteases rely on their active sites for substrate specificity, but these have inherent limitations that impact enzymatic efficiency and regulation. Exosites cofactors help overcome constraints by enhancing the protease's interactions, inhibition. Recent research Gangemi et al. highlights role of exosites in regulating inhibition protease neutrophil elastase serpin alpha-1-antitrypsin. Understanding mechanisms is crucial developing therapeutic applications. Advances computational analysis...

Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin tyrosine phosphorylated platelets spread on fibrinogen the phosphorylation Src kinases dependent. The of was reconstituted vanadate treated COS-7 cells coexpressing c-Src. sites were mapped to residues 100 1065. A phosphorylation-specific antibody directed against residue 1065 reacted with platelet but failed react from unstimulated lysates. Tyrosine located tail...

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, octanaldehyde from four human livers three kidneys were done to identify retinaldehyde their relationship other aldehydes. The tissue fractionation patterns liver kidney the same, indicating presence same in kidney. Moreover, both organs major NAD+-dependent activity copurified with propionaldehyde activities; was associated E1 isozyme (coded for byaldh1 gene) aldehyde dehydrogenase. A small amount E2...

We have previously shown that exosites in antithrombin outside the P6-P3′ reactive loop region become available upon heparin activation to promote rapid inhibition of target proteases, factor Xa and IXa. To identify these exosites, we prepared six antithrombin-α1-proteinase inhibitor chimeras which residues 224-286 310-322 circumscribe a surrounding on surface were replaced 10-16-residue segments with homologous α1-proteinase inhibitor. All bound high affinity similar wild-type, underwent...

We previously showed that conformational activation of the anticoagulant serpin, antithrombin, by heparin generates new exosites in strand 3 beta-sheet C, which promote reaction inhibitor with target proteases, factor Xa and IXa. To determine residues comprise exosites, we mutated 3C are conserved all vertebrate antithrombins. Combined mutations three surface-accessible residues, Tyr253,Glu255, Lys257, or just Tyr253 Glu255, but not any these alone, was sufficient to reproduce exosite...

Heparin activates the serpin, antithrombin, to inhibit its target blood-clotting proteases by generating new protease interaction exosites. To resolve effects of these exosites on initial Michaelis docking step and subsequent acylation conformational change steps antithrombin-protease reactions, we compared reactions catalytically inactive S195A active with site-specific fluorophore-labeled antithrombins that allow monitoring reaction steps. bound...

The integrin αIIbβ3 mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified tyrosine-phosphorylated from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence an internal 12-mer peptide derived this protein. was identical to human α-actinin sequences deposited Swiss Protein Database. α-Actinin, platelets, subsequently by four sequential chromatographic steps. Fractions were analyzed Western blotting...

Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which major serpin conformational change, resulting 70-Å translocation of the from its initial reactive center loop docking site to opposite pole serpin, kinetically traps acyl-intermediate complex. Although Michaelis and final trapped complexes have been well characterized structurally, intermediate stages involved this remarkable transformation are not understood. To better characterize such...

We have previously shown that residues Tyr-253 and Glu-255 in the serpin antithrombin function as exosites to promote inhibition of factor Xa IXa when is conformationally activated by heparin. Here we show functional can be engineered at homologous positions a P1 Arg variant alpha1-proteinase inhibitor (alpha1PI) does not require heparin for activation. The combined effect two increased association rate constant reactions alpha1PI with factors 11-14-fold, comparable their rate-enhancing...

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result very large increases the rates of inhibition factors IXa and Xa but not thrombin. These are accompanied CD, fluorescence, NMR spectroscopic changes. X-ray structures show that results extension helix D region 131–136 with coincident, possibly coupled, expulsion hinge reactive center loop. To examine importance extension, we have introduced strong helix-promoting mutations (YRKAQK to...

The subtilin leader segment of presubtilin was fused to alkaline phosphatase (AP), which used as a reporter polypeptide study the role in posttranslational modifications during conversion and translocation from cytoplasm Bacillus subtilis 168 extracellular medium. It observed that could be utilized by wild-type transporter, but secretion enhanced if transporter available. not cleaved away AP component precursor until had been transported cell wall, none released into medium after cleavage...

A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion native to latent (Larsson, H., Akerud, P., Nordling, K., Raub-Segall, E., Claesson-Welsh, L., Björk, I. (2001) J. Biol. Chem. 276, 11996-12002). Here, we show previously characterized a mixture modified, true are resolvable by heparin-agarose chromatography. Kinetic analyses revealed an intermediate in whose formation favored stabilizing...

Heparin allosterically activates the anticoagulant serpin, antithrombin, by binding through a sequence-specific pentasaccharide and inducing activating conformational changes in protein. Three basic residues of Lys114, Lys125, Arg129, have been shown to be hotspots for pentasaccharide, but molecular basis such hotspot has unclear. To determine whether this results from cooperative interactions, we analyzed effects single, double, triple mutations on activation antithrombin. Double-mutant...

The molecular determinants of substrate specificity and selectivity in the proprotein convertase (PC) family proteases are poorly understood. Here we demonstrate that natural serpin inhibitor, B8, is a specific selective inhibitor furin relative to other PCs constitutive protein secretion pathway, PC4, PC5, PACE4, PC7 (PC4–PC7, respectively), identify reactive-site (P6–P5′ residues) exosite elements contribute this through studies chimeras B8 α1PDX, an engineered furin. Kinetic revealed for...