Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

Metabolism and signaling intersect in the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the TCA cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand how metabolites function as covalent signals, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Building on previous studies, we applied a general reactivity probe to compile a dataset of cysteine residues sensitive to rescue of cellular FH activity. This revealed a broad upregulation of cysteine reactivity upon FH rescue, caused by an approximately equal proportion of transcriptional and posttranslational regulation in the rescue cell line. Gene ontology analysis highlights new targets and pathways potentially modulated by FH mutation. Comparison of the new dataset to literature studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation, and a foundation for future efforts to exploit this knowledge for cancer therapy.

, while the reactivity of only 17 cysteines were down-regulated to this extent 2 ( Fig. 2b, blue). This is consistent with our expectation of increased cysteine reactivity in FH rescue 3 (+/+) due to decreased accumulation of the electrophilic oncometabolite fumarate.

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Interestingly, the cysteines whose reactivity was most strongly increased upon FH rescue 6 predominantly mapped to the nucleus and cytosol (Fig. 2c), while the relatively few cysteines 7 whose reactivity was decreased by FH rescue were enriched in mitochondrial proteins (Fig. S1a).

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Hypothesizing that the latter effect may be due to compensatory upregulation of mitochondrial 9 proteins in FH-/-cells, we applied two complementary approaches to examine the relationship 10 between protein expression and cysteine reactivity in our dataset (Fig. 2d). First, we used 11 reductive dimethyl (ReDiMe) labeling to quantitatively assess changes in overall protein 12 abundance between the two cell lines (Table S3). 21 We obtained quantitative abundance data for 13 the parent proteins of one-third of cysteines whose relative reactivity was measured in UOK268 14 FH-/-and rescue cell lines (Table S4). Overall, relative cysteine reactivity and protein abundance 15 showed a strong correlation in the two HLRCC cell lines (Pearson's r = 0.57, Fig. S1b). Cysteines 16 exhibiting the greatest reactivity increases upon FH rescue (R≥2) also showed the most 17 augmented expression in this condition, consistent with the overall trend (Fig. S1c). Focusing on cysteine residues with increased reactivity upon FH-rescue (R≥2), we found 49 were also strongly 19 upregulated (≥2-fold decreased) at the protein level (Fig. 2d, Table S4), suggesting upregulated 20 protein expression. Conversely, the FH-dependent reactivities of 73 cysteines was relatively 21 unchanged, while an additional 35 shifted from R<2 to R>2 when correcting for protein abundance, consistent with the potential for these residues to be targets of FH-dependent 23 posttranslational modification (Fig. 2d, Table S4).

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To extend this analysis to FH-regulated (R≥2) cysteines whose proteomic abundance was not 26 sampled by ReDiMe we developed a second approach, examining cases where multiple IA-27 alkyne quantifiable cysteine residues were identified within a single polypeptide (Fig. 2d, Table   28 S5). Our hypothesis was that changes in expression levels should cause a consistent change in 29 reactivity across multiple quantified cysteine residues of a protein, while changes in cysteine post-

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Next, we explored changes in cysteine reactivity not explained by altered protein abundance 30 between the mutant and rescue UOK268 cell lines, which represent candidate sites of FH-31 dependent posttranslational modification (Fig. 4a) (Tables S4-6; Fig. 4a). Here, we focused on 32 119 "high confidence" residues which displayed an R≥2 following correction for protein abundance 33 or multiple cysteines (Fig. 2d, red), as well as an additional 236 "lower confidence" cysteines 34 whose parent proteins were not quantified by whole proteome LC-MS/MS analysis, and whose

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Motif analysis of cysteine-containing peptides whose reactivity was decreased by >2-fold by FH 18 inactivation found a high occurrence of flanking glutamate (E) and aspartate (D) residues, 19 independent of protein localization (Fig. 4g, Fig. S2, Table S10). These carboxylate amino acids 20 are not commonly found proximal to hyperreactive cysteines, and may contribute to fumarate 21 reactivity via hydrogen bonding or stabilization of surface-exposed alpha helices. 17 Overall, our survey of cysteine reactivity provides a novel resource for the identification of functional protein 23 activity changes involved the development, progression, or treatment of HLRCC.

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Concurrent with our initial chemoproteomic survey of FH-regulated cysteines in patient-derived   14 15

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Here we have reported a quantitative chemoproteomic analysis of cysteine reactivity in the it. Together, these data provide a fertile mechanistic resource from which to formulate biological 8 hypotheses regarding the development and progression of this genetically-defined kidney cancer.

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An unexpected finding of our study was that proteomes derived from distinct physiological 11 backgrounds demonstrate substantial heterogeneity in the adaptive response of reactive cysteine 12 residues to fumarate accumulation. This is typified by the distinct cohort of downregulated reactive

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Several recent studies have demonstrated that proteomic profiling of oncogene-induced 32 tumorigenesis can aid the design of new treatment strategies, 38-40 and two therapeutic hypotheses 33 are raised by our data. The first is that targeting pathways which demonstrate a compensatory 34 upregulation upon FH inactivation (either at the level of protein abundance or cysteine reactivity) 35 may serve to limit adaptation of cancer cells and trigger cell death. The second is that inhibiting 36 pathways found to be "damaged" by S-succination (R>2) or downregulated upon FH loss at the 37 whole proteome level may push essential pathways already poised on the precipice of failure over