Succinocysteine: a Post-Translational Modification and Biomarker of Complex Cellular Dysfunction

What is succination?

Protein succination is a post-translational modification formed by a reaction between the tricarboxylic acid cycle intermediate fumarate with protein cysteines to form S-(2-succino)cysteine (2SC). With many cysteines having key biological functions, their succination is important to understand for normal and dysfunctional cell processes.??

Cysteine predominantly exists in the thiolate form and can act as a reactive nucleophile. Cysteine succination can occur non-enzymatically and is formed by a Michael addition reaction between fumarate and the free thiol groups of protein cysteines at physiological pH. Fumarate is a weak electrophile, and its modification of thiols is highly pH dependent within physiological ranges. The thioether bond of 2SC is considered stable to acid hydrolysis and irreversible. Consequently, succination can be selective towards functional, low pKa cysteine residues in proteins, such as catalytic cysteine residues in enzymes.?

Why is succination important?

A wide range of proteins are subject to succination, including enzymes, adipokines, cytoskeletal proteins and ER chaperones and succination has been shown to have roles in regulatory biology. An increase in succination of adipocyte proteins is seen in diabetes mellitus and results from nutrient excess derived mitochondrial stress. Succination at critical cysteine residues can result in the inactivation of enzymatic activity or protein function in many biological processes and the succination of glutathione has been shown to increase oxidative stress and cellular senescence.

For example, the succination of key components of the iron-sulfur cluster biogenesis family of proteins, Iscu and Nfu1, lead to defects in iron-sulfur biosynthesis required for respiratory chain complexes. Succination of glutathione has been shown to increase oxidative stress and cellular senescence. The loss of fumarate hydratase (FH), the enzyme that catalyzes the reversible hydra fumarate's reversible hydration/dehydration tion/dehydration of fumarate to L-malate, contributes to the accumulation of fumarate and spontaneous succination. FH deficiency leads to the inactivation of the E3 ubiquitin ligase Keap1 by succination, which promotes the stabilization of NRF2 and activation of the antioxidant pathway. Keap1 also plays a key role in controlling tumorigenesis.

2SC – the biomarker of health

2SC is considered a biomarker for mitochondrial stress in obesity, insulin resistance and diabetes. The succination of adiponectin is increased in adipocytes and adipose tissue of type 2 diabetic mice. Adiponectin succination blocks the formation of oligomeric species and secreted forms of adiponectin, which contributes to reduced levels of plasma adiponectin in diabetes. Succination causes irreversible inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) resulting in the loss of activity in the muscle of diabetic rats. The elucidation of the succinated proteome will provide insight into the role of succination in regulatory biology and determine its effect on cellular dysfunction.

Dysregulation succination of the proteome is also an important diagnostic tool for certain cancers. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome is a genetic disorder in which mutant fumarate hydratase leads to an increased occurrence of cutaneous and uterine leiomyomas and renal cell carcinoma (RCC). HLRCC individuals have high incidence of leiomyoma formation, particularly in women aged 25 to 30. In contrast, RCC typically only affects around 25% of carriers. Unfortunately, RCC associated with HLRCC is particularly aggressive, with many patients not surviving 5 years after diagnosis. The presentation and histology of RCC cover a wide spectrum further compounding the issue of early HLRCC tumour identification. No specific biomarkers for HLRCC renal tumours are currently available. In HLRCC patients the functional loss of fumarate hydratase causes an increase in cellular levels of fumarate which can react with cysteine sulfhydryl groups to form 2SC. The anti-2SC antibody is uniquely positioned to detect an increase in succination levels due to an increase in fumarate levels. The anti-2SC antibody has been successfully shown to identify high 2SC samples within predicted HLRCC patients compared to control samples. 2SC is set to become a pivotal biomarker for the differentiation of HLRCC renal tumours. Further investigations in to 2SC immunohistochemistry could provide the lifeline these HLRCC patients are waiting for. Compared to control samples, the anti-2SC antibody has been successfully shown to accurately identify high 2SC samples within predicted HLRCC patient.

The anti-2SC antibody from Biosynth, staining for a brighter future

At Biosynth, we provide antibodies for a diverse range of life sciences. A highlight of our collection are our polyclonal anti-2SC antibodies which successfully detect succinated cysteine residues with high affinity. ?These antibodies are an essential tool for understanding the consequences of the loss of the tumour suppressor fumarate hydratase.

It acts as a surrogate for detecting the accumulation of fumarate which is a feature of fumarate hydratase deficient tumours. Furthermore, the anti-2SC antibody will be essential to identify new targets of succination driven by fumarate accumulation, not only in cancer but also in diabetes.

Anti-2SC antibody, 1mg/ml CRB2005017_3

The elucidation of the succinated proteome will provide insight into the role of succination in regulatory biology and determine its effect on cellular dysfunction.

Biosynth has more than 50,000 antibodies for research and development, as well as custom antibody production services. Find out more at www.biosynth.com