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2A protease of the pathogenic coxsackievirus B3 is key to the pathogenesis of inflammatory myocarditis and, therefore, an attractive drug target. However lack of a crystal structure impedes design of inhibitors. Here we predict 3D structure of CVB3 2Apro based on sequence comparison and homology modeling with human rhinovirus 2Apro. The two enzymes are remarkably similar in their core regions. However they have different conformations at the N-terminal. A large number of N-terminal hydrophobic residues reduce the thermal stability of CVB3 2Apro, as we confirmed by fluorescence, western blot and turbidity measurement. Molecular dynamic simulation revealed that elevated temperature induces protein motion that results in frequent movement of the N-terminal coil. This may therefore induce successive active site changes and thus play an important role in destabilization of CVB3 2Apro structure.

Aortic aneurysm is the leading cause of death in Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 gene. MFS aneurysm is characterized by weakening of the a...

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder presented with multiple cafe-au-lait spots, Lisch nodules,multiple nerve sheath tumors, and cardiovascular complications such as ischemia...

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder, characterized by multiple cafe-au-lait spots, iris harmartomas, multiple nerve sheath tumors. Patients also present with heart and cereb...

Vascular smooth muscle cell (VSMC) dysfunction is implicated in cardiovascular disease and can be attributed to dysregulation of Angiotensin-II (Ang-II) and transforming growth factor-β (TGF-β) sig...

Marfan syndrome (MFS) is an autosomal-dominant disorder of connective tissue caused by mutations in the fibrillin-1 (FBN1) gene. Mortality is often due to aortic dissection and rupture. We investigated the structural and functional properties of the heart and aorta in a [Fbn1C1039G/+] MFS mouse using high-resolution ultrasound (echo) and optical coherence tomography (OCT). Echo was performed on 6- and 12-month old wild type (WT) and MFS mice (n = 8). In vivo pulse wave velocity (PWV), aortic root diameter, ejection fraction, stroke volume, left ventricular (LV) wall thickness, LV mass and mitral valve early and atrial velocities (E/A) ratio were measured by high resolution echocardiography. OCT was performed on 12-month old WT and MFS fixed mouse hearts to measure ventricular volume and mass. The PWV was significantly increased in 6-mo MFS vs. WT (366.6 ± 19.9 vs. 205.2 ± 18.1 cm/s; p = 0.003) and 12-mo MFS vs. WT (459.5 ± 42.3 vs. 205.3 ± 30.3 cm/s; p< 0.0001). PWV increased with age in MFS mice only. We also found a significantly enlarged aortic root and decreased E/A ratio in MFS mice compared with WT for both age groups. The [Fbn1C1039G/+] mouse model of MFS replicates many of the anomalies of Marfan patients including significant aortic dilation, central aortic stiffness, LV systolic and diastolic dysfunction. This is the first demonstration of the direct measurement in vivo of pulse wave velocity non-invasively in the aortic arch of MFS mice, a robust measure of aortic stiffness and a critical clinical parameter for the assessment of pathology in the Marfan syndrome.

The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3beta, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3beta activity by LiCl augmented accumulation of beta-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. Beta-catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of beta-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and beta-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of beta-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3beta pathway via the beta-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.

We present results from a novel study of Ca2+ waves in vascular smooth muscle cells using a sarcoplasmic reticulum (SR)-targeted Ca2+ indicator that specifically binds to the luminal protein calsequestrin.Agonist-stimulated waves of elevated cytoplasmic calcium concentration regulate blood vessel tone, and vasomotion in vascular smooth muscle. Previous studies employing cytoplasmic calcium indicators revealed that these calcium waves are generated by a combination of inositol 1,4,5-trisphosphate (IP3) and calcium-induced calcium release (CICR) from the SR. Our findings confirm that these waves are due to regenerative CICR by the receptors for IP3 (IP3R).The main new finding from our bservations is a transient elevation in luminal SR Ca2+ concentration ([Ca2+]_SR) both at the site of wave initiation, just before regenerative Ca2+ release commences, and at the advancing wave front, during wave propagation. This strongly suggests a role of [Ca2+]_SR in activation of IP3R.In addition, we find that these depletion waves are characterized by a decreasing velocity as they progress. We developed a quantitative diffusional model to analyze this finding and conclude that the gradual decrease in the velocity of the SR Ca2+ depletion wave, observed in the absence of external calcium, indicates continuity of the lumen of the sarcoplasmicreticulum network.Finally, our observation that the depletion wave was arrested by the nuclear envelope may have implications for selective Ca2+ signalling.