• Site-specific mapping of a distinct type of oxiPTM
    Site-specific mapping of a distinct type of oxiPTM
  • MTRP enables HT target discovery of electrophiles
    MTRP enables HT target discovery of electrophiles
  • Proteome-wide assessment of drug bioactivation
    Proteome-wide assessment of drug bioactivation
  • Chemical diversity of 4-ONE modifications in cells
    Chemical diversity of 4-ONE modifications in cells
  • A potential protein degradation mechinary
    A potential protein degradation mechinary

Expanding the Redox Proteomics Toolbox

The nucleophilic thiol group allows cysteines to undergo a broad range of chemical modifications. Thiol-based protein oxidation (S-sulfenylation, S-sulfinylation, S-Glutathionylation, etc.) by exogenous and endogenous reactive oxygen species (ROS) is a crucial mechanism in cell signaling. To gain a better understanding of how these thiol modifications affect protein functions in normal or stressed biological systems, we should first know which cysteinyl thiols on a protein can be oxidized or modified. In other words, identification of protein targets of thiol oxidation is crucial to understanding of their roles in biology and disease. Our research programs develop two complementary site-centric chemoproteomic strategies to systematically quantify thiol reactivities and to globally map distinct types of thiol oxidation in native proteomes (Fig. 1). With these tools in hand, we redefine the hydrogen peroxide-dependent redoxome in human cells and generate the first site-centric S-sulfenylome and S-sulfinylome datasets. We also develop the first web portal database, called OXID, for sharing and integrating redox proteomics. These works not only expand the landscape of thiol redox proteome in human cells, but also suggest novel redox mechanisms of several proteins with key biological functions, such as SIRT6 and APIP. In collaboration with a diverse group of biologists worldwide, now we are applying our chemoproteomic toolbox to various model organisms, including M. musculus, C. elegans, D. melanogaster, and A. thaliana, and to study cysteine-mediated redox regulation in a range of physiological processes and adaptive responses.

Representative publications:

(1) Yang J, et al., The Expanding Landscape of The Thiol Proteome. Mol Cell Proteomics, 2016, 15: 1-11

(2) Fu L, et al., A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes. Nat Protoc. in press

(3) Fu L, et al. Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity with Cysteines Across Human Proteomes. Mol Cell Proteomics, 2017, 16:1815-1828 

(4) Fu L, et al., Proteome‐Wide Analysis of Cysteine S‐Sulfenylation Using a Benzothiazine‐Based Probe. Curr Protoc Protein Sci. 2019, 95(1): e76 

(5) Gupta V, et al. Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles. J Am Chem Soc, 2017, 139: 5588−5595

(6)  Akter S, et al. Chemical Proteomics Reveals New Targets of Cysteine Sulfinic Acid Reductase. Nat Chem Biol. 2018, 14:995-1004

(7)  Petrova B, et al. Dynamic Redox Balance Directs the Oocyte-to-Embryo Transition via Developmentally Controlled Reactive Cysteine Changes. Proc Natl Acad Sci U S A. 2018, 115:E7978-E7986

(8) Pei J, et al. Diurnal oscillations of endogenous H2O2 sustained by p66Shc regulate circadian clocks. Nat Cell Biol. 2019, 21(12): 1553-1564

Figure 1. Traditional chemistry-based strategy VERSUS  State-of-the-art site-centric chemoproteomics
  • Traditional chemistry-based strategy
    Traditional chemistry-based strategy
  • Our site-centric chemoproteomic strategy
    Our site-centric chemoproteomic strategy

Discovering Unexpected Protein PTMs Using Chemoproteomics  

We recently develop a generalized, quantitative chemoproteomic platform that can be broadly applicable to the analyses of  biorthogonal-chemically engineered PTMs. A key feature of this method is the use of light and heavy-labeled Azido-UV cleavable-biotin reagents (Fig. 2), which provides a means not only to site-specifically and quantitatively compare abundances of the protein PTMs, but also to minimize the false discovery rate in a large-scale proteomic analysis. In combination with the blind search tools like TagRecon, or pFind-alioth that enable the identification of all possible mass shifts on a detected peptide sequence, our chemoproteomic approach can also be applied to discover unexpected PTMs labeled by activity-based ‘clickable’ probes. Using this strategy, we successfully identify a previously unknown 4-oxo-2-nonenal (an endogenous lipid electrophile) derived pyrrole-adduction and discover a novel type of protein carbonyl product, N-terminal formyl protein degradants. We foresee that, in combination with new activity-based probes, our chemoproteomics-based strategy can be used to discover more unexpected PTMs with certain functional groups. 

