3. Predicted Existence of Extra-Mitochondrial TCA (eTCA) Cycle
As depicted in
Figure 1, the TCA cycle comprises eight enzymes (CS, aconitase, IDH, OGDH, succinyl-CoA synthetase, SDH, fumarase, and MDH). These enzymes are mainly distributed in the mitochondria, although most of these enzymes are also detected in the cytosolic region (
Table 1 and
Figure 2). Pyruvate, located at the interface between glycolysis and the TCA cycle, is an important intermediate. As mentioned above, the presence of two forms of CS, catalyzing the formation of acetyl-CoA, reinforces the importance of this interface. The mitochondrial TCA cycle has been extensively studied
[31], but even after these extensive studies, the roles of cytosolic TCA cycle enzymes, including CS, are not well understood.
Figure 2. TCA cycle-related enzymes. The TCA cycle is the second stage of cellular respiration. This cycle occurs in the matrix of mitochondria and is catalyzed by eight enzymes. Interestingly, enzymes function in cytosolic forms, such as eCS, aconitase, isocitrate dehydrogenase, fumarase, malate dehydrogenase. Particularly, isocitrate dehydrogenase and fumarase were reported as human disease-related enzymes.
Aconitase catalyzes the isomerization of citrate and isocitrate. In mammals, the ACO1 (cytosolic aconitase), also known as iron regulatory protein 1 (IRP1), plays a role in sensing cellular iron homeostasis
[32]. Cytosolic aconitase, upon losing an iron-sulfur cluster, becomes IRP1
[32]. Cytosolic aconitase belongs to a family of RNA-binding proteins that modulate iron metabolism in vertebrates, contributing to optimal cell growth
[33].
Fumarase (also known as fumarate hydratase) is an enzyme found in both the mitochondria and the cytoplasm, and is extensively found in microorganisms, plants, and animals
[34]. In mitochondria, fumarase catalyzes the reversible formation of l-malate from fumarate. In plants, cytosolic fumarase is also involved in fumarate formation
[35]. Plants inhabiting cold environments are adapted to cold and freezing temperatures. Cytosolic fumarase-mediated accumulation of fumarate is essential for adaptation of
Arabidopsis thaliana to cold
[36].
Molecular cues of fumarases have been obtained from microorganisms
[37]. In microorganisms, fumarases are divided into two classes, I and II, with distinct properties. Bacteria have three fumarase genes:
fumA,
fumB, and
fumC. Their products, FUMA, FUMB, and FUMC, are biochemically divided into two distinct classes. Class I fumarases, FUMA and FUMB, are homologous to fumarases identified in
Euglena. FUMA and FUMB are differentially regulated; FUMA functions in the TCA cycle, while FUMB supplies fumarate as an anaerobic electron acceptor. The class II fumarase, FUMC, is homologous to fumarases identified in
Bacillus subtilis,
Saccharomyces cerevisiae, and mammals. Class II fumarases are structurally conserved, with highly homologous sequences across species.
Human fumarase exists in both cytosolic and mitochondrial forms with extended N-terminus, differing only in the translation initiation site
[34]; however, its role in the cytoplasm is unclear. Recent evidence has demonstrated that fumarase functions as a tumor suppressor in mammals
[34]. Fumarase functions in the mitochondria, but in recent studies, it has emerged as a participant in the response to DNA double-strand breaks in the nucleus
[34]. In humans, fumarase deficiency causes the formation of kidney tumors in hereditary leiomyomatosis and renal cell carcinoma (HLRCC)
[18]. HLRCC is a rare genetic disease with smooth muscle growth on the skin and uterus, and is associated with a risk of developing kidney (renal) cancer. A mutation in a gene encoding fumarase is believed to cause all known cases of HLRCC. Specifically, the cytosolic form of fumarase is involved in the onset of this disease. Cytosolic fumarase plays a role in repairing DNA double-strand breaks, both through its movement from the cytoplasm to the nucleus, and enzymatic activity
[38]. In other words, when fumarase is absent from cells, the DNA repair mechanism is impaired, but the administration of a high concentration of fumarate reverses this abnormality
[34]. This result raises the possibility that fumarate moves actively or passively across organelles, including the mitochondria and the nucleus, and possibly other organelles.
Cytoplasmic and mitochondrial forms of isocitrate dehydrogenases (IDH1 and IDH2) dehydrate isocitrate to form oxalosuccinate
[10]. Point mutations in both
IDH1 and
IDH2 have been frequently associated in the pathogenesis of a subset of gliomas, mainly low-grade gliomas and secondary glioblastomas
[10]. Somatic mutations in
IDH1 cause disturbances in cell metabolism, a common feature of gliomas
[10]. In addition, a novel inactivating mutation in
IDH has been discovered in high-grade astrocytomas
[39].
MDH is an enzyme that converts l-malate to oxaloacetate. In eukaryotic cells, MDH has two isoforms, MDH1 and MDH2
[29]. MDH1 is a cytoplasmic protein that transports malate into mitochondria, whereas MDH2 is a mitochondrial protein, which is part of the TCA cycle. In honeybees, three alleles encode cytosolic MDH: F, M, and S
[40]. These alleles have temperature-dependent fitness benefits.
On the other hand, the cytoplasmic forms of the three enzymes, OGDH, succinyl-CoA synthetase, and SDH, remain unidentified.
Generally, ATP production using the TCA cycle occurs under aerobic conditions. Notably, cancer cells rely on glycolysis for ATP production, even under aerobic conditions
[41], referred to as the Warburg effect (aerobic glycolysis)
[20][21]. Although cancer cells mainly generate ATP via this process, its functions remain unclear
[42]. As citrate synthesis is essential for a switch from glycolysis to the TCA cycle, dysfunction or gain-of-function of CS could be a possible cause for the Warburg effect.
Due to the lack of MTS in eCS, it is predominantly located in the sperm acrosome and not in the mitochondria. Therefore, eCS may be involved in energy production for sperm function via the eTCA cycle independently. The findings obtained from oximetric and biochemical analyses of retinal rod outer segments support the possible existence of the eTCA
[43]. Additionally,
eCs-KO male mice exhibited decreased fertility with aging (>6-month-old), suggesting an increase in eCS contribution for sperm function in older mice. This implies the possibility of the existence of the TCA cycle in the extra-mitochondrial space
[43].