A chemoproteomic portrait of the oncometabolite fumarate

Abstract

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.

Discovery of protein modifications using high resolution differential mass spectrometry proteomics

“…Recently, LPS-stimulated macrophages were reported to produce itaconate, an electrophilic dicarboxylic acid that is similar to succinate and fumarate TCA cycle intermediates. Itaconate can chemically react with nucleophilic amino acids (Strelko et al 2011;Cordes et al 2016;Kulkarni et al 2019;Mills et al 2018). Indeed, we confirmed the generation of free itaconate and the expression of Acod1 (Irg1) aconitate decarboxylase enzyme responsible for its production in RAW264.7 cells upon LPS stimulation using small molecule mass spectrometry and Western immunoblotting, respectively (Figure 5a-c, Supplementary Figure 7).…”

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4–MyD88 Complex

“…It has also been reported that endogenous fumarate production through the citric acid cycle is associated with oncogenesis (57,58). We have demonstrated that other methyl fumarate-bearing small molecules (59) are vulnerable to enzymatic hydrolysis by carboxylesterases, but it is unclear whether DMF can be hydrolyzed twice to reveal the free-acid fumarate oncometabolite in appreciable concentrations.…”

Defective Function of α-Ketoglutarate Dehydrogenase Exacerbates Mitochondrial ATP Deficits during Complex I Deficiency

“…We identified and confirmed the succination sites of mitochondrial voltage-dependent anion channels (VDAC) 1 and 2 (Piroli et al, 2016). Since an acidic environment favors this modification (Kulkarni et al, 2019); mitochondrial matrix acidification due to reduced electron transport chain activity may contribute to increased succination. In this study, we further pursued and identified dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the TCA cycle.…”

“…Succination is a post-translational modification of protein cysteine thiols due to their non-enzymatic and irreversible modification by fumarate, generating S-2-succinocysteine (2SC) ( Figure S1A, Alderson et al, 2006;Frizzell et al, 2009;Merkley et al, 2014). Elevations in protein succination have been described in several tissues and conditions where fumarate is elevated, and is most frequently associated with reduced protein function (Kulkarni et al 2019;Manuel et al, 2017;Piroli et al 2014;Ternette et al 2014). We were the first to demonstrate increased protein succination of select proteins in the most pathologically affected regions, e.g.…”