The succinated proteome

Abstract

The post-translational modifications (PTMs) of cysteine residues include oxidation, S-glutathionylation, S-nitrosylation, and succination, all of which modify protein function or turnover in response to a changing intracellular redox environment. Succination is a chemical modification of cysteine in proteins by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in 3T3 adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes in mice. Increased succination of proteins is also detected in the kidney of a fumarase deficient conditional knock-out mouse which develops renal cysts. A wide range of proteins are subject to succination, including enzymes, adipokines, cytoskeletal proteins, and ER chaperones with functional cysteine residues. There is also some overlap between succinated and glutathionylated proteins, suggesting that the same low pKa thiols are targeted by both. Succination of adipocyte proteins in diabetes increases as a result of nutrient excess derived mitochondrial stress and this is inhibited by uncouplers, which discharge the mitochondrial membrane potential (ΔΨm) and relieve the electron transport chain. 2SC therefore serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes, and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic PTM of proteins by proteomics approaches.

Keywords: chemical modification of proteins; cysteine; diabetes; fumarate; succination.

FIGURE 1

Formation of 2-(S-succino)cysteine (2SC) by reaction of fumarate with cysteine residues in protein.

FIGURE 2

Detection of succinated proteins in adipocytes. A: Total cell lysates (30 μg protein) from adipocytes cultured in 5 or 30 mM glucose for 4 days were separated by 1D PAGE. 2SC-modified proteins were detected using polyclonal anti-2SC antibody. M indicates marker lane and numbers at right indicate molecular weights of marker proteins. B–E: Further separation of 150 μg protein from adipocytes grown in 5 or 30 mM glucose was analyzed by 2D gel electrophoresis across a 4–7 pH range. Duplicate gels were either stained with Sypro Ruby to observe total protein (B,C) or transferred to PVDF and immunoblotted with anti-2SC antibody to detect succinated proteins (D,E). Protein succination was increased in adipocytes cultured in 30 mM glucose (E) versus 5 mM (D) although there were no major changes observed in the Sypro total protein profile (B,C). Reprinted with permission from Frizzell et al. (2012), copyright 2012 the Biochemical Society.

FIGURE 3

Pathway leading to succination of proteins. This scheme outlines a proposed sequence of events resulting from high glucose concentration (glucotoxicity) in type 2 diabetes including the glucose-induced increase in ATP/ADP, ΔΨ_m, NADH/NAD⁺, and fumarate. Fumarate reacts directly with protein to form 2SC. Alternatively, protein is oxidized by ROS, generating a sulfenic acid, which is reversed by GSH. In this scheme, irreversible succination competes with reversible glutathionylation of protein. Accumulation of succinated and glutathionylated chaperone proteins in the endoplasmic reticulum inhibits protein folding, assembly, post-translational modification, transport and secretion, leading to ER stress. Excessive ER stress, exceeding the capacity of the Unfolded Protein Response (UPR), leads to apoptosis. Reprinted with permission from Frizzell, Lima, and Baynes (2011), copyright 2011 Informa Healthcare.

FIGURE 4

Distinguishing succination (C₄O₄H₄, 116.011 Da) from double carbamidomethylation (2 × C₂H₃NO, 2 × 57.021 Da = 114.042 Da). A: Collision-induced dissociation spectrum of the doubly charged peptide YMACCLLYR from mouse alpha-tubulin, from LC-ESI-MS analysis of a tryptic digest of mouse adipocyte proteins alkylated with iodoacetamide. The peptide is modified either by succination on the second cysteine residue, or by carbamidomethylation on both of the two cysteine residues. The b-ion series is shown in red and the y-ion series in dark blue. A doubly charged y-ion series is shown in light blue. Alternative peak assignments supporting double carbamidomethylation are shown in green. The spectrum was recorded in the ion trap mass analyzer of a Thermo Scientific LTQ Orbitrap mass spectrometer. B: Summary of observed fragmentation and database search scores for the succinated peptide. C: Results for the doubly carbamidomethylated peptide. Xcorr is the Xcorr score from Sequest (Eng, McCormack, & Yates, 1994); MS-GF spectral probability is the probability that a better-scoring peptide exists for this spectrum, calculated by the MS-GF software (Kim, Gupta, & Pevzner, 2008; Kim et al., 2010). Note that while the succinated peptide has better MS-GF and Xcorr scores, the precursor mass information supports the identification of the peptide as the doubly carbamidomethylated species. In addition, the prominent peak at m/z ~725 is only explained by double carbamidomethylation. The “deisotoping correction” is automatically applied on our pipeline in cases where the difference between theoretical and observed mass is very close to exactly 1 or 2 Da, and corrects for whole-Dalton errors in the observed mass that arise from occasional failures of the DeconMSN (Mayampurath et al., 2008) algorithm in identifying the monoisotopic mass. Here, the automatically applied correction is inappropriate, since the ~2 Da shift comes from the mass difference between succination and double carbamidomethylation.

FIGURE 5

Protein succination in diabetes and cancer. Protein succination increases in the diabetic adipocyte when glucotoxicity driven mitochondrial stress leads to an increase in mitochondrial membrane potential (ΔΨ_m) and NADH:NAD⁺, resulting in increased fumarate. The rise in fumarate leads to increased protein succination on a number of targets including GAPDH, adiponectin, and protein disulfide isomerase (PDI) resulting in decreased GAPDH activity, reduced adiponectin secretion and increased endoplasmic reticulum stress (ER stress), respectively. Independent of glucotoxicity, cancers which are derived from deletion or mutation of fumarate hydratase (FH) also result in elevated fumarate levels and subsequent protein succination. Aconitase 2 and Keap1 are succinated targets in FH-deficient cells, resulting in Nrf2 activation and reduced Aconitase 2 activity, both of which are associated with oncogenesis in this model.