Date of Award

2012

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Pure sciences, Biological sciences, Nitric oxide, Protein disulfide, Vascular function

Supervisor

Bulent Mutus

Rights

info:eu-repo/semantics/openAccess

Abstract

Nitric oxide (NO) is a ubiquitous signaling molecule that diffuses freely across the cell membrane to activate several cellular signaling pathways. S-nitrosothiols (RSNO), the storehouse of NO and protein disulfide isomerase (PDI) catalyze release of NO from RSNO under physiological conditions. The objectives of the present study are to determine the role of NO signaling in hypoxic vasodilatation and to develop methods for detecting NO signaling.

We studied the role of red blood cell (RBC) PDI in hypoxic vasodilation. The data presented demonstrate that nitrite support S-nitrosation of PDI under oxygenated conditions; also PDI has high affinity for RBC surface under oxygenated conditions. We show interaction of PDI with hemoglobin and RBC resident proteins like band 3 and Glut1 supporting the hypothesis that PDI can act as an acceptor of NO-equivalents from NOx-modified RBC resident proteins.

We have shown that sinapinic acid can denitrosate RSNO generating a free thiol but it does not reduce disulfide bridges thus can be used for detecting S-nitrosated proteins. Furthermore, we have shown that sinapinic acid detects basal protein S-nitrosothiols and produces nearly identical S-nitrosoprotein patterns as ascorbate in the biotin switch analysis.

Thiols are susceptible to NO-dependent and independent oxidative modification. In this work we present a RFP-PDI-construct as a probe to monitor thiol redox status at the cell surface. Our results show increase in RFP-PDI fluorescence with oxidative stress. Also, RFP-PDI forms covalent mixed disulfides with cell surface protein thiols that are susceptible to extracellular reductants.

The decrease in synthesis and availability of NO in endothelial cells is one of the risk factors leading to endothelial dysfunction. Our next objective was to investigate the possible link between endothelial dysfunction and ER stress. Our results show that under conditions of ER stress, neutral sphingomyelinase (NSMase2) the enzyme that control plasma membrane cholesterol levels was dysfunctional as a result of nitration of its tyrosine residues.

We also studied plasma membrane cholesterol and effect of ticagrelor (AZD6140) on platelet P2Y12 receptor. Herein, we show that ticagrelor disrupts P2Y12 receptor clustering in lipid rafts and inhibits platelet aggregation. Interestingly, we found that differential effect of ticagrelor in normal and type-2 diabetic (T2D) platelets correlates to the differences in cholesterol levels of platelets.

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