Succination of Protein Thiols during Adipocyte Maturation

Abstract

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased >5-fold during adipogenesis in medium containing 30 mM glucose, producing a >10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N ⑀ -(carboxymethyl)lysine and N ⑀ -(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one-and two-dimensional electrophoresis and ϳ60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.The adipocyte is increasingly recognized as a dynamic cell that readily adapts to the changing nutritional status of the body. In a state of over-nutrition, the adipocyte responds by synthesizing and storing triglycerides, a process that may eventually lead to obesity and insulin resistance and then to diabetes.Chemical modification of proteins by advanced glycoxidation and lipoxidation end products (AGE/ALEs) 3 is increased in diabetes and is strongly implicated in the development of diabetic complications (1-3). However, relatively little is known about the chemical modification of proteins in adipocytes or adipose tissue or the possible role of chemical modifications of proteins in the regulation of adipocyte metabolism during adipogenesis or diabetogenesis.In general, AGE/ALEs are derivatives of lysine and arginine residues, formed by reaction of the amino or guanidino groups on protein with electrophilic intermediates in carbohydrate and lipid autoxidation or metabolism (4). In contrast to extracellular proteins, the lower pK a sulfhydryl group of cysteine residues is a more likely target for modification by electrophiles on intracellular proteins. We recently described S-(2-succinyl)cysteine (2SC) as a novel chemical modification of cysteine residues in proteins, formed by a...

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

“…Polyvinylidene difluoride (PVDF) membranes and ECL Plus chemiluminescent substrate were from GE Healthcare (Piscataway, NJ). The preparation of the polyclonal anti-2SC antibody has been described previously (2). The following commercial antibodies were used: ␣-tubulin DM1A, VDAC2 9412 from Cell Signaling Technology, Inc.…”

“…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). In the adipocyte the increase in fumarate and succinated proteins develops as a direct result of mitochondrial stress induced by nutrient excess.…”

“…Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel bio- We previously identified the formation of S-(2-succino)cysteine (2SC) 1 (protein succination) as a result of the irreversible reaction of fumarate with reactive cysteine thiols (1,2). 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).…”

“…These data suggest that fumarate is a novel bio- We previously identified the formation of S-(2-succino)cysteine (2SC) 1 (protein succination) as a result of the irreversible reaction of fumarate with reactive cysteine thiols (1, 2). 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).…”

A computational analysis of S-(2-succino)cysteine sites in proteins

“…Originally detected in plasma proteins [10], including albumin, this post-translational Cys modification type (referred as protein succination) has been described in models of diabetes/obesity, fumarate hydratase (FH)-related diseases, and Leigh syndrome. Increased levels of succinated proteins have been found in murine 3T3-L1 adipocytes cultured in high glucose medium (30 mM, compared with a physiological level of 5 mM), as well as in tissues from streptozotocin-treated rats, db/db (leptin receptor deficient), ob/ob (leptin deficient), and diet-induced obese mice [11][12][13][14]. It has been proposed that nutrient excess results in elevated ATP:ADP, NADH:NAD + , and mitochondrial membrane potential, and that the increased NADH:NAD + inhibits oxidative phosphorylation, leading to a persistent accumulation of mitochondrial intermediates including FA, which, in turn, causes protein succination [15].…”