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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2023)

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.

    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2021)
    As optimum temperature is essential for all living organisms, heat shock represents a challenging problem for their survival. Therefore, cellular response to heat shock is among the most extensively investigated stress response pathways; however, how the human proteome responds to heat shock has not been comprehensively investigated. In this study, we employed stable isotope labeling by amino acids in cell culture (SILAC), together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, to fulfill an in-depth analysis of the alterations in the human proteome in M14 human melanoma cells in response to heat shock stress. We found that, after heat shock, 284 and 278 out of the 4319 quantified proteins were with substantially diminished and elevated expressions, respectively. We also examined the alterations in human kinome after heat shock by using our recently developed targeted proteomic method relying on parallel-reaction monitoring. Our results showed that the expression levels of 11 and 22 kinase proteins were increased and decreased, respectively, by at least 1.5-fold upon heat shock. By interrogating publicly available RNA-seq and m6A sequencing data, we observed that the elevated expression of more than 30 proteins, including CHEK1 and CCND3 kinases, could occur via an m6A-mediated mechanism. Furthermore, our results from single-base elongation and ligation-based quantitative polymerase chain reaction (qPCR) amplification (SELECT) and luciferase reporter assays revealed that heat shock gave rise to elevated m6A levels at A280 and A286 sites in the 5-untranslated region of HSPH1 mRNA, thereby leading to increased translation of HSPH1 protein. Together, our discovery and targeted proteomic methods revealed the reprogramming of human proteome and kinome upon heat shock stress and provided insights into cellular responses toward heat shock stress.
      Cover page: Quantitative Proteomic Analysis Revealed Broad Roles of N6-Methyladenosine in Heat Shock Response.
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2022)
      Aha1 is a co-chaperone of heat shock protein 90 (HSP90), and it stimulates the ATPase activity of HSP90 to promote the folding of its client proteins. By employing ascorbate peroxidase (APEX)-based proximity labeling and proteomic analysis, we identified over 30 proteins exhibiting diminished abundances in the proximity proteome of HSP90 in HEK293T cells upon genetic depletion of Aha1. Dicer1 is a top-ranked protein, and we confirmed its interactions with HSP90 and Aha1 by immunoprecipitation followed by western blot analysis. Genetic depletion of Aha1 and pharmacological inhibition of HSP90 both led to reduced levels of Dicer1 protein. Additionally, HSP90 and Aha1 bind preferentially to newly translated Dicer1. Reconstitution of Aha1-depleted cells with wild-type Aha1 substantially rescued Dicer1 protein level, and a lower level of restoration was observed for complementation with the HSP90-binding-defective Aha1-E67K, whereas an Aha1 mutant lacking the first 20 amino acids-which abolishes its chaperone activity-failed to rescue Dicer1 protein level. Moreover, knockdown of Aha1 and inhibition of HSP90 led to diminished levels of mature microRNAs (miRNAs), but not their corresponding primary miRNAs. Together, we uncovered a novel mechanism of HSP90 and Aha1 in regulating the miRNA pathway through promoting the folding of Dicer1 protein, and we also demonstrated that Aha1 modulates this process by acting as an autonomous chaperone and a co-chaperone for HSP90.
        Cover page: HSP90 and Aha1 modulate microRNA maturation through promoting the folding of Dicer1.
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2020)
        Small GTPases are essential signaling molecules for regulating glucose uptake in adipose tissues upon insulin stimulation, and this regulation maintains an appropriate range of glycemia. The involvement of small GTPases in adipogenesis, however, has not been systemically investigated. In this study, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled peptides, to identify differentially expressed small GTPase proteins during adipogenesis of cultured murine cells. We were able to quantify the relative levels of expression of 55 and 49 small GTPases accompanied by adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells, respectively. When compared with analysis conducted in the data-dependent acquisition (DDA) mode, the MRM-based proteomic platform substantially increased the coverage of the small GTPase proteome. Western blot analysis further corroborated the MRM quantification results for selected small GTPases. Interestingly, overall a significant number of small GTPases were down-regulated during adipogenesis. Among them, the expression levels of Rab32 protein were consistently lower in differentiated adipocytes than the corresponding undifferentiated precursors in both cell lines. Overexpression of Rab32 in 3T3-L1 and C3H10T1/2 cells prior to adipogenesis induction suppressed their differentiation. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome during adipogenesis, and we reveal a previously unrecognized role of Rab32 in adipogenic differentiation.
          Cover page: Targeted Proteomic Analysis of Small GTPases in Murine Adipogenesis.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2022)
          Mutations in the SOD1 (superoxide dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase-based proximity labeling, coupled with LC-MS/MS analysis, we uncovered 43 and 24 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of wild-type SOD1. Immunoprecipitation followed by western blot analysis indicated the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over the wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type protein. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to the diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, that is, through aberrant binding toward XPO5.
            Cover page: Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Modulate miRNA Biogenesis through Aberrant Interactions with Exportin 5.
