Mitochondrial Factors in the Cell Nucleus

Citation: González-Arzola, K.; Díaz-Quintana, A. Mitochondrial Factors in the Cell Nucleus. Int. J. Mol. Sci. 2023,24, 13656. https:// doi.org/10.3390/ijms241713656 Academic Editor: Martin Van der Laan Received: 18 July 2023 Revised: 31 August 2023 Accepted: 31 August 2023 Published: 4 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Review Mitochondrial Factors in the Cell Nucleus Katiuska González-Arzola 1,2 and Antonio Díaz-Quintana 2,3,* 1Centro Andaluz de Biología Molecular y Medicina Regenerativa—CABIMER, Consejo Superior de Investigaciones Científicas—Universidad de Sevilla—Universidad Pablo de Olavide, 41092 Seville, Spain 2Departamento de Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, 41012 Seville, Spain 3Instituto de Investigaciones Químicas—cicCartuja, Universidad de Sevilla—C.S.I.C, 41092 Seville, Spain *Correspondence: [email protected]; Tel.: +34-954489631 Abstract: The origin of eukaryotic organisms involved the integration of mitochondria into the ancestor cell, with a massive gene transfer from the original proteobacterium to the host nucleus. Thus, mitochondrial performance relies on a mosaic of nuclear gene products from a variety of genomes. The concerted regulation of their synthesis is necessary for metabolic housekeeping and stress response. This governance involves crosstalk between mitochondrial, cytoplasmic, and nuclear factors. While anterograde and retrograde regulation preserve mitochondrial homeostasis, the mitochondria can modulate a wide set of nuclear genes in response to an extensive variety of conditions, whose response mechanisms often merge. In this review, we summarise how mitochondrial metabolites and proteins—encoded either in the nucleus or in the organelle—target the cell nucleus and exert different actions modulating gene expression and the chromatin state, or even causing DNA fragmentation in response to common stress conditions, such as hypoxia, oxidative stress, unfolded protein stress, and DNA damage. Keywords: mito-nuclear crosstalk; stress response; oxidative stress; hypoxia; unfolded stress response ; DNA damage 1. Introduction The mitochondria aare double-membrane organelles where aerobic respiration takes place in eukaryotes. This set of coupled biochemical reactions causes the oxidation of pyruvate to CO 2 , yielding reducing power that fuels oxidative phosphorylation (OXPHOS), in which the chemiosmotic coupling of exergonic electron-transfer reactions drives ATP synthesis. Mitochondria were discovered during the nineteenth century. However, their association with biochemical reactions reported previously—the tricarboxylic acid cycle (TCA), fatty acid oxidation, OXPHOS, and part of the urea cycle, occurred a century later, with the development of cell-fractionation techniques [ 1 , 2 ]. Subsequently, the characterization of most biochemical pathways led to the recognition of mitochondria as the organelles housing the above processes, in addition to other key reactions in essential pathways, such as heme-group [ 3 ] and iron-sulphur-cluster syntheses [ 4 ], steroidogenesis [ 5 ], and the one-carbon cycle [ 6 ], which are essential for thymine synthesis from uracil and, therefore, for DNA biosynthesis. The latter routes sustain cellular biosynthetic processes, and the TCA cycle itself supports them by generating reducing power and metabolic intermediates, such as oxalacetate—whose cytoplasmic trans-amination yields aspartate, which is essential to adenosine and pyrimidine nucleotide biosynthesis [ 7 ]—and succinyl-CoA, a precursor of δ -aminolaevulinic acid and a key substrate for porphyrin synthesis. The convergence of catabolic and biosynthetic routes makes mitochondria key organelles. This organelle was recognised as a key metabolic hub, in which catabolic pathways meet to fuel cell activity and building blocks are produced to support biosynthetic routes. Warburg found hepatocarcinoma cells running aerobic glycolysis, suggesting a mitochondrial connection to pathologies [ 8 ] and illustrating the ability of the organelle to take Int. J. Mol. Sci. 2023,24, 13656. https://doi.org/10.3390/ijms241713656 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023,24, 13656 2 of 38 part in coordinated responses [ 9 ]. Further, mitochondria were found to modulate intracellular Ca 2+ concentrations [ 10 ]. In the 1970s, calcium was unveiled as a second messenger in many processes, playing key roles in diverse cell processes [ 11 ]. These findings provided a rationale involving mitochondrial signalling to explain a wide assortment of pathologies, beyond the alteration of single biochemical reactions. In the 1990s, mitochondria were found to take part in apoptosis. Subsequent well-known milestones are the discovery of the release of cytochrome c(Cyt-c) to the cytoplasm [ 12 ] and the translocation of the apoptosis-induction factor (AIF) to the nucleus [ 13 , 14 ]. More recently, a vast body of works has highlighted mitochondria as a core of cell signals that influence health and aging. A variety of cytosolic sensors of mitochondria-derived signals are known to aim at the nucleus to shift transcriptional patterns [ 15 ]. Here, we summarise the role of distinct mitochondrial factors that target the cell nucleus to govern diverse cell responses to stress. 