UNDERSTANDING THE RAL GTPASES: THE REGULATION BY PROTEIN LYSINE FATTY ACYLATION, SIRT2, AND INTERACTING PROTEINS
Spiegelman, Nicole Alexis
The RAS superfamily of small GTPases are molecular switches that control a wide range of cellular processes. Detailed understanding of these small GTPases is important for elucidating cell signaling mechanisms and identifying new disease treatment strategies. My thesis is centered on understanding two highly similar small GTPases, RalA and RalB. They are the two members of the Ras-like (Ral) GTPases in the Ras subfamily. Despite their similar biochemical and structurally properties, the Ral GTPases can exhibit diverging roles. My thesis work provides important insights into the differential regulation and function of RalA and RalB. In one study (Chapter 2), I discovered that RalA and RalB are differentially regulated by lysine fatty acylation. Lipidation, such as cysteine palmitoylation and prenylation, is a key regulatory mechanism for small GTPases. Recently it has been reported that lysine fatty acylation also regulates several small GTPases. My graduate work shows that RalB, but not RalA, is regulated by lysine fatty acylation. RalB lysine fatty acylation is reversible and can be removed by SIRT2. Lysine fatty acylation promotes RalB GTP binding and cell migration. To further understand the differential functions of RalA and RalB, in a second study (Chapter 3), we used a quantitative proteomic approach (stable isotope labeling of amino acids in cell culture or SILAC) to identify the interacting proteins of RalA and RalB. This study revealed many interacting proteins that can bind to RalA, RalB, or both in a nucleotide dependent manner. These interacting proteins provide important insights that will guide future studies. For example, among these interacting proteins identified, we confirmed that RalB selectively interacts with ERK2 and can decrease its nuclear localization. This may explain why RalB could suppress anchorage-independent growth while RalA does not. As protein lysine fatty acylation is becoming a more abundant protein post translational modification, we wanted to identify tools that could help us better understand the roles of lysine fatty acylation in signaling and cancer. Histone Deacetylases (HDACs), such as SIRT2, can act as protein lysine defatty-acylases (have the ability to hydrolyze long chain fatty acyl groups from lysine residues). Thus, SIRT2 inhibitors could be useful tools to study lysine fatty acylation. However, most reported SIRT2 inhibitors were not tested against its defatty-acylation activity. Through an in-depth comparison of four SIRT2 inhibitors (AGK2, SirReal2, Tenovin-6 and TM), we found that TM was the most potent and selective SIRT2 inhibitor (Chapter 4). However, TM could not efficiently inhibit defatty-acylation. In an attempt to find a tool compound that could be used to study sirtuin regulated de-fatty acylation, we identified a TM analogue, JH-T4 (Chapter 5). JH-T4 can more potently inhibit the defatty-acylation activity of SIRT2, although it can also inhibit SIRT1 and SIRT3. I was interested in developing chemical proteomic tools to identify lysine fatty-acylated proteins. Through our proteomic studies aimed at identifying HDAC lysine fatty acylation substrates, we found that JAM-C has DHHC7-regulated cysteine palmitoylation (Chapter 6). However, current proteomic methods cannot provide site identification. This makes profiling lysine fatty acylated proteins challenging. To overcome this, I attempted to develop a chemical proteomic platform that would allow direct site detection of protein lipidation, including lysine fatty acylation with limited success (Chapter 7).
Cerione, Richard A.; Aye, Yimon
Chemistry and Chemical Biology
Ph. D., Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis