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Scholarly Works (12 results)

  • Thesis
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UC Riverside Electronic Theses and Dissertations (2022)

Recent advances in mass spectrometry instrumentation and sample preparation methods have enabled robust identification and quantification of proteins at the entire proteome level. The focus of this dissertation is placed on two groups of proteins, the TBC domain-containing proteins as well as epitranscriptomic reader, writer and eraser (RWE) proteins. In Chapter 2, I utilized a shotgun quantitative proteomic method to assess, at the global proteome scale, differential protein expression in a matched pair of primary/metastatic melanoma cell lines (i.e. WM-115/WM-266-4). I found TBC1D7 may play a role in melanoma cell invasion. In Chapter 3, I established a liquid chromatography–parallel-reaction monitoring (LC-PRM) method for high-throughput profiling of approximately 150 epitranscriptomic RWE proteins. I employed this LC-PRM method coupled with stable isotope labeling by amino acids in cell culture (SILAC) to examine the differences in expression levels of the proteins in two matched pairs of radioresistant/wild type (MDA-MB-231/C5 and MCF-7/C6) breast cancer cells. This method allows for the quantifications of 70% and 65% of the epitranscriptomic RWE proteome. Among them, TRMT1 (an m2,2G writer) may assume a crucial role in enhancing breast cancer radioresistance. In Chapter 4, I further applied this LC-PRM method to assess the expression of epitranscriptomic RWE proteins in modulating colorectal cancer (CRC) metastasis. I was able to quantify 74% of the epitranscriptomic RWE proteome; among them, 48 and 5 were up- and down-regulated by over 1.5-fold in metastatic SW620 relative to primary SW480 CRC cells, respectively. In Chapter 5, I modified the LC-PRM method by employing a mixture of 48 stable isotope-labeled (SIL) peptides representing RWE proteins as internal or surrogate standards. I utilized this method to explore potential crosstalk between N6-methyladenosine (m6A) and other modified ribonucleosides by assessing the epitranscriptomic RWE proteome in ALKBH5-/-, FTO-/-, METTL3-/- cells, and their isogenic parental HEK293T. NOP2, PUS3, TGS1 and RBMX were altered by more than 1.5-fold in the opposite directions in ALKBH5-/- and METTL3-/- cells relative to isogenic HEK293T cells. Together, the research described in this dissertation documented the power of quantitative proteomics in revealing new functions of cellular proteins in modulating cancer metastasis, radioresistance, and the epitranscriptome.

    Cover page: Quantitative Proteomic Profiling of TBC Domain-Containing Proteins and Epitranscriptomic Reader, Writer and Eraser Proteins
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2022)
    Epitranscriptomic reader, writer, and eraser (RWE) proteins recognize, install, and remove modified nucleosides in RNA, which are known to play crucial roles in RNA processing, splicing, and stability. Here, we established a liquid chromatography-parallel-reaction monitoring (LC-PRM) method for high-throughput profiling of a total of 152 epitranscriptomic RWE proteins. We also applied the LC-PRM method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to quantify these proteins in two pairs of matched parental/radioresistant breast cancer cells (i.e., MDA-MB-231 and MCF-7 cells and their corresponding radioresistant C5 and C6 clones), with the goal of assessing the roles of these proteins in radioresistance. We found that eight epitranscriptomic RWE proteins were commonly altered by over 1.5-fold in the two pairs of breast cancer cells. Among them, TRMT1 (an m2,2G writer) may play a role in promoting breast cancer radioresistance due to its clinical relevance and its correlation with DNA repair gene sets. To our knowledge, this is the first report of a targeted proteomic method for comprehensive quantifications of epitranscriptomic RWE proteins. We envision that the LC-PRM method is applicable for studying the roles of these proteins in the metastatic transformation of cancer and therapeutic resistance of other types of cancer in the future.
      Cover page: Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Accompanied with Radioresistance in Breast Cancer Cells.
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2022)
      ALKBH4 is a versatile demethylase capable of catalyzing the demethylation of monomethylated lysine-84 on actin and N6 -methyladenine in DNA. In this study, we conducted a quantitative proteomic experiment to reveal the altered expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results showed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein.
        Cover page: Quantitative proteomics revealed new functions of ALKBH4.