            • 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 (2020)
              N6-Methyladenosine (m6A) in messenger RNA (mRNA) regulates its stability, splicing, and translation efficiency. Here, we explored how the expression levels of small GTPase proteins are regulated by m6A modulators. We employed a high-throughput scheduled multiple-reaction monitoring (MRM)-based targeted proteomic approach to quantify systemically the changes in expression of small GTPase proteins in cells upon genetic ablation of METTL3 (the catalytic subunit of the major m6A methyltransferase complex), m6A demethylases (ALKBH5 and FTO), or m6A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBH5 resulted in substantially diminished and augmented expression, respectively, of a subset of small GTPase proteins, including RHOB and RHOC. Our results also revealed that the stability of RHOB mRNA is significantly increased in cells depleted of METTL3, suggesting an m6A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBH5 also displayed higher discrepancies between protein and mRNA expression in paired primary/metastatic melanoma or colorectal cancer cells than those that are not. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome regulated by epitranscriptomic modulators of m6A, and our study suggests the potential of an alternative therapeutic approach to target the currently undruggable small GTPases.
                Cover page: Proteome-wide Interrogation of Small GTPases Regulated by N6-Methyladenosine Modulators.
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2023)
                The small GTPase superfamily of proteins are crucial for numerous cellular processes, including early development. The roles of these proteins in osteogenic differentiation, however, remained poorly explored. In this study, we employed a high-throughput targeted proteomic method, relying on scheduled liquid chromatography-multiple-reaction monitoring (LC-MRM) coupled with synthetic stable isotope-labeled peptides, to interrogate systematically the temporal responses of the entire small GTPase proteome during the course of osteogenic differentiation of H9 human embryonic stem cells. Our results demonstrated that the method offers high quantification accuracy, reproducibility, and throughput. In addition, the quantification results revealed altered expression of a large number of small GTPases accompanied with osteogenic differentiation, especially those involved with autophagy. We 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). Together, this study represents a novel application of a state-of-the-art analytical method, i.e., targeted quantitative proteomics, for revealing the progressive reprogramming of the small GTPase proteome during osteogenic differentiation of human embryonic stem cells, and our results revealed KRAS as a new regulator for osteogenesis.
                  Cover page: Targeted Proteomic Profiling Revealed Roles of Small GTPases during Osteogenic Differentiation
                  • Article
                  • Peer Reviewed
                  UC Riverside Previously Published Works (2023)
                  Osteogenesis is modulated by multiple regulatory networks. Recent studies showed that RNA modifications and their reader, writer, and eraser (RWE) proteins are involved in regulating various biological processes. Few studies, however, were conducted to investigate the functions of RNA modifications and their RWE proteins in osteogenesis. By using LC-MS/MS in parallel-reaction monitoring (PRM) mode, we performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins throughout the entire time course of osteogenic differentiation in H9 human embryonic stem cells (ESCs). We found that approximately half of the 127 detected RWE proteins were down-regulated during osteogenic differentiation, and they included mainly proteins involved in RNA methylation and pseudouridylation. Protein-protein interaction (PPI) network analysis unveiled significant associations between the down-regulated epitranscriptomic RWE proteins and osteogenesis-related proteins. Gene set enrichment analysis (GSEA) of publicly available RNA-seq data obtained from osteogenesis imperfecta patients suggested a potential role of METTL1 in osteogenesis through the cytokine network. Together, this is the first targeted profiling of epitranscriptomic RWE proteins during osteogenic differentiation of human ESCs, and our work unveiled potential regulatory roles of these proteins in osteogenesis. LC-MS/MS data were deposited on ProteomeXchange (PXD039249).
                    Cover page: Temporal Profiling of Epitranscriptomic Modulators during Osteogenic Differentiation of Human Embryonic Stem Cells.
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2017)
                    The ATP-sensitive potassium channel (KATP) functions as a metabo-electric transducer in regulating insulin secretion from pancreatic β-cells. The pancreatic KATP channel is composed of a pore-forming inwardly-rectifying potassium channel, Kir6.2, and a regulatory subunit, sulphonylurea receptor 1 (SUR1). Loss-of-function mutations in either subunit often lead to the development of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). PHHI is a rare genetic disease and most patients present with immediate onset within the first few days after birth. In this study, we report an unusual form of PHHI, in which the index patient developed hyperinsulinemic hypoglycemia after 1 year of age. The patient failed to respond to routine medication for PHHI and underwent a complete pancreatectomy. Genotyping of the index patient and his immediate family members showed that the patient and other family members with hypoglycemic episodes carried a heterozygous novel mutation in KCNJ11 (C83T), which encodes Kir6.2 (A28V). Electrophysiological and cell biological experiments revealed that A28V hKir6.2 is a dominant-negative, loss-of-function mutation and that KATP channels carrying this mutation failed to reach the cell surface. De novo protein structure prediction indicated that this A28V mutation reoriented the ER retention motif located at the C-terminal of the hKir6.2, and this result may explain the trafficking defect caused by this point mutation. Our study is the first report of a novel form of late-onset PHHI that is caused by a dominant mutation in KCNJ11 and exhibits a defect in proper surface expression of Kir6.2.
                      Cover page: A new familial form of a late-onset, persistent hyperinsulinemic hypoglycemia of infancy caused by a novel mutation in KCNJ11.
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