2. Mito-Nuclear Crosstalk: An Outcome of Evolution A large number of genes—ca. 1705, including both nuclear and mitochondrial, according to the Human MitoCarta [ 16 ]—encode the functionality of mitochondrial responses and their ability to adapt to shifting physiological and pathological conditions. Mitochondria contain multiple copies of a small circular chromosome (mtDNA)—16.569 bp in humans—which limits the loss of this genetic information due to stress, organelle fission, and mitophagy. However, this multi-copy mtDNA comprises 13 protein-coding genes only, together with 22 tRNA genes, two encoding mitochondrial rRNAs and a D-loop region corresponding to a long-noncoding RNA (lncRNA) [ 17 ]. Recent reports suggest a large variety of non-coding RNAs (ncRNAs), including small noncoding (snc)-, and circular (c-) RNAs derived from this genome, although their biological relevance remains unclear [ 18 ]. Additionally, purported short open reading frames (ORFs) encode at least eight small mitochondria-derived peptides, some of which have emerged as strong metabolic regulators [ 19 ]. This tiny chromosomal size results from an intense gene transfer from the parasitic α -proteobacterium to the host and redundancy elimination along the evolution of the endosymbiotic α -proteobacterial ancestry towards a fully integrated organelle [ 20 ]. Only some of the nuclear DNA genes encoding mitochondrial proteins derive from the transfer from the endosymbiont, with another non-negligible part derived from other prokaryotes [ 20 – 22 ]. Phylogenetic analyses have shown that only 394 of the nuclear genes encoding mitochondrial proteins have a mitochondrial origin, and they were present in proto-mitochondria [ 22 ]. This implies that large mitochondrial macromolecular assemblies are indeed mosaics of subunits encoded in distinct genomes. This requires the tight regulation of transcriptional activities in both genomes to synchronise the syntheses of their components [ 23 ]. Furthermore, this makes the cell nuclei the main target for signal transduction into transcriptional activity to initiate cellular processes and govern mitochondrial functionality. However, this brings into question how cells respond to each shift in mitochondrial performance. This engages the generation of mitochondrial signals to communicate with cell nuclei, as well as a set of response elements in the latter to trigger cell reaction. Some processes for which human mitochondria are endowed according to gene ontology (GO) searches—like apoptosis, induction or carboxylic-acid metabolism, intracellular transport, or control of cell-redox homeostasis—seemed not to be fully covered by the protomitochondrion proteome [ 20 ]. This suggests that host cells furnish mitochondria with gears to fully integrate them along their evolution, in addition to providing essential components to optimise their function as “powerhouses”. As proposed by Chandel, a metabolite could be the earliest indicator of mitochondrial operation for the first eukaryotes [ 15 ]. Acetate was anticipated as its active derivative— acetyl-CoA—is the substrate for protein acetylation. When targeted to histones, this modification is a key regulator of cell cycle [ 24 , 25 ]. However, new metagenomic analyses— reconstructing the metabolism of the last Asgard common ancestor (LACA)—suggest instead that the host produced acetate, which fed the a-proteobacterium as the substrate [ 26 ]. In yeasts, nuclear-cytoplasmic acetyl-CoA synthetase is the main driver of the furnishing of Int. J. Mol. Sci. 2023,24, 13656 3 of 38 cells with “acetylation power” [ 27 ]. In mammal cells, nuclear-cytoplasmic citrate-lyase is the enzyme that transforms excess citrate, produced by the TCA cycle in mitochondria, into acetyl-CoA for later use as a substrate for signalling [ 25 ]. Nevertheless, other metabolites may play a part, given the parasitic nature of the pre-mitochondrial ancestor and the use of amino acids as carbon sources by a-proteobacteria, such as Rickettsia, to yield dicarboxylic acids [ 28 ]. These compounds— α -ketoglutarate ( α− KG), succinate, fumarate— can also influence the state of chromatin by regulating the levels of histone modification (see Section 3.5). Further, mitochondrial activity yields reactive oxygen species (ROS) by enzymatic and electron-transport reactions [ 29 ]. The ROS levels in cells are tightly regulated by an assortment of mechanisms [ 29 – 33 ], and they play a regulatory role in a broad set of cell processes in mammals, including cell proliferation and mobility [ 34 – 36 ]. Therefore, ATP and AMP can be suitable markers of the energetic status of a cell—AMP is already known to regulate AMP-activated protein kinases (AMPKs, see Section 3). Although this is a speculative point, this could be the case for the TACK—Thaumarchaeota (Nitrososphaerota), Crenarchaeota (Thermoproteota), and Korarchaeota—group of archaea host cells. It is likely that ATP became a signal for mitochondrial-quality control once the relationship evolved and the “guest’s” ATP transporter switched polarity during the transition from parasitic α -proteobacterium to proto-mitochondria. Indeed, it is a key metabolite in the retrograde response (see Section 3) in yeasts [37]. In addition to metabolites, gene-encoded products have also been proposed as putative communicators between the α -proteobacterium and the ancestral archaeal host, as in quorum sensing [38]. 3. From Crosstalk to Integral Signalling During their evolution, eukaryotic cells have developed housekeeping strategies to maintain mitochondrial functionality and adapt their replication and turnover to physiological needs [ 39 ]. The forms of the collection of these regulatory processes can be classified into two groups: one governing the flux of information from the cell nucleus to the mitochondria—called anterograde signalling—and a second, known as retrograde regulation. Nevertheless, both types of control pathway are intimately coupled and knotted to each other. Moreover, they merge themselves with signalling pathways from other cell compartments. In retrograde regulation, upstream signals starting in the mitochondria report their status to the cell nucleus to influence gene-transcription and protein-synthesis profiles. This keeps mitochondrial performance under surveillance, to adapt biochemical routes to the failure of the respiratory electron transport chain (ETC), which is responsible for OXPHOS, and to restore organelle activity. Thus, both the above products of mitochondrial activity and others target primary signal transducers to either elicit or modulate cell responses. One example of retrograde regulation is governed by ATP resulting from OXPHOS [ 40 ]. In yeasts, ATP targets retrograde response 2 (RTG2) in the cytoplasm, a phosphatase shifting the hyperphosphorylation of the RTG1–RTG3 complex—a transcription factor belonging to the basic helix–loop–helix leucine-zipper family—to its active form by releasing its bound inhibitor, the MKS1–BMH1/2 complex, which is responsible for RTG3 hyperphosphorylation. Upon RTG2 binding, MKS1 dissociates, and