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2023)
        RNA contains more than 170 types of chemical modifications, and these modified nucleosides are recognized, installed and removed by their reader, writer, and eraser (RWE) proteins, respectively. Here, we employed a parallel-reaction monitoring (PRM)-based targeted proteomic method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to examine comprehensively the differential expression of epitranscriptomic RWE proteins in a matched pair of primary/metastatic colorectal cancer (CRC) cells, namely SW480/SW620. We were able to quantify 113 nonredundant epitranscriptomic RWE proteins; among them, 48 and 5 were up- and down-regulated by >1.5-fold in SW620 over SW480 cells, respectively. Some of those proteins with marked up-regulation in metastatic CRC cells, including NAT10, hnRNPC, and DKC1, were documented to assume important roles in the metastasis of CRC and other types of cancer. Interrogation of the Clinical Proteomic Tumor Analysis Consortium data revealed the involvement of DUS1L in the initiation and metastatic transformation of CRC. It can be envisaged that the PRM method can be utilized, in the future, to identify epitranscriptomic RWE proteins involved in the metastatic transformations of other types of cancer.
          Cover page: Parallel-reaction monitoring revealed altered expression of a number of epitranscriptomic reader, writer, and eraser proteins accompanied with colorectal cancer metastasis.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2023)
          Trimethylation of lysine 36 on histone H3 (H3K36me3), an epigenetic mark associated with actively transcribed genes, plays an important role in multiple cellular processes, including transcription elongation, DNA methylation, DNA repair, etc. Aberrant expression and mutations of the main methyltransferase for H3K36me3, i.e., SET domain-containing 2 (SETD2), were shown to be associated with various cancers. Here, we performed targeted profiling of 154 epitranscriptomic reader, writer, and eraser (RWE) proteins using a scheduled liquid chromatography-parallel-reaction monitoring (LC-PRM) method coupled with the use of stable isotope-labeled (SIL) peptides as internal standards to investigate how H3K36me3 modulates the chromatin occupancies of epitranscriptomic RWE proteins. Our results showed consistent changes in chromatin occupancies of RWE proteins upon losses of H3K36me3 and H4K16ac and a role of H3K36me3 in recruiting METTL3 to chromatin following induction of DNA double-strand breaks. In addition, protein-protein interaction network and Kaplan-Meier survival analyses revealed the importance of METTL14 and TRMT11 in kidney cancer. Taken together, our work unveiled cross-talks between histone epigenetic marks (i.e., H3K36me3 and H4K16ac) and epitranscriptomic RWE proteins and uncovered the potential roles of these RWE proteins in H3K36me3-mediated biological processes.
            Cover page: Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Regulated by H3K36me3.
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2022)
            Radiation therapy benefits more than 50% of all cancer patients and cures 40% of them, where ionizing radiation (IR) deposits energy to cells and tissues, thereby eliciting DNA damage and resulting in cell death. Small GTPases are a superfamily of proteins that play critical roles in cell signaling. Several small GTPases, including RAC1, RHOB, and RALA, were previously shown to modulate radioresistance in cancer cells. However, there is no systematic proteomic study on small GTPases that regulate radioresistance in cancer cells. Herein, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled (SIL) peptides, to identify differentially expressed small GTPase proteins in two pairs of breast cancer cell lines, MDA-MB-231 and MCF7, and their corresponding radioresistant cell lines. We identified 7 commonly altered small GTPase proteins with over 1.5-fold changes in the two pairs of cell lines. We also discovered ARFRP1 as a novel regulator of radioresistance, where its downregulation promotes radioresistance in breast cancer cells. Together, this represents the first comprehensive investigation about the differential expression of the small GTPase proteome associated with the development of radioresistance in breast cancer cells. Our work also uncovered ARFRP1 as a new target for enhancing radiation sensitivity in breast cancer.
              Cover page: Targeted Proteomic Analysis of Small GTPases in Radioresistant Breast Cancer Cells.
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2022)
              N6-Methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m6A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m6A and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m6A eraser proteins (i.e., ALKBH5, FTO) and the catalytic subunit of the major m6A writer complex (i.e., METTL3) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5. Analysis of previously published m6A mapping results revealed the presence of m6A in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m6A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.
                Cover page: Targeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled Peptides.
