Noncanonical Role of Telomerase in Regulation of Microvascular Redox Environment With Implications for Coronary Artery Disease.

Telomerase reverse transcriptase (TERT) (catalytic subunit of telomerase) is linked to the development of coronary artery disease (CAD); however, whether the role of nuclear vs. mitchondrial actions of TERT is involved is not determined. Dominant-negative TERT splice variants contribute to decreased mitochondrial integrity and promote elevated reactive oxygen species production. We hypothesize that a decrease in mitochondrial TERT would increase mtDNA damage, promoting a pro-oxidative redox environment. The goal of this study is to define whether mitochondrial TERT is sufficient to maintain nitric oxide as the underlying mechanism of flow-mediated dilation by preserving mtDNA integrity.Immunoblots and quantitative polymerase chain reaction were used to show elevated levels of splice variants α- and ß-deletion TERT tissue from subjects with and without CAD. Genetic, pharmacological, and molecular tools were used to manipulate TERT localization. Isolated vessel preparations and fluorescence-based quantification of mtH2O2 and NO showed that reduction of TERT in the nucleus increased flow induced NO and decreased mtH2O2 levels, while prevention of mitochondrial import of TERT augmented pathological effects. Further elevated mtDNA damage was observed in tissue from subjects with CAD and initiation of mtDNA repair mechanisms was sufficient to restore NO-mediated dilation in vessels from patients with CAD. The work presented is the first evidence that catalytically active mitochondrial TERT, independent of its nuclear functions, plays a critical physiological role in preserving NO-mediated vasodilation and the balance of mitochondrial to nuclear TERT is fundamentally altered in states of human disease that are driven by increased expression of dominant negative splice variants.

Effect of acute high-intensity resistance exercise on optic nerve sheath diameter and ophthalmic artery blood flow pulsatility.

Exertional hypertension associated with acute high-intensity resistance exercise (RE) increases both intravascular and intracranial pressure (ICP), maintaining cerebrovascular transmural pressure. Carotid intravascular pressure pulsatility remains elevated after RE. Whether ICP also remains elevated after acute RE in an attempt to maintain the vessel wall transmural pressure is unknown. Optic nerve sheath diameter (ONSD), a valid proxy of ICP, was measured in 20 participants (6 female; 24 ± 4 yr, 24.2 ± 3.9 kg m(-)(2)) at rest (baseline), following a time-control condition, and following RE (5 sets, 5 repetition maximum bench press, 5 sets 10 repetition maximum biceps curls) using ultrasound. Additionally, intracranial hemodynamic pulsatility index (PI) was assessed in the ophthalmic artery (OA) by using Doppler. Aortic pulse wave velocity (PWV) was obtained from synthesized aortic pressure waveforms obtained via a brachial oscillometric cuff and carotid pulse pressure was measured by using applanation tonometry. Aortic PWV (5.2 ± 0.5-6.0 ± 0.7 m s(-1), P < 0.05) and carotid pulse pressure (45 ± 17-59 ± 19 mm Hg, P < 0.05) were significantly elevated post RE compared with baseline. There were no significant changes in ONSD (5.09 ± 0.7-5.09 ± 0.7 mm, P > 0.05) or OA flow PI (1.35 ± 0.2-1.38 ± 0.3, P > 0.05) following acute RE. In conclusion, during recovery from acute high-intensity RE, there are increases in aortic stiffness and extracranial pressure pulsatility in the absence of changes in ICP and flow pulsatility. These findings may have implications for alterations in cerebral transmural pressure and cerebral aneurysmal wall stress following RE.

Annexin A6 is a scaffold for PKCα to promote EGFR inactivation.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.

Redox control of ß2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

BACKGROUND: ß(2) -Glycoprotein I (ß(2) GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of ß(2) GPI in thrombus formation is unknown. We have recently shown that ß(2) GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin-1 and protein disulfide isomerase, and that reduction of ß(2) GPI can take place on the platelet surface. METHODS: ß(2) GPI, reduced by thioredoxin-1, was labeled with the selective sulfhydryl probe N(a)-(3-maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced ß(2) GPI for von Willebrand factor (VWF) and the effect of reduced ß2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin-activated VWF was studied in the presence of reduced ß(2) GPI. RESULTS: We demonstrate that the Cys288-Cys326 disulfide in domain V of ß(2) GPI is the predominant disulfide reduced by thioredoxin-1. Reduced ß(2) GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. ß(2) GPI reduced by thioredoxin-1, in comparison with non-reduced ß(2) GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. CONCLUSIONS: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol-dependent interaction of ß(2) GPI with VWF may contribute to the redox regulation of platelet adhesion.