it is subsequently degraded [ 41 ]. Furthermore, RTG2 also participates in chromatin remodelling, as part of the Saga-like (SLIK) complex, which has histone acetyltransferase (HAT) activity [ 42 ]. Notably, a variety of cell-response mechanisms, such as those mediated by the target of rapamycin complex- 1 (TORC1) or RAS-cAMP signalling—among others—modulate this apparently simple retrograde control [ 40 ]. Thus, ATP default—in mtDNA-depleted (r0 − , petite) yeasts, for instance—promotes the transcription of genes, allowing the glyoxylate cycle and anaplerotic reactions to complement disrupted TCA cycles, among other responses [40]. A highly conserved cytoplasmic signalling pathway is the AMPK (formerly sucrose non-fermenting 1, SNF1) route, involving ADP and AMP as elicitors [ 43 ]. This pathway is active when OXPHOS is unable to support efficient ATP synthesis, which leads to increases Int. J. Mol. Sci. 2023,24, 13656 4 of 38 in both AMP and ADP. In mammals, the activation of AMPK requires its phosphorylation by either the Ser/Thr liver kinase B1 (LKB1, a.k.a. STK11) [ 44 ]—in neurons—or the calcium/calmodulin-dependent protein kinase 2 (CAMKK2) [ 45 , 46 ]. Thus, AMPK is additionally upregulated by other factors, such as cytoplasmic calcium levels, overall energetic homeostasis, and different sources of stress by cytoplasmic signalling pathways affecting post-translational modifications (PTMs) cascades, chromatin remodelling, and gene-expression patterns. In mammals, the G-protein pathway suppressor 2 (GPS2) has been suggested as a functional analogue of RTG2 [ 47 ]. The GPS2 is a mainly nuclear-cytoplasmic protein, acting as the master regulator of a variety of processes, such as inflammation [ 48 ], insulin signalling—via the AKT (protein kinase B) route [ 49 ]—and cholesterol regulation [ 50 ], among others. Knockout GPS2 is lethal to mice embryos. The GPS2 controls the expression of nuclear-encoded mitochondrial genes, in addition to others involved in Golgi, endoplasmic reticulum (ER) and lysosomal function, cytoskeletal performance, or chromosomal management [ 47 ]. Notably, there are two minor populations of GPS2 in the mitochondria: one in the matrix and another in the outer mitochondrial membrane (OMM). These two species also differ in their PTMs, with the OMM species purportedly displaying SUMOylation. Upon ETC decoupling, these mitochondrial species migrate to the cell nucleus, causing a new transcriptional response that is different from others, which are mediated by the protein. The mechanism leading to this differential gene-expression pattern is puzzling, since it is unclear that PTMs are key for the translocation of these minor GPS2 species. Therefore, the specific roles played by these mitochondrial species within the organelles need further clarification. Research on a wide variety of pathologies has revealed a highly complex landscape of retrograde regulation routes and their effects [ 39 , 51 – 56 ]. In addition, many toxicants target the mitochondria—either in a direct manner or indirectly—causing their failure, again affecting organs with strong energy needs [ 57 ]. The causes of these conditions can be of external origin—such as bacterial toxins—or internal, such as hypoxia, stroke and reperfusion, inflammatory processes, and cell-state transitions such as the endothelial–mesenchymal transition (EMT) and tumour development, among others. Often, severe mitochondrial alterations exert pleiotropic effects within and outside the organelle, leading to common dysfunctional features, such as mtDNA damage and copy-number decrease, the loss of protonmotive force ( ∆Ψ ) and Ca 2+ retention capability, OXPHOS and TCA impairment, protein-import loss, increased ROS production, altered levels of metabolites, mitochondrial segmentation, etc. Upon their detection, a cascade of signal transduction takes place, causing protein PTMs, chromatin remodelling, and changes in gene-expression patterns. The redundancy of signal mediators and feedback regulation causes these response networks to show dual stability, which converts a continuous range of stimuli in a binary response [ 58 ]. Thus, mitochondrial dysfunction generates responses that lead to either the recovery of physiological cell operation or pathology, according to the concept of mitohormesis. For instance, an acute stimulus causes the unfolded protein response (UPR) to trigger apoptosis, while a mild stimulus leads to adaptation [59]. Although different conditions cause initially distinct mitochondrial responses, the signalling pathways may blend into a more general response. For instance, the reaction to hypoxia affects inflammatory responses through ROS signalling, as well as affecting the ER and mitochondrial UPRs (UPR ER and UPR mt , respectively) [ 60 , 61 ]. Moreover, a single mitochondrial factor can partake in or modulate responses to distinct stimuli by alternative routes. Nevertheless, in each, we can distinguish one or more mitochondrial factors acting as messengers to other organelles and cell nuclei. 3.1. Adaptation to Oxygen Levels and Mitochondrial Regulation of Nuclear-Gene Transcription Although it poses the risk of oxidative damage, oxygen is an essential substrate for OXPHOS. Hypoxia is a common stress in pluricellular organisms, resulting from the imbalance between the supply and the demand of oxygen [ 62 ]. In cell cultures during Int. J. Mol. Sci. 2023,24, 13656 5 of 38 normoxia—with proper airing—oxygen levels are ca. 21% (160 mmHg). However, oxygen concentrations drop to an average steady-state level of 6.1% under physiological conditions, in which arterial blood irrigates peripheric tissues (physoxia) [ 62 , 63 ]. Physiological hypoxia are situations in which cell responses can stimulate a return to homeostasis. This is related to the physoxic levels in each tissue, laying on the range of 2% oxygen. Finally, the persistence of poor oxygenation leads to pathological hypoxia, often with O2levels below 1%. 