Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Towards this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel a FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome, and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4C's known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease. Communication pubs.acs.org/JACS
1Hereditary cancer disorders often provide an important window into novel mechanisms supporting 2 tumor growth and survival. Understanding these mechanisms and developing biomarkers to identify 3 their presence thus represents a vital goal. Towards this goal, here we report a chemoproteomic 4 map of the covalent targets of fumarate, an oncometabolite whose accumulation marks the genetic 5 cancer predisposition syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). First, 6 we validate the ability of known and novel chemoproteomic probes to report on fumarate reactivity 7 in vitro. Next, we apply these probes in concert with LC-MS/MS to identify cysteine residues sensitive 8 to either fumarate treatment or fumarate hydratase (FH) mutation in untransformed and human 9 HLRCC cell models, respectively. Mining this data to understand the structural determinants of 10 fumarate reactivity reveals an unexpected anti-correlation with nucleophilicity, and the discovery of 11 a novel influence of pH on fumarate-cysteine interactions. Finally, we show that many fumarate-12 sensitive and FH-regulated cysteines are found in functional protein domains, and perform 13 mechanistic studies of a fumarate-sensitive cysteine in SMARCC1 that lies at a key protein-protein 14 interface in the SWI-SNF tumor suppressor complex. Our studies provide a powerful resource for 15 understanding the influence of fumarate on reactive cysteine residues, and lay the foundation for 16 future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy. Introduction 1 2 A major finding of modern cancer genomics has been the unexpected discovery of driver mutations 3 in primary metabolic enzymes. [1][2][3][4][5] Many of these lesions cause the characteristic accumulation of 4 "oncometabolites," endogenous metabolites whose accretion can directly drive malignant 5 transformation. 6 For example, mutation of fumarate hydratase (FH) in the familial cancer 6 susceptibility syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC) leads to high 7 levels of intracellular fumarate. 7-8 Fumarate has been hypothesized to promote tumorigenesis both 8 by reversibly inhibiting dioxygenases involved in epigenetic signaling, [9][10][11][12][13][14] as well as by interacting 9 with proteins covalently as an electrophile, forming the non-enzymatic posttranslational modification 10 cysteine S-succination ( Fig. 1). [15][16] This latter mechanism is unique to fumarate, and has been 11 proposed to contribute to the distinct tissue selectivity, gene expression profiles, and clinical 12 outcomes observed in HLRCC relative to other oncometabolite-driven cancers. [17][18] Consistent with 13 a functional role, recent studies have found that S-succination of Keap1 can activate NRF2-mediated 14 transcription in HLRCC. 19 Furthermore, global immunohistochemical staining of S-succination has 15 been applied to assess stage and progression of FH-deficient tumors, 20 suggesting the utility of this 16 modification as a biomarker...
Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene‐expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell‐permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3‐dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.
Metabolites regulate protein function via covalent and noncovalent interactions. However, manipulating these interactions in living cells remains a major challenge. Here, we report a chemical strategy for inducing cysteine S-succination, a nonenzymatic post-translational modification derived from the oncometabolite fumarate. Using a combination of antibody-based detection and kinetic assays, we benchmark the in vitro and cellular reactivity of two novel S-succination "agonists," maleate and 2-bromosuccinate. Cellular assays reveal maleate to be a more potent and less toxic inducer of S-succination, which can activate KEAP1-NRF2 signaling in living cells. By enabling the cellular reconstitution of an oncometabolite-protein interaction with physiochemical accuracy and minimal toxicity, this study provides a methodological basis for better understanding the signaling role of metabolites in disease.
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