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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2021)

Advances in mass spectrometry (MS) have greatly facilitated protein identification and quantification in complex sample matrices, it can combined with chemical proteomic method to selectively label, identify, and characterize target proteins in the entire human proteome which has been widely used to study functional subgroups of proteins, and provided valuable resources for systematic study of biology pathways. Cell signaling pathways can be largely effected by biological small molecules through their binding to target important proteins, so the discovery and understanding of molecular interactions between small molecules and their targets is critical in drug discovery. However, some of the observed effects of small molecule cannot be attributed to its interactions with their known binding proteins. Thus, there are likely other yet identified ligand-binding proteins. With this in mind, we focused on the discovery of novel binding proteins for several important biological small molecules. Our first study described a strategy used the synthesized affinity chemical lysophosphatidic acid(LPA) probe conjugated with LC-MS/MS analysis to enrich and identify putative LPA-binding proteins in the human proteome, and we further validated that ANXA5 and PGK1 can bind directly with LPA. We next relies on SILAC-based metabolic labeling, biotin-As probe pull-down and LC-MS/MS analysis, achieved the proteome-wide identification of arsenic-binding proteins in human cells and quantitatively characterized the arsenic-protein interactions, revealed HSPA4 as a potential new molecular target underlying the proteotoxic effects of arsenic exposure. Lastly, we employed isotope-coded ATP acyl-phosphate probes, in conjunction with a multiple-reaction monitoring (MRM)-based targeted proteomic method, for proteome-wide identifications of endogenous kinases that can bind to two N6-modified ATP derivatives. Each study shows MS-based chemical proteomic approaches can serve as a efficient tool for the discovery of ligand binding proteins and also provides valuable database for cell signaling mechanism study.

    Cover page: Chemical Proteomic Approaches Toward Understanding Cell Signaling and Arsenic Toxicity
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2022)
    Arsenic is a widespread environmental contaminant, and long-term exposure to arsenic in drinking water is known to be associated with the development of many human diseases. Identification of arsenic-binding proteins is important for understanding the mechanisms underlying the toxic effects of arsenic species. Here, we developed a chemoproteomic strategy, relying on the use of a biotin-As(III) probe, stable isotope labeling by amino acids in cell culture, and liquid chromatography-tandem mass spectrometry analysis, to identify quantitatively As(III)-binding proteins. Over 400 proteins were enriched from the lysate of HEK293T cells with streptavidin beads immobilized with the biotin-As(III) probe. Competitive labeling experiments in the presence or absence of p-aminophenylarsenoxide (PAPAO) revealed 51 candidate As(III)-binding proteins, including several molecular chaperones and cochaperones, that is, HSPA4, HSPA4L, HSPH1, HOP1, FKBP51, and FKBP52. We also validated, by employing western blot analysis, the ability of HSPA4, a member of heat shock protein 70 (HSP70) family, in binding with PAPAO and sodium arsenite in vitro. Together, our work led to the identification of a number of new As(III)-interaction proteins, and our results suggest that As(III) may perturb proteostasis partly through binding directly with molecular chaperones.
      Cover page: Chemoproteomic Approach for the Quantitative Identification of Arsenic-Binding Proteins.
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2019)
      Lysophosphatidic acid (LPA) is an endogenous cell signaling molecule, and dysregulation of LPA signaling pathways is accompanied by several types of cancer. Herein, we developed a chemical proteomic method for the proteome-wide identification of LPA-binding proteins. The method involves the synthesis of a desthiobiotin-conjugated LPA acyl phosphate probe for the covalent labeling, enrichment, and subsequent LC-MS/MS identification of LPA-binding proteins at the proteome-wide level. By conducting labeling reactions at two different probe concentrations (10 and 100 μM) in conjunction with an SILAC (stable isotope labeling by amino acids in cell culture)-based workflow, we characterized the LPA-binding capabilities of these proteins at the entire proteome scale, which led to the identification of 86 candidate LPA-binding proteins in HEK293T cells. Moreover, we validated that two of these proteins, annexin A5 and phosphoglycerate kinase 1, can bind directly with LPA. Together, we developed a novel LPA probe for the identification and characterizations of LPA-binding proteins from the entire human proteome. The method should be adaptable for the identification of other lipid-binding proteins.
        Cover page: Chemical Proteomic Profiling of Lysophosphatidic Acid-Binding Proteins
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2021)
        Poly- and perfluoroalkyl substances (PFASs) are widely used in industrial products and consumer goods. Due to their extremely recalcitrant nature and potential bioaccumulation and toxicity, exposure to PFASs may result in adverse health outcomes in humans and wildlife. In this study, we developed a chemoproteomic strategy, based on the use of isotope-coded desthiobiotin-perfluorooctanephosphonic acid (PFOPA) probe and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, to profile PFAS-binding proteins. Targeted proteins were labeled with the desthiobiotin-PFOPA probe, digested with trypsin, and the ensuing desthiobiotin-conjugated peptides were enriched with streptavidin beads for LC-MS/MS analysis. We were able to identify 469 putative PFOPA-binding proteins. By conducting competitive binding experiments using low (10 μM) and high (100 μM) concentrations of stable isotope-labeled PFOPA probes, we further identified 128 nonredundant peptides derived from 75 unique proteins that exhibit selective binding toward PFOPA. Additionally, we demonstrated that one of these proteins, fatty acid-binding protein 5 (FABP5), could interact directly with PFASs, including perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), and perfluorobutanesulfonic acid (PFBS). Furthermore, desthiobiotin-labeled lysine residues are located close to the fatty acid-binding pocket of FABP5, and the binding affinity varies with the structures of PFASs. Taken together, we developed a novel chemoproteomic method for interrogating the PFAS-interacting proteome. The identification of these proteins sets the stage for understanding the mechanisms through which exposure to PFASs confers adverse human health effects.