3.1.1. Response Mediated by Cytoplasmic and Nuclear Factors: HIF1α Both anterograde and retrograde routes govern hypoxia responses. One signal route involves the oxygen-dependent activity of the hypoxia-inducible factors 1 (HIF-1) and 2 (HIF-2). These are basic heteromeric transcription factors containing a basic DNA-binding helix–turn–helix (bHLH) Per-Arnt-Sim (PAS) monomer (HIF1 α , Figure 1A) [ 64 ]—similar to its paralogs, HIF2 α [ 65 ] and HIF3 α [ 66 ]—and a second unit, the aryl hydrocarbon receptor nuclear translocator (ARNT) domain (HIF1 β ), which also contains a basic helix-turn-helix, PAS [ 64 ]. The HIF-1 α is ubiquitous, whereas the other isoforms are rather cell-specific, and differ in their specific interactions [ 67 ]. In addition, HIF-1 is constitutively expressed, and it is normally located in the nucleoplasm, so it cannot be considered a mitochondrial factor. Nevertheless, some reports indicate that a minor fraction (1–5%) of the protein may be located in mitochondria under oxidative stress or hypoxia [ 68 , 69 ]. Notably, the α - subunit includes an oxygen-dependent degradation region (ODD) containing a N-terminal trans-activation domain (N-TAD) comprising two PEST—proline, glutamic, serine, and threonine—sequences, which are responsible for proteasomal degradation in the presence of oxygen [70,71]. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 40 3.1. Adaptation to Oxygen Levels and Mitochondrial Regulation of Nuclear-Gene Transcription Although it poses the risk of oxidative damage, oxygen is an essential substrate for OXPHOS, . Hypoxia is a common stress in pluricellular organisms, resulting from the imbalance between the supply and the demand of oxygen [62]. In cell cultures during normoxia—with proper airing—oxygen levels are ca. 21% (160 mmHg). However, oxygen concentrations drop to an average steady-state level of 6.1% under physiological conditions, in which arterial blood irrigates peripheric tissues (physoxia) [62,63]. Physiological hypoxia are situations in which cell responses can stimulate a return to homeostasis. This is related to the physoxic levels in each tissue, laying on the range of 2% oxygen. Finally, the persistence of poor oxygenation leads to pathological hypoxia, often with O2 levels below 1%. 3.1.1. Response Mediated by Cytoplasmic and Nuclear Factors: HIF1α Both anterograde and retrograde routes govern hypoxia responses. One signal route involves the oxygen-dependent activity of the hypoxia-inducible factors 1 (HIF-1) and 2 (HIF-2). These are basic heteromeric transcription factors containing a basic DNA-binding helix–turn–helix (bHLH) Per-Arnt-Sim (PAS) monomer (HIF1α, Figure 1A) [64]—similar to its paralogs, HIF2α [65] and HIF3α [66]—and a second unit, the aryl hydrocarbon receptor nuclear translocator (ARNT) domain (HIF1β), which also contains a basic helixturn-helix, PAS [64]. The HIF-1α is ubiquitous, whereas the other isoforms are rather cellspecific, and differ in their specific interactions [67]. In addition, HIF-1 is constitutively expressed, and it is normally located in the nucleoplasm, so it cannot be considered a mitochondrial factor. Nevertheless, some reports indicate that a minor fraction (1–5%) of the protein may be located in mitochondria under oxidative stress or hypoxia [68,69]. Notably, the α-subunit includes an oxygen-dependent degradation region (ODD) containing a N-terminal trans-activation domain (N-TAD) comprising two PEST—proline, glutamic, serine, and threonine—sequences, which are responsible for proteasomal degradation in the presence of oxygen [70,71]. Figure 1. Proteins involved in the response to oxygen levels. (A) Schema of HIF1α and HIF1β. bHLH stands for basic helix–loop (turn)–helix domain, PAS stands for Per-Arnt-Sim domains, and ODD is the oxygen-dependent degradation region. Key sequence stretches (PEST) and residues (P402, R532, and P562) for HIF1α regulation in the ODD. Representation also shows the location of the two Figure 1. Proteins involved in the response to oxygen levels. ( A ) Schema of HIF1 α and HIF1 β . bHLH stands for basic helix–loop (turn)–helix domain, PAS stands for Per-Arnt-Sim domains, and ODD is the oxygen-dependent degradation region. Key sequence stretches (PEST) and residues (P402, R532, and P562) for HIF1 α regulation in the ODD. Representation also shows the location of the two transactivation domains (N- and C-TAD). ( B ) Representative members of the coiled-coil helix coiled-coil helix domain in this work (see page 7). CHCH domains are in blue, with yellow bars showing the approximate locations of CX 9 C motifs. MTS stands for mitochondrial targeting sequence, TMD is a transmembrane domain, DUF stands for domain of unknown function. Red acyl groups indicate glycine myristylation sites. Int. J. Mol. Sci. 2023,24, 13656 6 of 38 The degradation of HIF1 α requires the post-translational hydroxylation of proline residues by prolyl-4-hydroxylases (PHDs)—Fe 2+ -dependent dioxygenases using oxygen and α -KG as substrates, and ascorbate as an electron donor [ 72 ]. This regulatory mechanism is conserved among metazoans. The hydroxylation of PEST enables interactions with the von Hippel Lindau (pVHL) E3 ligase component, ubiquitination, and proteasomal degradation. The Michaelis–Menten constant (K M ) values of the PHDs for O 2 are ca. 230 mM—ca 6.3% O 2 in solution, matching the levels in physoxia [ 62 , 63 , 73 ]. Active HIF1 α binds to the β -subunit, and the active heterodimer promotes transcription from core DNA sequence (G/ACGTG) in hypoxia response elements (HREs) coupled to several target genes [ 74 , 75 ]. Notably, PHDs are inhibited by TCA metabolites, such as fumarate (Ki 50–80 µM ), succinate—apyruvate dehydrogenase (PDH)-reaction product (inhibitory constant, Ki, ca. 400 µ M)—and citrate, with a lower affinity [ 76 ]. Thus, mutations in TCA enzymes, such as succinate dehydrogenase (SDH) or fumarate hydratase (FH), can lead to anomalous, pseudo-hypoxic cell responses, as observed in different types of cancer. The HIF1 α activity is further regulated, in a pVHL-independent manner, by hydroxylation in its carboxyl terminal-trans-activation domain (C-TAD) by asparaginyl hydroxylase (FIH), which prevents binding to p300, the cAMP-response element-binding protein (CREB)-binding protein (CBP), or other transcriptional coactivators [ 77 , 78 ]. These proteins bind the C-TAD of HIF1 α through their first cysteine–histidine-rich domain (CH1, a.k.a. TAZ1) [ 