Fumarase: From the TCA Cycle to DNA Damage Response and Tumor Suppression.

Fumarase is an enzyme of the tricarboxylic acid (TCA) cycle in mitochondria, but in recent years, it has emerged as a participant in the response to DNA double strand breaks (DSBs) in the nucleus. In fact, this enzyme is dual-targeted and can be also readily detected in the mitochondrial and cytosolic/nuclear compartments of all the eukaryotic organisms examined. Intriguingly, this evolutionary conserved cytosolic population of fumarase, its enzymatic activity and the associated metabolite fumarate, are required for the cellular DNA damage response (DDR) to double-strand breaks. Here we review findings from yeast and human cells regarding how fumarase and fumarate may precisely participate in the DNA damage response. In yeast, cytosolic fumarase is involved in the homologous recombination (HR) repair pathway, through its function in the DSB resection process. One target of this regulation is the resection enzyme Sae2. In human cells, fumarase is involved in the non-homologous end joining (NHEJ) repair pathway. Fumarase is phosphorylated by the DNA-dependent protein kinase (DNA-PK) complex, which induces the recruitment of fumarase to the DSB and local generation of fumarate. Fumarate inhibits the lysine demethylase 2B (KDM2B), thereby facilitating the dimethylation of histone H3, which leads to the repair of the break by the NHEJ pathway. Finally, we discuss the question how fumarase may function as a tumor suppressor via its metabolite substrate fumarate. We offer a number of models which can explain an apparent contradiction regarding how fumarate absence/accumulation, as a function of subcellular location and stage can determine tumorigenesis. Fumarate, on the one hand, a positive regulator of genome stability (its absence supports genome instability and tumorigenesis) and, on the other hand, its accumulation drives angiogenesis and proliferation (thereby supporting tumor establishment).

Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2.

One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the main DSB repair pathways. Fumarase is a mitochondrial enzyme which functions in the tricarboxylic acid cycle. Intriguingly, the enzyme can be readily detected in the cytosolic compartment of all organisms examined, and we have shown that cytosolic fumarase participates in the DNA damage response towards DSBs. In human cells, fumarase was shown to be involved in NHEJ, but it is still unclear whether fumarase is also important for the HR pathway. Here we show that the depletion of cytosolic fumarase in yeast prolongs the presence of Mre11 at the DSBs, and decreases the kinetics of repair by the HR pathway. Overexpression of Sae2 endonuclease reduced the DSB sensitivity of the cytosolic fumarase depleted yeast, suggesting that Sae2 and fumarase functionally interact. Our results also suggest that Sae2 and cytosolic fumarase physically interact in vivo. Sae2 has been shown to be important for the DSB resection process, which is essential for the repair of DSBs by the HR pathway. Depletion of cytosolic fumarase inhibited DSB resection, while the overexpression of cytosolic fumarase or Sae2 restored resection. Together with our finding that cytosolic fumarase depletion reduces Sae2 cellular amounts, our results suggest that cytosolic fumarase is important for the DSB resection process by regulating Sae2 levels.

Critical role for IL-1ß in DNA damage-induced mucositis.

ß-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of ß-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1ß as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1ß is induced by DNA damage via an NF-κB-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1ß receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1ß prevents epithelial tight junction dysfunction and alleviates mucositis in ß-TrCP-deficient mice. IL-1ß antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

Spermatogenesis rescue in a mouse deficient for the ubiquitin ligase SCF{beta}-TrCP by single substrate depletion.

beta-TrCP, the substrate recognition subunit of a Skp1-Cul1-F-box (SCF) ubiquitin ligase, is ubiquitously expressed from two distinct paralogs, targeting many regulatory proteins for proteasomal degradation. We generated inducible beta-TrCP hypomorphic mice and found that they are surprisingly healthy, yet have a severe testicular defect. We show that the two beta-TrCP paralogs have a nonredundant role in spermatogenesis. The testicular defect is tightly associated with cell adhesion failure within the seminiferous tubules and is fully reversible upon beta-TrCP restoration. Remarkably, testicular depletion of a single beta-TrCP substrate, Snail1, rescued the adhesion defect and restored spermatogenesis. Our studies highlight an unexpected functional reserve of this central E3, as well as a bottleneck in a specific tissue: a single substrate whose stabilization is incompatible with testicular differentiation.