Year of Publication

2016

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Molecular and Cellular Biochemistry

First Advisor

Dr. Sidney W. Whiteheart

Abstract

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants containing phosphomimetics showed enhanced initial fusion rates. These results, combined with previous work in vivo, confirm that SNAP-23 phosphorylation is involved in regulating membrane fusion, and that IKK-mediated signaling contributes to platelet exocytosis.

To address the role(s) of acylation, I sought to determine how syntaxin-11 and SNAP-23 are associated with plasma membrane. Using metabolic labeling, I showed that both proteins contain thioester-linked acyl groups which turn over in resting cells. Mass spectrometry mapping showed that syntaxin-11 is modified on C275, 279, 280, 282, 283 and 285, while SNAP-23 is modified on C79, 80, 83, 85, and 87. To probe the effects of acylation, I measured ADP/ATP release from platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. Cerulenin pretreatment inhibited t-SNARE acylation and platelet function while palmostatin B had no effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state of platelet proteins is important for their function. Thus my work indicates that the enzymes controlling protein acylation could be valuable targets for modulating platelet exocytosis in vivo.

Digital Object Identifier (DOI)

http://dx.doi.org/10.13023/ETD.2016.044

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