79 , 80 ], which was initially considered essential for the transcription of HIF target genes. Furthermore, CH1 participates in roughly 48% of HIF-mediated transcriptional activities, including those involving the participation of histone deacetylase 7 (HDAC7) [ 79 , 81 ]. This is surprising, since CBP and p300 have a bromodomain to bind acetylated histones and the CH2 domain shows histone acetyltransferase (HAT) activity. Authors suggested that acetylation and deacetylation could take place at separate times. According to the Reactome site [82], more than a hundred genes display HRE elements. Oxygen levels further regulate HIF1 activity through the expression of the HIF3 α locus. First, the promoter of this gene displays an HRE sequence targeted by HIF1 itself [ 83 ]. Next, transcripts from this gene are subjected to an alternative splicing mechanism, which is oxygen-dependent [ 84 ]. The alternative transcript encodes a PAS protein (IPAS) that binds to and inhibits HIF1 α . Further, oxygen also regulates the expression of the CBP/p300-interacting trans-activator 2 (CITED2) via cytoplasmic Fork-head box O-3 (FOXO-3) activation [ 85 ]. In its turn, CITED2 is an allosteric competitor for CH1 binding to HIF1 α [ 86 ]. Additionally, HIF1 α controls the expression of a plethora of ncRNAs with effects on a broad set of processes, many of which control HIF1 α expression and activity at different levels [87,88]. The dependence of HIF activation on the activity of PHDs mixes the anterograde character of this route with retrograde signals. This may result from bottleneck in HIF regulation created by PHD kinetics, as highlighted in the literature [ 89 ]. First, mitochondrial dysfunction—e.g., OXPHOS impairment—leads to the inhibition of complex II (CII or succinate dehydrogenase, SDH) and, therefore, of the TCA cycle. This leads to succinate depletion and α -KG accumulation. The first is one of the products of PHD-catalysed reactions and an inhibitor of the enzyme, while the second is its substrate, thereby exerting contrasting effects on PHD activity, on HIF1 α stability, and on HIF assembly. Indeed, mutations in FH and SDH promote HIF-hydroxylation activity [ 90 ]. This results in the expression of genes shifting energy metabolism and favouring glycolysis, affecting cell proliferation and survival and iron homeostasis, among other processes, depending on the cellular context. Similarly, the iron cofactor of PHDs is sensitive to ROS, and it eventually requires ascorbate to exit non-functional oxidised states [ 91 ]. In addition, since HIF is sensitive to oxygen, ROS, and the redox state, its interactions with a wide variety of coactivators are also affected [ 62 ]. Thus, oxidative stress mediates another retrograde regulation of HIF-mediated response to mitochondrial dysfunction. To our knowledge, cytochrome coxidase (complex IV, CIV, a.k.a. COX) is the only enzyme in cells that uses oxygen directly for energy transduction. During hypoxia, its Int. J. Mol. Sci. 2023,24, 13656 7 of 38 activity transiently increases, before declining as glycolysis takes over cell metabolism. A transcriptional screening revealed three genes showing early and transient upregulation upon hypoxia [ 92 ]. One of these genes encodes the hypoxia-inducible gene-domain-family member 1A (HIGD1A)—a homologue of yeast respiratory super-complex factor 1 (RCF1)— whose product binds CIV to promote super-complex formation [ 92 , 93 ]. The HIGD1A is transiently induced under hypoxic conditions and increases the activity of complex IV, leading to increased oxygen consumption and subsequent mitochondrial ATP synthesis, thereby improving cell viability during hypoxia [ 92 ]. Recent data show that HIGD1A and its homolog, HIGD2A, participate in the assembly of CIII and CIV [ 94 ]. Further, its expression was previously detected in a hypoxic brain by microarray screening [ 95 ]. In addition, HIGD1A is already known to protect cells from low glucose and hypoxia [ 96 ]. The HIGD1A gene is activated by HIF in a CH1-independent manner [ 97 ]. In glioma cells, residual HIGD1A levels are maintained separately from HIF induction, and linked to the activity of the DNA methyl transferase-1 (DNMT1) on HIGD1A HREs [ 98 ]. Although HIGD1A is more present in the brain, the expression of several of its homologs takes place in other organs. All its homologs are targeted to the intermembrane spaces of the mitochondria and exert a protective effect, impairing the release of Cyt-cinto the cytoplasm, among other effects [ 99 ]. In cardiomyocytes, this protective effect also involves an increase in CIV activity [ 92 ]. Notably, Cyt-cphosphorylation modulates improvements in CIV performance by HIGD1A [ 100 , 101 ]. Nevertheless, co-immunoprecipitation experiments suggest that HIGD1A is rather in contact with complex III (CIII) [98]. 3.1.2. Mitochondrial Factors and Responses to Oxygen Levels The hypoxia response also involves alternative—albeit complementary—pathways, acting under less severe conditions. A mitochondrial gene whose product is usually located in the intermembrane space (IMS)—the mitochondria nuclear retrograde regulator 1 (MNRR1, a.k.a., coiled-coil-helix-coiled-coil-helix domain 2, or CHCHD2)—was detected through a computational gene-expression search for MIA40 interactors across the NCBI Gene Expression Omnibus database [ 102 ]. This protein, together with the mitochondrial intermembrane-space import and assembly protein 40 (MIA40, a.k.a., CHCHD4, a key factor in mitochondrial protein import), CHCHD10, and others, presents helix–turn–helix domains with twin CX 9 C motifs, enabling the formation of interhelical disulphide bonds characteristic of the CHCHD-protein family (Figure 1B) [ 103 ]. The MNRR1 uses the MIA40 import system to enter mitochondria, despite presenting a mitochondrial targeting sequence in its N-terminus [ 104 ]. Mild hypoxia impairs this import pathway, so MNRR1 accumulates in the cell nucleus (Figure 2) [ 105 ]. Notably, MIA40 mediates imports linked to cysteine oxidation, suggesting that the conformation of nuclear MNRR1 differs from that of its nuclear species. In fact, the substitution of cysteine residues from the CX 9 C motifs by serine yield the retention