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2018)
                Small GTPases of the Ras superfamily are master regulators of intracellular trafficking and constitute essential signaling components in all eukaryotes. Aberrant small GTPase signaling is associated with a wide spectrum of human diseases, including cancer. Here, we developed a high-throughput, multiple reaction monitoring-based workflow, coupled with stable isotope labeling by amino acids in cell culture, for targeted quantification of approximately 100 small GTPases in cultured human cells. Using this method, we investigated the differential expression of small GTPases in three pairs of primary and metastatic melanoma cell lines. Bioinformatic analyses of The Cancer Genome Atlas data and other publicly available data as well as cell-based assays revealed previously unrecognized roles of RAB38 in promoting melanoma metastasis. Diminished promoter methylation and the subsequent augmented binding of transcription factor MITF contributed to elevated expression of RAB38 gene in metastatic versus primary melanoma cells. Moreover, RAB38 promoted invasion of cultured melanoma cells by modulating the expression and activities of matrix metalloproteinases-2 and -9. Together, these data establish a novel targeted proteomic method for interrogating the small GTPase proteome in human cells and identify epigenetic reactivation of RAB38 as a contributing factor to metastatic transformation in melanoma.Significance: A novel quantitative proteomic method leads to the discovery of RAB38 as a new driver of metastasis in melanoma. Cancer Res; 78(18); 5431-45. ©2018 AACR.
                  Cover page: A targeted quantitative proteomic approach assesses the reprogramming of small GTPases during melanoma metastasis
                  • Article
                  • Peer Reviewed
                  UC Riverside Previously Published Works (2020)
                  Kinases are involved in numerous critical cell signaling processes, and dysregulation in kinase signaling is implicated in many types of human cancers. In this study, we applied a parallel-reaction monitoring (PRM)-based targeted proteomic method to assess kinome reprogramming during melanoma metastasis in three pairs of matched primary/metastatic human melanoma cell lines. Around 300 kinases were detected in each pair of cell lines, and the results showed that Janus kinase 3 (JAK3) was with reduced expression in the metastatic lines of all three pairs of melanoma cells. Interrogation of The Cancer Genome Atlas (TCGA) data showed that reduced expression of JAK3 is correlated with poorer prognosis in melanoma patients. Additionally, metastatic human melanoma cells/tissues exhibited diminished levels of JAK3 mRNA relative to primary melanoma cells/tissues. Moreover, JAK3 suppresses the migration and invasion of cultured melanoma cells by modulating the activities of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). In summary, our targeted kinome profiling method provided by far the most comprehensive dataset for kinome reprogramming associated with melanoma progression, which builds a solid foundation for examining the functions of other kinases in melanoma metastasis. Moreover, our results reveal a role of JAK3 as a potential suppressor for melanoma metastasis.
                    Cover page: A Targeted Quantitative Proteomic Method Revealed a Substantial Reprogramming of Kinome during Melanoma Metastasis
                    • Article
                    • Peer Reviewed
                    UC Riverside Previously Published Works (2021)
                    Metastasis is a major obstacle in the therapeutic intervention of melanoma, and several GTP-binding proteins were found to play important roles in regulating cancer metastasis. To assess systematically the regulatory roles of these proteins in melanoma metastasis, we employed a targeted chemoproteomic method, which relies on the application of stable isotope-labeled desthiobiotin-GTP acyl phosphate probes in conjunction with scheduled multiple-reaction monitoring (MRM), for profiling quantitatively the GTP-binding proteins. Following probe labeling, tryptic digestion, and affinity pull-down of desthiobiotin-conjugated peptides, differences in expression levels of GTP-binding proteins in two matched pairs of primary/metastatic melanoma cell lines were measured using liquid chromatography-MRM analysis. We also showed that among the top upregulated proteins in metastatic melanoma cells, AK4 promotes the migration and invasion of melanoma cells; overexpression of AK4 in primary melanoma cells leads to augmented migration and invasion, and reciprocally, knockdown of AK4 in metastatic melanoma cells results in repressed invasiveness. In summary, we examined the relative expression levels of GTP-binding proteins in two pairs of primary/metastatic melanoma cell lines. Our results confirmed some previously reported regulators of melanoma metastasis and revealed a potential role of AK4 in promoting melanoma metastasis.
                      Cover page: Targeted Quantitative Profiling of GTP-Binding Proteins Associated with Metastasis of Melanoma Cells.
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