Protein Methylation and Quantifications of Nucleotide-Binding Proteins
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Protein Methylation and Quantifications of Nucleotide-Binding Proteins

  • Author(s): Bade, David
  • Advisor(s): Wang, Yinsheng
  • et al.
No data is associated with this publication.
Abstract

Post-translational modifications (PTMs) constitute an evolutionally conserved and ubiquitous mechanism for regulating protein structure and function. The first part of this dissertation focuses on the discovery of an epitranscriptomic mechanism of regulation N-terminal methyltransferase 1 (NTMT1) expression, and the identification of novel substrates of α-N-methylation and histidine methylation. The goal of the second half of this dissertation is to discover nucleotide-binding proteins (i.e. small GTPases and kinases) as drivers and suppressors for cancer metastasis and acquired radioresistance. In Chapter Two, we discovered that NTMT1 expression is regulated by reader/writer/eraser proteins of N6-methyladenosine (m6A). We further identified Mortality Factor 4 Like 1 (MRG15) as a novel α-N-methylated protein. We subsequently demonstrated that NTMT1 is responsible for α-N-methylation of MRG15, and this methylation is also regulated through an m6A-based epitranscriptomic mechanism. In Chapter Three, we interrogated a series of publicly available mass spectrometry datasets with MaxQuant to identify novel protein substrates for α-N-methylation. Our results uncovered 219 instances of N-terminal methylation in 196 proteins. We then identified, through affinity purification and mass spectrometry analysis, that Vesicle-associated membrane protein 4 (VAMP4) is α-N-methylated. We subsequently confirmed that NTMT1 is the primary enzyme responsible for the N-terminal methylation of VAMP4. In Chapter Four, we analyzed publicly available mass spectrometry datasets to identify novel protein substrates for histidine methylation. Using this method, we uncovered 33 instances of histidine methylation among 26 proteins. We subsequently used affinity purification and mass spectrometry analysis to confirm the histidine methylation of Pre-mRNA-splicing factor RBM22 (RBM22). In Chapter Five, we employed stable isotopic labeling by amino acids in cell culture (SILAC) and parallel-reaction monitoring (PRM) to monitor the differential expression of kinases in MCF-7 and MDA-MB-231 breast cancer cells and their corresponding radioresistant C6 and C5 clones. Using this method, we were able to identify and quantify the relative expression levels of 300 and 281 kinases in C5/MDA-MB-231 and C6/MCF-7 pairs of breast cancer cells, respectively. We further identified transcription initiation factor TFIID subunit 9 (TAF9) as a driver of radioresistance in breast cancer cell lines. In Chapter Six, we employed a proteomic method, based on multiple-reaction monitoring (MRM), for quantitative profiling of GTP-binding proteins using isotope-coded GTP probes. After probe labeling, tryptic digestion, and affinity enrichment of labeled peptides, the expression level differences of GTP-binding proteins in two matched pairs of primary/metastatic melanoma cell lines (WM-115/WM-266-4 and IGR-39/IGR-37) were investigated with LC-MRM analysis. Among the most upregulated proteins, adenylate kinase 4 (AK4) was identified as a potential driver of melanoma metastasis. We further demonstrated that AK4 expression is necessary for melanoma cell invasion and migration.

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This item is under embargo until April 23, 2023.