UC Riverside

Recent advances in proteomics, including instrumentation, data acquisition approaches, and sample preparation methods, allow us to investigate the proteome of interest with high sensitivity, selectivity, and throughput. The focus of this dissertation is placed on the applications of modern proteomics to systematically interrogate an important subset of the proteome. The research reported in this dissertation is divided into two parts, i.e., applying multiple-reaction monitoring (MRM)-based targeted proteomics to systematically quantify the expression of the entire small GTPase superfamily; and employing ascorbate peroxidase (APEX)-based proximity labeling to characterize the interacting proteins of METTL3/METTL14, the catalytic heterodimer of the major N6-methyladenosine (m6A) methyltransferase complex. I utilized MRM-based targeted proteomics coupled with scheduled-liquid chromatography (LC) and synthetic stable isotope-labeled (SIL) peptides to systematically quantify the differential expression of small GTPase upon the adipogenesis of murine mesenchymal stem cells (MSCs), including 3T3-L1 and C3H10T1/2 cells. The results revealed Rab32 as a novel adipogenesis regulator, where its downregulation is required for successful adipogenic differentiation in MSCs. In addition, I further employed the LC-MRM method to interrogate systematically the temporal responses of the entire small GTPases proteome during the course of osteogenic differentiation of H9 human embryonic stem cells. The quantification results revealed altered expression of a large number of small GTPases accompanied with osteogenic differentiation, especially those involved in autophagy. The results also documented a previously unrecognized role of KRAS in osteogenesis, where it regulates the accumulation of extracellular matrix for mineralization through attenuating the activity of secreted matrix metalloproteinase-9 (MMP9). To understand how m6A in mRNA regulates the expression of small GTPases, I also adopted the LC-MRM platform to assess the differential expression of small GTPases in cells upon genetic depletion of m6A reader, writer, and eraser proteins. The results demonstrated that depletion of METTL3 and ALKBH5 (m6A demethylase) resulted in substantially altered expression of a subset of small GTPase proteins, including RhoB and RhoC. The results further demonstrated that the mRNA stability of RhoB is significantly augmented in METTL3-/- cells, suggesting a m6A-mediated decay of RhoB transcript. To investigate the protein interactomes of METTL3/METTL14, I employed APEX-based proximity labeling followed by LC-MS/MS analysis to examine systematically the interacting proteins of the METTL3 and METTL14 in HEK293T cells. The results revealed a subset of histone modification writer and eraser enzymes to be enriched in the proximity proteome of METTL3, including histone acetyltransferase 1 (HAT1). Consistently, the acetylation level of H3K9 diminished in METTL3-/- cells, which could be restored upon complementation with wild-type, but not catalytically inactive METTL3. We also discovered that the METTL3-catalyzed m6A in chromatin-associated RNAs (caRNAs) promotes the chromatin occupancy of the long α isoform of HAT1 (HAT1 α), thereby stimulating H3K9Ac, especially at loci of LTR and LINE retrotransposons. The HAT1 occupancy in chromatin was diminished upon genetic depletion of METTL3 or METTL14 while its global expression was unaltered, suggesting that METTL3-containing methyltransferase complex regulates chromatin localization of HAT1 without altering its expression level. Results from our in vitro acetylation assay demonstrated the ability of HAT1 α to catalyze H3K9Ac in nucleosomes. Moreover, HAT1’s chromatin occupancy and its function in acetylating H3K9 in chromatin requires its nuclear localization and is promoted by YTHDC1, an m6A reader protein that also interacts with METTL3. Together, the research presented in this dissertation displayed the robustness of modern proteomics to systematically investigating the proteome of interest with excellent sensitivity, selectivity, and throughput, and its power in offering new biological insights.