Representative publications:

(1) Sun R, et al. Chemoproteomics Reveals Chemical Diversity and Dynamics of 4-Oxo-2-Nonenal Modifications in Cells. Mol Cell Proteomics, 2017, 16:1789-1800 (Editors' Highlight)

(2) Yang J, et al. Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells. Anal Chem. 2015, 87: 2535-41 (Editors' Highlight)

(3) Tian C, et al. Chemoproteomics Reveals Unexpected Lysine/Arginine-Specific Cleavage of Peptide Chains as a Potential Protein Degradation Machinery. Anal Chem. 2017, 90: 793-800

Figure 2. Site-centric quantitative chemoproteomic workflow for global profiling of unexpected protein PTMs (ABP: Activity-based Probe)

Deconvoluting Protein Targets of Thiol Reactive Chemicals

Although our chemoproteomic platforms are originally designed for global profiling of protein oxidation and other PTMs, they can be easily adapted to  globally map the cellular targets of thiol-reactive electrophiles, such as electrophilic natural products, reactive drug metabolites (RDMs), and targeted covalent inhibitors (e.g., afatinib, ibrutinib, neratinib, dacomitinib, and other FDA-approved irreversible kinase inhibitors). For example, the QTRP analysis of a pathogenic bacterium (Pseudomonas syringae) revealed ~100 potential cysteine targets of the Arabidopsis defense compound sulforaphane. Among these QTRP hits, C209 of HrpS (a key transcription factor controlling bacterial type III secretion system gene expression) was found to be essential for bacterial sensitivity to sulforaphane in vitro and sensitivity to plant defenses in vivo. In another example, this platform was used to determine the reactivity of thousands of proteomic cysteines, including active sites of most major CYP450 isoforms, toward RDMs of diclofenac formed in human liver microsomes in vitro and mouse liver in vivo48. Unlike traditional biochemical assays for assessing bioactivation potential of drugs, notably, this platform does not require prior information about metabolic transformation of the tested drugs. Moreover, it can provide a global view of site-specific target profile of potential RDMs, thereby enabling rapid risk assessment of drug-drug interactions and drug toxicity.

Moreover, building upon QTRP, we also develop a chemoproteomic method called multiplexed thiol reactivity profiling (MTRP) that enables high-throughput target identification of thiol reactive substances. Using the MTRP method, we reveal CSE1L/CAS (Also known as Exportin-2) as one of major functional targets of gambogic acid, a potent anti-tumor natural product from Garcinia hanburyi. MTRP also allow us to perform a quantitative comparison of seven structurally diversified alpha,beta-unsaturated gamma-lactones. The analysis not only provides insights into the relative proteomic reactivity and target preference of diverse structural scaffolds coupled to a common reactive motif, but also reveals a variety of potential druggable targets with liganded cysteines. 

We believe that both QTRP and MTRP approaches would greatly facilitate the analysis of target profile and engagement of other thiol reactive covalent ligands and drugs. Meanwhile, we aim to continuingly optimize these approaches to further facilitate the analysis of target profile and engagement of other bioactive chemicals targeting functionally important cysteines, especially targeted covalent inhibitors for anti-cancer treatment (e.g. Afatinib, Ibrutinib, Neratinib). Ultimately, our goal is to advance our chemoproteomic technology into the clinical setting to understand covalent drug-target interactions and to identify new therapeutically relevant targets. For instance, we were now carrying out a large-scale survey of target landscape of clinical covalent drugs with potential cysteine reactivity, which might provide insights into the relative proteomic reactivity and target preference of diverse structural scaffolds, thereby expanding the “druggable” targets in the human proteome. More interestingly, this analysis may help us find drugs that can be repurposed for treatment of additional diseases. 

Representative publications:

(1) Wang W, et al., An Arabidopsis Secondary Metabolite Directly Targets Expression of the Bacterial Type III Secretion System to Inhibit Bacterial Virulence. Cell Host Microbe. 2020, 27(4): 601-613.
(2) Tian C, et al., Multiplexed Thiol Reactivity Profiling for Target Discovery of Electrophilic Natural Products. Cell Chem Biol. 24(11):1416-1427
(3) Sun R, et al., A Chemoproteomic Platform to Assess Bioactivation Potential of Drugs. Chem Res Toxicol. 2017, 30: 1797-1803

Grants:

1.  Redox proteomics (NSFC:21922702)
2
. Harnessing quantitative chemoproteomics to reveal the target landscape of clinical covalent drugs(81973279)
3. Proteome-wide discovery of the substrates of sulfiredoxin and their roles in regulating oxidative susceptibility of tumor cells(NSFC:31770885)

4. Quantitative chemoproteomic analysis of dynamic and tumor-specific target profile of gambogic acid and its global regulatory network Study(NSFC: 81573395)

5.  Oxidative aggregation of APIP and its role in biological regulation (NSFC: 31500666)

6.  Oxidation of APIP and its role in redox regulation of apoptosis (BNSF:5162009)

7.  Beijing Nova Program (No. Z171100001117014)

8.  Youth talent support program of Beijing (No. 201700021223ZK16)