Endosomal localization of phospholipase D 1a and 1b is defined by the C-termini of the proteins, and is independent of activity.

The factors regulating the activity of cellular phospholipase D (PLD) have been well characterized; however, the cellular distribution of specific PLD isoforms and the factors defining localization are less clear. Two specific PLD1 isoforms, PLD1a and PLD1b, are shown in the present study to be localized in endosomal compartments with early endosomal autoantigen 1, internalizing epidermal growth factor receptor (ErbB1) and lysobisphosphatidic acid. Novel C-terminal splice variants of PLD1, PLD1a2 and PLD1b2, do not exhibit this endosomal localization. Studies using catalytically inactive and C-terminal deletion mutants of the four PLD1 isoforms led to the conclusion that the C-terminus plays an important part in the catalytic activity of PLD1, but that the endosomal localization of PLD1a and PLD1b is defined by the C-terminus and not catalytic activity.

Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility.

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.

Sac phosphatase domain proteins.

Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae.

SAC1 encodes a regulated lipid phosphoinositide phosphatase, defects in which can be suppressed by the homologous Inp52p and Inp53p phosphatases.

The yeast protein Sac1p is involved in a range of cellular functions, including inositol metabolism, actin cytoskeletal organization, endoplasmic reticulum ATP transport, phosphatidylinositol-phosphatidylcholine transfer protein function, and multiple-drug sensitivity. The activity of Sac1p and its relationship to these phenotypes are unresolved. We show here that the regulation of lipid phosphoinositides in sac1 mutants is defective, resulting in altered levels of all lipid phos- phoinositides, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. We have identified two proteins with homology to Sac1p that can suppress drug sensitivity and also restore the levels of the phosphoinositides in sac1 mutants. Overexpression of truncated forms of these suppressor genes confirmed that suppression was due to phosphoinositide phosphatase activity within these proteins. We have now demonstrated this activity for Sac1p and have characterized its specificity. The in vitro phosphatase activity and specificity of Sac1p were not altered by some mutations. Indeed, in vivo mutant Sac1p phosphatase activity also appeared unchanged under conditions in which cells were drug-resistant. However, under different growth conditions, both drug sensitivity and the phosphatase defect were manifest. It is concluded that SAC1 encodes a novel lipid phosphoinositide phosphatase in which specific mutations can cause the sac1 phenotypes by altering the in vivo regulation of the protein rather than by destroying phosphatase activity.

Mutations in the Saccharomyces cerevisiae gene SAC1 cause multiple drug sensitivity.

Wild-type yeast Saccharomyces cerevisiae are surprisingly resistant to a wide range of drugs and agents. We had previously isolated novobiocin-sensitive mutants to aid the study of the intracellular target for this drug. Characterization of one of these mutants, mds1, revealed that it was sensitive not only to novobiocin but also to a wide range of drugs. The nature of this multiple drug-sensitive phenotype was shown to be different from that of previously isolated multiple drug-sensitive mutants. We have shown that the multiple drug-sensitivity of mds1 is due to mutations within the gene SAC1 and have identified a variety of mutations within the gene from the Mds1 strain. SAC1 encodes a protein which has been previously implicated in the correct function of the actin cytoskeleton, in inositol metabolism, in ATP transport in the endoplasmic reticulum and in Sec14p (PI-TP) function. We have shown that multiple drug-sensitivity is a new phenotype seen in some, but not all, of the previously characterized sac1 mutants. Based on our findings, we propose a mechanism by which Sac1p could affect drug resistance and also mediate other effects on cell growth.

Platelet serotonin uptake during myocardial ischemia.

Platelets are believed to play a role in the pathologic sequelae of acute myocardial ischemia. Several of the compounds generated and released by activated platelets during cardiac ischemia may contribute to the production of cellular necrosis (free oxygen radicals), coronary vasoconstriction (thromboxane, serotonin, catecholamines), and perpetuation of the platelet aggregatory reaction (thromboxane, serotonin, adenosine diphosphate). In the present study, it was demonstrated that platelet serotonin uptake function is markedly depressed following the induction of myocardial ischemia in cats. The possible mechanism through which the depression occurs and the resulting pathologic sequelae are discussed. The results of the present study illustrate another mechanism by which platelets can potentially mediate the severity and perpetuation of cellular events during acute myocardial ischemic insult.