          Cover page: Chemoproteomic Approach toward Probing the Interactomes of Perfluoroalkyl Substances
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2021)
          Kinases catalyze the transfer of the γ-phosphate group from adenosine triphosphate (ATP) to their protein and small-molecule substrates, and this phosphorylation is a crucial element of multiple cell signaling pathways. Herein, we employed isotope-coded ATP acyl-phosphate probes, in conjunction with a multiple-reaction monitoring (MRM)-based targeted proteomic method for proteome-wide identifications of endogenous kinases that can bind to two N6-modified ATP derivatives, N6-methyl-ATP (N6-Me-ATP), and N6-furfuryl-ATP (a.k.a. kinetin triphosphate, KTP). We found that, among the ∼300 quantified kinases, 27 and 18 are candidate kinases that can bind to KTP and N6-Me-ATP, respectively. Additionally, GSK3α and GSK3β are among the kinases that can bind to both ATP analogues. Moreover, the in vitro biochemical assay showed that GSK3β could employ N6-Me-ATP but not KTP as the phosphate group donor to phosphorylate its substrate peptide. Molecular modeling studies provided insights into the differences between N6-Me-ATP and KTP in enabling the GSK3β-mediated phosphorylation. Together, our chemoproteomic approach led to the identification of endogenous kinases that can potentially be targeted by the two ATP analogues. The approach should be generally applicable for assessing endogenous kinases targeted by other ATP and purine analogues.
            Cover page: Proteome-Wide Characterizations of N 6‑Methyl-Adenosine Triphosphate- and N 6‑Furfuryl-Adenosine Triphosphate-Binding Capabilities of Kinases
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2020)
            Isoprenoid pyrophosphates are involved in protein prenylation and assume regulatory roles in cells; however, little is known about the cellular proteins that can interact with isoprenoid pyrophosphates. Here, we devised a chemical proteomic strategy, capitalizing on the use of a desthiobiotin-geranyl pyrophosphate (GPP) acyl phosphate probe for the enrichment and subsequent identification of GPP-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining stable isotope labeling by amino acids in cell culture (SILAC) and competitive labeling with low vs high concentrations of GPP probe, with ATP vs GPP acyl phosphate probes, or with the GPP probe in the presence of different concentrations of free GPP, we uncovered a number of candidate GPP-binding proteins. We also discovered, for the first time, histone deacetylase 1 (HDAC1) as a GPP-binding protein. Furthermore, we found that the enzymatic activity of HDAC1 could be modulated by isoprenoid pyrophosphates. Together, we developed a novel chemical proteomic method for the proteome-wide discovery of GPP-binding proteins, which sets the stage for a better understanding about the biological functions of isoprenoids.
              Cover page: Chemical Proteomic Profiling of the Interacting Proteins of Isoprenoid Pyrophosphates.
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2017)
              Arsenic is a ubiquitous environmental contaminant with widespread public health concern. Epidemiological studies have revealed that chronic human exposure to arsenic in drinking water is associated with the prevalence of skin, lung, and bladder cancers. Aberrant histone modifications (e.g., methylation, acetylation, and ubiquitination) were previously found to be accompanied by arsenic exposure; thus, perturbation of epigenetic pathways is thought to contribute to arsenic carcinogenesis. Arsenite is known to interact with zinc finger motifs of proteins, and zinc finger motif is present in and indispensable for the enzymatic activities of crucial histone-modifying enzymes especially the MYST family of histone acetyltransferases (e.g., TIP60). Hence, we reasoned that trivalent arsenic may target the zinc finger motif of these enzymes, disturb their enzymatic activities, and alter histone acetylation. Herein, we found that As3+ could bind directly to the zinc-finger motif of TIP60 in vitro and in cells. In addition, exposure to As3+ could lead to a dose-dependent decrease in TIP60 protein level via the ubiquitin-proteasome pathway. Thus, the results from the present study revealed, for the first time, that arsenite may target cysteine residues in the zinc-finger motif of the TIP60 histone acetyltransferase, thereby altering the H4K16Ac histone epigenetic mark. Our results also shed some new light on the mechanisms underlying the arsenic-induced epigenotoxicity and carcinogenesis in humans.
                Cover page: Arsenite Binds to the Zinc Finger Motif of TIP60 Histone Acetyltransferase and Induces Its Degradation via the 26S Proteasome.
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