of this protein in the cell nucleus [ 104 ]. However, there are some intriguing aspects in the localization of these proteins, such as the folding state of the nuclear population. If it is folded, it is questionable whether this folding is due to the activity of extramitochondrial chaperones. Knocking MNRR1 down decreases CIV-turnover rates by ca. 40% in cells [ 104 ]. The MNRR1 co-purifies with COX, a interaction that is enabled by ABL-related gene (ABL2) phosphorylation within the IMS [ 106 ]. The MNRR1 levels increase in the mitochondria in response to the dissipation of ∆Ψ —not ∆ pH—across the IMM through a hypoxiainsensitive mechanism involving PTMs [ 107 ]. Recently, MNRR1 was shown to modulate mitochondrial fusion by controlling the processing of the optic atrophy 1 product (OPA1) by YME1 Like 1 ATPase (YME1L1) protease. First, the YME1L1-encoding gene is under the control of an oxygen-responsive element (ORE) targeted by MNRR1 [ 104 , 108 ]. In addition, MNRR1 binds to complement component 1 Q subcomponent-binding protein (C1QBP), which modulates YME1L1 protease activity [109]. Int. J. Mol. Sci. 2023,24, 13656 8 of 38 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 40 YME1 Like 1 ATPase (YME1L1) protease. First, the YME1L1-encoding gene is under the control of an oxygen-responsive element (ORE) targeted by MNRR1 [104,108]. In addition, MNRR1 binds to complement component 1 Q subcomponent-binding protein (C1QBP), which modulates YME1L1 protease activity [109]. Figure 2. Transcriptional regulation of nuclear genes by mitochondrial proteins in response to perturbations in oxygen metabolism. All these proteins, except MOTS-c, are nuclear-encoded. Inhibition of the import system by low oxygen or oxidative stress leads to the accumulation of otherwise mitochondrial factors in the cell nucleus, activating different responses according to target promoter and modulator. ORE stands for oxygen-responsive element; ARE stands for antioxidant response element. Proteins of the CHCHD family, except MIA40, are abbreviated as D3, D6, and D10. MOTS- c transcription is fostered by oxidative stress, and the transcript is translated in the cytoplasm. TIM and TOM stand for the inner and outer membrane-transport systems, respectively. MICOS stands for mitochondrial contact site and cristae-organizing system, which, together with the F1 FO-ATP synthase, and optic atrophy 1 (OPA1) protein, facilitates formation, maintenance, and stability of cristae membranes. Hypoxia elicits MNRR1 expression, which, in turn, activates tissue-specific COX subunit 4 isoform 2 (COX4I2) transcription in the cell nucleus. This involves binding to a 13- base-pair ORE in its promoter [108,110]. These elements comprise four out of five nucleotides in the HRE sequence—the fifth being T in place of G—but seem to be unresponsive to HIF1α regulation [108]. The MNRR1 competes with the CXX5 repressor to bind recombination-signal-binding protein for the immunoglobulin kappa J region (RBPJκ) transcription factor, which then activates transcription from ORE [108]. Moreover, the MNRR1 gene has an ORE in its own promoter; thus, it is activated by its own product. In addition, MNRR1 is also under transcriptional regulation by the cAMP-responsive element-binding protein (CREB 3-like protein 3), a fact that links MNRR1 expression to both the UPRER, and the UPRmt [105]. These processes contribute to quality control (QC) tasks that are key for cell survival. Furthermore, MNRR1 lifetimes depend on the proteolytic activity of YME1L1 and OMA1 zinc metallopeptidase [111]. Analyses with KO models indicate that UPRER causes UPRmt in an MNRR1-dependent manner, upstream of known UPRmt effectors such as the basic Leu-zipper activating transcription factor 5 (ATF5)—the mammal homologue of C. elegans ATFS-1. In mitochondrial myopathy encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) cybrids, MNRR1 levels are low, and UPRmt is defective [105]. The dysfunction of another protein in this family, CHCHD10 (Figure 1B), emerged recently as a relevant factor in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and ALS-frontotemporal dementia (ALS-FTLD) [112–114], among other neurodegenerative diseases. The CHCHD10 and MNRR1 proteins display 58% sequence identity, and they probably evolved from a gene-duplication event [115]. More recently, it was proposed that this protein modulates mitochondrial functionality in several tissues by affecting mitochondrial morphology, the steadiness of mitochondrial cristae, CIV activity, mtDNA stability, and cell survival under stress. Furthermore, CHCHD10 is a 14 Figure 2. Transcriptional regulation of nuclear genes by mitochondrial proteins in response to perturbations in oxygen metabolism. All these proteins, except MOTS-c, are nuclear-encoded. Inhibition of the import system by low oxygen or oxidative stress leads to the accumulation of otherwise mitochondrial factors in the cell nucleus, activating different responses according to target promoter and modulator. ORE stands for oxygen-responsive element; ARE stands for antioxidant response element. Proteins of the CHCHD family, except MIA40, are abbreviated as D3, D6, and D10. MOTS-c transcription is fostered by oxidative stress, and the transcript is translated in the cytoplasm. TIM and TOM stand for the inner and outer membrane-transport systems, respectively. MICOS stands for mitochondrial contact site and cristae-organizing system, which, together with the F 1 F O -ATP synthase, and optic atrophy 1 (OPA1) protein, facilitates formation, maintenance, and stability of cristae membranes. Hypoxia elicits MNRR1 expression, which, in turn, activates tissue-specific COX subunit 4 isoform 2 (COX4I2) transcription in the cell nucleus. This involves binding to a 13-base-pair ORE in its promoter [ 108 , 110 ]. These elements comprise four out of five nucleotides in the HRE sequence—the fifth being T in place of G—but seem to be unresponsive to HIF1 α regulation [ 108 ]. The MNRR1 competes with the CXX5 repressor to bind recombination-signal-binding protein for the immunoglobulin kappa J region (RBPJ κ ) transcription factor, which then activates transcription from ORE [ 108 ]. Moreover, the MNRR1 gene has an