Fumarase

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Introduction

Fumarase is an enzyme involved in the tricarboxylic acid (TCA) cycle in mitochondria, but evidence in recent years suggests that it is also a participant in DNA double strand breaks (DSBs) in the nucleus. In fact, this enzyme is dual-targeted and can be easily detected in the mitochondrial and cytosolic/nuclear compartments of all eukaryotes. This evolutionary conserved cytosolic fumarase, its enzymatic activity and the related metabolite fumarate are required for the cellular DNA damage response (DDR) to double-strand breaks. In yeast, cytoplasmic fumarase is involved in the homologous recombination (HR) repair pathway. In human cells, it 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 recruitment of fumarate to DSBs and local production of fumarate. Fumarate inhibits lysine demethylase 2B (KDM2B), thereby promoting the dimethylation of histone H3 and repairing breaks through the NHEJ pathway.

Fumarase, Its Canonical Function and Subcellular Locations

Fumarase is a member of class II fumarase enzymes that are conserved from prokaryotes to humans. In S. cerevisiae, fumarase is encoded by the FUM1 gene and forms a homotetramer with a molecular weight of approximately 200 kDa. It is responsible for catalyzing the hydration of fumarate to L-malate and the reverse dehydration reaction. Fumarase is present in mitochondria and is involved in the tricarboxylic acid (TCA) cycle. In addition to mitochondria, fumarate can also be found in the cytosolic compartment, where its cytoplasmic localization is highly conserved. This dual localization is called "echoforms," indicating repetitious forms of the same protein in the cell. There are a number of known mechanisms that regulate the subcellular distribution of fumarase in eukaryotes. Both the cytoplasmic and mitochondrial fumarase echoforms of S. cerevisiae are encoded by the FUM1 gene. During translation, a subset of the FUM1 translation products, which are partially translocated, fold outside mitochondria, and a mechanism known as reverse translocation prevents full mitochondrial import. Upon translation termination, these folded translation products remain in the cytosol, which constitutes the cytoplasmic fumarase population.

The human homolog of fumarase, termed fumarate hydratase (FH) is expressed by a single gene. The fumarase gene promoter contains multiple transcription start sites from which two sets of fumarase mRNAs are transcribed. The first group includes transcripts that are translated into proteins containing the fumarate mitochondrial targeting sequence (MTS), and the second group is translated into fumarase proteins lacking this sequence. Subsequently, these two versions of the protein constituted the mitochondrial and cytoplasmic echoforms of fumarase, respectively.

Figure 1. Mechanisms of fumarase dual targeting in different organisms (Leshets, M.; et al. 2018)

The Paradoxical Nature of the Mechanism By Which Fumarase Acts as a Tumor Suppressor

There are two proposed models for the activity of fumarase as a tumor suppressor. First, the loss of fumarase block the TCA cycle in mitochondria, leads to the accumulation of fumarate, which stabilizes HIF. The second model suggests that the loss of fumarate reduces the ability of cells to produce fumarate, thereby compromising genomic stability. In both models, fumarate is the key effector molecule, which induces an oncogenic effect in the first model and acts as an oncogenic inhibitor in the second model. In both models, the inactivation of fumaric acid in cells is thought to result in the accumulation of fumarate due to blockage of the TCA cycle and an inability to generate fumarate for the DDR. This discrepancy raises the question of how these two apparently contradictory models can be reconciled.

The first plausible answer to this question might depend on the possibility that, in order to function in the DDR, fumarase must produce high concentrations of fumarate near the cellular DNA near the DSB. The second possibility is an extension of the first, argues that the two mechanisms by which fumarase acts as a tumor suppressor occur at different stages of tumor development. In the first stage, biallelic inactivation of fumarase in a single tumor cell disrupts the ability to produce fumarate in the proximity of the cellular DNA, thereby reducing genome stability. In the second stage, the proliferation of fumarase-deficient cells may form a tightly-located cell population in which the fumarate concentration required for HIF stabilization can be achieved.

Figure 2. Two stage model of fumarase depletion leading to tumorigenesis (Leshets, M.; et al. 2018)