Proteomic Analysis of Palmitoylated Platelet Proteins
Authors: Dowal Louisa M. , Yang Wei , Peters Christian G , Freeman Michael , Steen Hanno , Flaumenhaft Robert 
Published: October 2019

Abstract 2017

Protein palmitoylation is a dynamic process that regulates membrane targeting of proteins and protein-protein interactions. It is unique among the fatty acid modifications as it is reversible, and its reversibility suggests that it can participate in the regulation of cell signaling. We have previously demonstrated a critical role for protein palmitoylation in platelet activation and have begun to characterize the palmitoylation machinery in platelets. We have now employed a novel proteomic approach termed Palmitoyl Protein Identification and Site Characterization (PalmPISC) to define the platelet “palmitoylome.” Using acyl biotin exchange (ABE) chemistry, we have purified palmitoylated proteins from membranes of resting platelets and identified them using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectral counting analysis identified 131 putative palmitoylated proteins including 58 novel palmitoylated proteins. Components of the G protein signal transduction pathways (15% of palmitoylated proteins) and membrane fusion proteins (10% of palmitoylated proteins) were highly represented. Platelets undergo a dramatic phenotypic change upon activation and platelet proteins are known to undergo activation-dependent palmitoylation. Changes in the palmitoylation state of proteins during platelet signaling may be reflective of the activation process. We have compared changes in protein palmitoylation in resting and thrombin-activated platelets to identify proteins that undergo activation-dependent palmitoylation or depalmitoylation. To quantify these changes by mass spectrometry, we employed iTRAQ labeling and identified 32 proteins that increase or decrease their palmitoylation upon activation. We have focused our initial efforts on one of these proteins, Triggering Receptor Expressed on Myloid cells (TREM)-like transcript-1 (TLT-1), an immunoglobulin domain-containing receptor expressed exclusively in platelets and megakaryocytes. We have validated that platelet TLT-1 is palmitoylated using [3H]palmitate labeling and have identified the site of TLT-1 palmitoylation as juxtamembrane Cys196, which is adjacent to an ITIM domain. Our iTRAQ results reveal that TLT-1 exhibits a 2-fold decrease in palmitoylation upon activation. A decrease in TLT-1 palmitoylation upon Par1-mediated activation was confirmed using an ABE strategy, which detects total protein palmitoylation. In contrast, there is a 2.5-fold increase in [3H]palmitate labeling of TLT-1 upon activation of platelets, indicating increased turnover of palmitate with activation. These observations suggest that activation-dependent depalmitoylation of TLT-1 occurs more rapidly than activation-dependent palmitoylation and underscores the importance of measuring both total palmitoylation and palmitate turnover in assessing activation-dependent palmitoylation. This global analysis of platelet protein palmitoylation provides a platform to inform future investigations identifying the role of palmitoylation in the function of specific platelet proteins. Identification of proteins that undergo activation-dependent palmitoylation or depalmitoylation will enable studies of the platelet protein palmitoylation machinery.

Disclosures:

No relevant conflicts of interest to declare.