ORE in its own promoter; thus, it is activated by its own product. In addition, MNRR1 is also under transcriptional regulation by the cAMP-responsive element-binding protein (CREB 3-like protein 3), a fact that links MNRR1 expression to both the UPR ER, and the UPR mt [ 105 ]. These processes contribute to quality control (QC) tasks that are key for cell survival. Furthermore, MNRR1 lifetimes depend on the proteolytic activity of YME1L1 and OMA1 zinc metallopeptidase [ 111 ]. Analyses with KO models indicate that UPR ER causes UPR mt in an MNRR1-dependent manner, upstream of known UPR mt effectors such as the basic Leu-zipper activating transcription factor 5 (ATF5)—the mammal homologue of C. elegans ATFS-1. In mitochondrial myopathy encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) cybrids, MNRR1 levels are low, and UPRmt is defective [105]. The dysfunction of another protein in this family, CHCHD10 (Figure 1B), emerged recently as a relevant factor in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and ALS-frontotemporal dementia (ALS-FTLD) [ 112 – 114 ], among other neurodegenerative diseases. The CHCHD10 and MNRR1 proteins display 58% sequence identity, and they probably evolved from a gene-duplication event [115]. More recently, it was proposed that this protein modulates mitochondrial functionality in several tissues by affecting mitochondrial morphology, the steadiness of mitochondrial cristae, CIV activity, mtDNA stability, and cell survival under stress. Furthermore, CHCHD10 is a 14 kDa protein that is also located in the IMS—weakly bound to the IMM [ 116 ]. Together with mitofilin, CHCHD3, and CHCHD6, it has been proposed, CHCHD10 takes part in the mitochondrial contact site and cristae organizing system (MICOS) system, which determines the IMM topology and the formation of membrane-contact sites [ 117 ]. However, the relevance of Int. J. Mol. Sci. 2023,24, 13656 9 of 38 CHCHD10 to MICOS stability is a controversial issue. Mutagenesis directed by CHCHD10 affects mitochondrial IMM shapes in fibroblasts [ 114 ], also observed in specific FTD-ALS patients [ 113 ], but not in KO mice tissues [ 113 ]. Fibroblast mitochondria from ALS patients displaying CHCHD10 mutations have altered nucleoids, with limit repair ability, since the recovery after H 2 O 2 treatment is only 60% compared to when wild-type controls are used. The permeabilization of the OMM (MOMP) is also affected in these patients. Furthermore, CHCHD10 expression is higher in tissues with high energy demand, such as the skeletal muscles, heart, liver, kidneys, and substantia nigra [ 116 ]. With the exception of the heart, CHCHD10 KO hardly affects OXPHOS performance, but instead increases the iron levels within the mitochondria. As with other twin CX 9 C-motif-containing proteins, the entry of CHCHD10 into the mitochondria is MIA40 (CHCHD4)-dependent, and Cys-to-Ser mutations impair its import into the IMS. In addition, MNRR1 expression takes place at O 2 levels as low as 4% [ 108 ], while CHCHD10 transcription occurs in a narrow interval at higher concentrations (8%), proposed to be the physioxic level. As with MNRR1, CHCHD10 can be detected mainly in mitochondrial cristae and, then, in the nucleus [ 118 ]. The functions of both proteins are tightly linked, with CHCHD10 acting as a scaffold to enable MNRR1 phosphorylation within mitochondria, and forming an MNRR1-mediated complex with CIV, stimulating its activity. In the cell nucleus, contrary to MNRR1, CHCHD10 acts as a repressor in the ORE sequences by interacting with the CXX5 repressor, and morbid mutations of CHCHD10 impair these interactions [ 119 ]. Nevertheless, CHCHD10 KO does not affect MNRR1-transcription levels [ 118 ]. Thus, at physioxic concentrations [ 50 ], transcription from ORE-containing promoters is inhibited by CHCHD10. Again, CHCHD10 protein levels are proteolytically controlled by YME1L1 and OMA1 proteases, suggesting their participation in UPR mt (see Section 3.3). Uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) cause the accumulation of both MNRR1 and CHCHD10, mainly in the mitochondria, but also in the cell nucleus and, probably, in cytosol [ 107 , 111 ]. As with MNRR1, in the mitochondria, such increases are due to decreases in their proteolysis. Notably, the MIA40/ERV1 (CHCHD4/ALR in humans) import system is non-responsive to ∆Ψ changes, whereas the mitochondrial proteases, OMA1 and YME1L1, are degraded upon IMM depolarization [120]. In addition, CHCHD10 binds TAR DNA-binding protein 43 (TDP43) in the cell nucleus upon IMM depolarization [ 118 ]. This protein belongs to the family of heterologous ribonucleoproteins (hnRNPS) and is key to RNA metabolism. In addition, TDP43 acts both in the cell nucleus—regulating transcription, splicing, miRNA biogenesis, and the binding of lncRNAs—and within cytosol, influencing mRNA stability, trafficking, and translation [ 121 ]. The binding of CHCHD10 modifies the cellular location of TDP43, targeting it to the cell nucleus. Several ALS-FTD-associated CHCHD10 mutations (R15L, S59L) result in TDP43 accumulation in cytosol, a substantial amount (>35%) of which is located in mitochondria and speckles, with a decrease in TDP43 content in stress granules [ 118 , 122 ]. This resembles the effect of a reported ALS-related gain-of-function mutation (S55L), which causes the aggregation of CHCHD10 itself within mitochondria and triggers the integration of UPRmt [114]. To summarise, there is a tight link between O 2 levels, ETC operation, IMM protonmotive force, and other features of the cellular functional state, such as the UPR. The responses to their changes are partly mediated by the performance of several mitochondrial proteins as activatable transcription factors in the cell nucleus. 