Oxidative stress plays a critical role in the pathogenesis of atherosclerosis including the formation of lipid laden macrophages and the development of inflammation. However, oxidative stress-induced molecular signaling that regulates the development of vascular calcification has not been investigated in depth. Osteogenic differentiation of vascular smooth muscle cells (VSMC) is critical in the development of calcification in atherosclerotic lesions. An important contributor to oxidative stress in atherosclerotic lesions is the formation of hydrogen peroxide from diverse sources in vascular cells. In this study we defined molecular signaling that is operative in the H 2 O 2 -induced VSMC calcification. We found that H 2 O 2 promotes a phenotypic switch of VSMC from contractile to osteogenic phenotype. This response was associated with an increased expression and transactivity of Runx2, a key transcription factor for osteogenic differentiation. The essential role of Runx2 in oxidative stress-induced VSMC calcification was further confirmed by Runx2 depletion and overexpression. Inhibition of Runx2 using short hairpin RNA blocked VSMC calcification, and adenovirus-mediated overexpression of Runx2 alone induced VSMC calcification. Inhibition of H 2 O 2 -activated AKT signaling blocked VSMC calcification and Runx2 induction concurrently. This blockage did not cause VSMC apoptosis. Taken together, our data demonstrate a critical role for AKT-mediated induction of Runx2 in oxidative stress-induced VSMC calcification.Atherosclerosis is characterized by the presence of atherosclerotic lesions in the arterial intima that leads to narrowing of the vessel lumen. Vascular calcification, the presence of calcium deposits in the vessel wall, is a feature of advanced atherosclerosis and reduces elasticity and compliance of the vessel wall (1). Hence, the extent of calcification is a key risk factor in the pathogenesis of the disease. Several cell types, such as endothelium, monocytes, and vascular smooth muscle cells (VSMC), 5 are involved in different stages of lesion development. VSMC contribute to the development of atherosclerotic lesions through increased migration, proliferation, secretion of matrix components, osteogenic differentiation, and the associated calcification (1). During this process, the differentiated VSMC undergo de-differentiation, and subsequently osteogenic transition that results in vascular calcification (2).Many factors that have been linked to an increased prevalence of vascular calcification are associated with elevated oxidative stress, including hypercholesterolemia, hypertension, diabetes mellitus, and dialysis-dependent end stage renal disease (3-6). Pro-oxidant events in atherosclerosis include the production of reactive oxygen species (ROS) and nitrogen species by vascular cells (7). Of particular interest is hydrogen peroxide (H 2 O 2 ), which is a cell-permeable ROS that has emerged as a key mediator of intracellular signaling (8 -10). H 2 O 2 is produced in vascular cells by multiple enzyma...

Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAX proteins to the plasma membrane. Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT. The deduced amino acid sequence of mammalian pc-CMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter. The human gene complemented a ste14 mutant. pcCMT mRNAs were ubiquitously expressed in human tissues. An anti-pc-CMT antiserum detected a 33-kDa protein in myeloid cell membranes. Ectopically expressed recombinant pc-CMT had enzymatic activity identical to that observed in neutrophil membranes. Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum. Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane.A number of signaling molecules, including Ras and G proteins, are targeted to the inner leaflet of the plasma membrane by a sequence of posttranslational modifications of a C-terminal CAAX 1 motif that include prenylation, proteolysis, and carboxyl methylation (1). In some cases palmitoylation of an upstream cysteine is also required (2). These modifications render otherwise hydrophilic proteins hydrophobic, promoting association with membranes. The relative contributions of prenylation, proteolysis, and carboxyl methylation to membrane targeting are not well understood. Whereas neutralization of the negative charge on the ␣-carboxyl group by methyl esterification adds to overall hydrophobicity, particularly for farnesylated proteins, this modification contributes little to the affinity of geranylgeranylated proteins for membranes (3). Although processed CAAX proteins can associate with phospholipid vesicles in vitro (4), it is not known whether membrane proteins participate in prenylcysteine membrane association in vivo. The Saccharomyces cerevisiae mating pheromone, a-factor, is a CAAX-processed polypeptide, and both its secretion via the Ste6p transporter (5) and its engagement of the Ste3p G protein-linked receptor (6) are dependent on prenylcysteine carboxyl methylation, suggesting a role for this modification in protein-protein interactions. A cycle of prenylcysteine carboxyl methylation is associated with neutrophil activation (7), and inhibitors of this enzyme block signal transduction in neutrophils (7), macrophages (8), and platelets (9), suggesting that, like bacterial chemotaxis (10), some eukaryotic processes may be regulated by reversible carboxyl methylation.Because prenylcysteine carboxyl methylation cannot be abolished by mutation of the substrate without also eliminating prenylation, elucidation of the role of carboxyl methylation will require characterization and disruption of the methyltransferase. Until recently, the only prenylcysteine carboxyl methyltransfe...

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (⌬F508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ⌬F508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ⌬F508 CFTR mRNA in the vicinity of the mutation. The consequence of ⌬F508 CFTR mRNA "misfolding" is decreased translational rate. A synonymous single nucleotide variant of the ⌬F508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ⌬F508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ⌬F508 CFTR misfolding. Therefore, we propose that mRNA "misfolding" contributes to ⌬F508 CFTR protein misfolding and consequently to the severity of the human ⌬F508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ⌬F508 homozygous CF patients.

Ethanol (EtOH) is the most widely abused substance in the United States, and it contributes to well-documented harmful (at high dosages) and beneficial (at low dosages) changes in inflammatory and immune responses. Lipid rafts have been implicated in the regulation and activation of several important receptor complexes in the immune system, including the TLR4 complex. Many questions remain about the precise mechanisms by which rafts regulate the assembly of these receptor complexes. Results summarized in this review indicate that EtOH acts by altering the LPS-induced redistribution of components of the TLR4 complex within the lipid raft and that this is related to changes in actin cytoskeleton rearrangement, receptor clustering, and subsequent signaling. EtOH provides an example of an immunomodulatory drug that acts at least in part by modifying lipid rafts, and it could represent a model to probe the relationships between rafts, receptor complexes, and signaling.

The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR) mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

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These data confirm our previous observations, suggest a novel mechanism of EtOH action that involves interference with receptor clustering, and indicate a potential role of actin filaments in the formation of receptor patches, subsequent activation of macrophages by LPS, and production of TNF-alpha.

Athletes in interceptive sports are superior to nonathletes in their visuomotor skills. They also have broader access to various visual and complex visuo-oculomotor abilities than nonathletes. This likely allows athletes to more effectively coordinate visual and oculomotor abilities under demanding conditions when some visual cues are degraded. The present findings are consistent with a pyramid of sports vision and suggest a top-down process for athlete screening and training.