Aberrant succination of proteins in fumarate hydratase‐deficient mice and HLRCC patients is a robust biomarker of mutation status

Abstract

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.

Acetoacetate Accelerates Muscle Regeneration and Ameliorates Muscular Dystrophy in Mice

“…Collectively, these findings support the notion that AA is capable of functioning as a signaling molecule in regulating muscle cell function. Interestingly, recent reports have demonstrated that abnormally accumulated fumarate plays a novel role in the irregular modification of cellular proteins (succination of KEAP1) and Nrf2 (NFE2-related factor 2) signaling in fumarate hydratase-deficient cells and that these functions are independent of its ability to inhibit ␣-ketoglutarate-dependent dioxygenases (17,(73)(74)(75). Thus, our findings and the results of prior studies collectively indicate that AA could regulate cellular activity independent of its energetic role.…”

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome

“…Increased succination in biological samples is restricted to situations where fumarate concentrations are significantly increased, for example, 2SC levels are specifically increased in cancers derived from fumarate hydratase mutations (6,8), and in the metabolically overwhelmed adipocyte in type 2 diabetes (4,5,9). Glucotoxicity driven mitochondrial stress in the adipocyte prevents the oxidation of NADH, facilitating the accumulation of fumarate because of the inhibition of the Krebs cycle NAD ϩ -dependent dehydrogenases (9).…”

“…Fumarate concentrations are increased during adipogenesis and adipocyte maturation (2,3), and the excess of glucose and insulin leads to augmented protein succination in the adipose tissue of type 2 diabetic mice (4,5). Protein succination is also specifically increased in fumarate hydratase deficient hereditary leiomyomatosis and renal cell carcinoma (HLRCC), because of the decreased conversion of fumarate to malate (6,7). In both cases, intracellular fumarate concentrations are elevated; in fumarate hydratase deficient cells, the fumarate concentration is about 5 mM (8), whereas fumarate levels increase up to fivefold in adipocytes grown in the presence of high (30 mM) versus normal (5 mM) glucose concentrations (2).…”

Mitochondrial metabolic remodeling in response to genetic and environmental perturbations

“…The high concentrations of fumarate produced in FH‐deficient cells have been shown to result in reversal of part of the urea cycle, producing argininosuccinate and disrupting arginine metabolism within FH‐deficient cells 80, 81. In addition, fumarate has been shown to react with enzymes and other intracellular metabolites in a process known as succination 82, 83, 84. This posttranslational modification has been shown to inhibit mitochondrial aconitase activity in FH‐deficient cells, resulting in further truncation of the TCA cycle82 (Figure 2).…”