3.2. Dealing with Oxidative Stress Oxygen reactive species (ROS) and the products of their reactions with other cellular or mitochondrial components also act as signal triggers. The fortuitous electron leakage associated with the activity of the ETC—and TCA cycle enzymes, among others—makes the mitochondria the main generators of these species [ 29 , 123 ]. The activity of diverse enzymes in mitochondria, cell membranes, peroxisomes, the endoplasmic reticulum, and even the Int. J. Mol. Sci. 2023,24, 13656 16 of 38 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 16 of 40 associated proteins do translocate to the nucleus under DNA damage. We review this process below. Figure 3. Protein trafficking from mitochondria to nucleus during DNA damage. Mitochondrial proteins can be targeted to the cell nucleus to modulate the chromatin dynamics and/or take part in DNA repair. In the nucleus, apoptosis-induction factor (AIF) promotes chromatin condensation and DNA fragmentation, while endonuclease G (ENDOG) increases g-H2AX levels and favours DNA repair. AIF translocation to the nucleus is mediated by cyclophilin A (CYPA). The metabolism-re- lated enzymes fumarate hydratase (FH) and hexokinase 2 (HK2) can be located in the nucleus, where FH promotes dimethylation of histone H3 to recruit DNA-dependent protein kinase (DNAPK), thereby enhancing non-homologous end-joining (NHEJ), whereas HK2 favours chromatin opening, which results in increased expression levels of p53 binding protein 1 (53BP1) and RAD51 recombinase, promoting NHEJ and homologous recombination (HR), respectively. Nuclear translocation of HK2 is exerted by importin IPO5. Hypoxia-inducible gene-domain-family member 1A (HIGD1A) moves into the nucleus in response to double-strand breaks (DSBs) with the help of nucleoporin NUP93. Next, nuclear HIGD1A recruits the replication protein A (RPA) and the 9-1-1 complex, thereby prompting HR. B-cell lymphoma 2 (BCL-2) protein accumulates in the nucleus and interacts with the Ku70-Ku80 heterodimer to inhibit NHEJ. Nuclear BCL-2 also docks apurinic/apyrimidinic endonuclease (APE1)—hampering base excision repair (BER)—as well as poly (ADP-ribose) polymerase-1 (PARP-1), thereby abolishing NHEJ, HR BER, and nucleotide excision repair (NER) pathways. BCL-2 and breast cancer tumour suppressor 1 (BRCA1) co-localise to mitochondria, resulting in the decrease in nuclear BRCA1, and subsequently inhibiting HR. Cytochrome c (Cyt-c) migrates to the cell nucleus following DSBs, where it displaces the acetylated histone H2A from the chromatin, thereby producing chromatin condensation. Furthermore, nuclear Cyt-c interacts with suppressor of variegation, enhancer of zeste, and Trithorax (SET), or nucleosome assembly protein 1 (NAP1)-related protein 1 (NRP1), inhibiting their nucleosome-assembly abilities. Dynamin-related protein 1 (DRP1) is translocated into the nucleus by human homologue of yeast Rad23 protein A (hHR23A), where it hinders DNA-damage repair. Under ionizing radiation, CR6- Figure 3. Protein trafficking from mitochondria to nucleus during DNA damage. Mitochondrial proteins can be targeted to the cell nucleus to modulate the chromatin dynamics and/or take part in DNA repair. In the nucleus, apoptosis-induction factor (AIF) promotes chromatin condensation and DNA fragmentation, while endonuclease G (ENDOG) increases g-H2AX levels and favours DNA repair. AIF translocation to the nucleus is mediated by cyclophilin A (CYPA). The metabolism-related enzymes fumarate hydratase (FH) and hexokinase 2 (HK2) can be located in the nucleus, where FH promotes dimethylation of histone H3 to recruit DNA-dependent protein kinase (DNA-PK), thereby enhancing non-homologous end-joining (NHEJ), whereas HK2 favours chromatin opening, which results in increased expression levels of p53 binding protein 1 (53BP1) and RAD51 recombinase, promoting NHEJ and homologous recombination (HR), respectively. Nuclear translocation of HK2 is exerted by importin IPO5. Hypoxia-inducible gene-domain-family member 1A (HIGD1A) moves into the nucleus in response to double-strand breaks (DSBs) with the help of nucleoporin NUP93. Next, nuclear HIGD1A recruits the replication protein A (RPA) and the 9-1-1 complex, thereby prompting HR. B-cell lymphoma 2 (BCL-2) protein accumulates in the nucleus and interacts with the Ku70-Ku80 heterodimerto inhibit NHEJ. Nuclear BCL-2 also docks apurinic/apyrimidinic endonuclease (APE1)— hampering base excision repair (BER)—as well as poly (ADP-ribose) polymerase-1 (PARP-1), thereby abolishing NHEJ, HR BER, and nucleotide excision repair (NER) pathways. BCL-2 and breast cancer tumour suppressor 1 (BRCA1) co-localise to mitochondria, resulting in the decrease in nuclear BRCA1, and subsequently inhibiting HR. Cytochrome c(Cyt-c) migrates to the cell nucleus following DSBs, where it displaces the acetylated histone H2A from the chromatin, thereby producing chromatin condensation. Furthermore, nuclear Cyt-cinteracts with suppressor of variegation, enhancer of zeste, and Trithorax (SET), or nucleosome assembly protein 1 (NAP1)-related protein 1 (NRP1), inhibiting their nucleosome-assembly abilities. Dynamin-related protein 1 (DRP1) is translocated into the nucleus by human homologue of yeast Rad23 protein A (hHR23A), where it hinders DNA-damage repair. Under ionizing radiation, CR6-interacting factor 1 (CRIF1) moves to the nucleus, where it mediates the nuclear transport of cyclin-dependent kinase 2 (CDK2) to help with DNA-damage repair. Moreover, nuclear CRF1 negatively regulates cell growth through its interaction with growth arrest and DNA-damage-inducible protein 45 (GADD45). TCA: tricarboxylic acid cycle. Int. J. Mol. Sci. 2023,24, 13656 17 of 38 3.4.1. AIF and DNA damage Treatment with DNA-damage inducers, such as doxorubicin, actinomycin D, or topoisomerase I inhibitor camptothecin, causes AIF translocation to the nucleus [ 142 ]. Furthermore DNA-damage-induced t AIF translocation and apoptosis have been shown to depend on the presence of p53 and a pro-apoptotic member of the B-cell lymphoma-2 (BCL-2) family, BCL-2-associated X protein (BAX) [ 142 , 151 ]. The translocation of t AIF to the nucleus is apparently controlled by CYPA and heat-shock protein 70 (HSP70) through the direct binding of these two proteins to t AIF [ 223 ]. In addition, CYPA is a positive regulator of the cytosolic-to-nuclear redistribution of t AIF [ 153 ], whereas HSP70 blocks this translocation, thereby suppressing t AIF-mediated apoptosis [ 224 ]. Remarkably, the nuclear accumulation of t AIF is hampered by the expression of the E3 ubiquitin ligase parkin—a protein that promotes dopaminergic neuron survival—in cells exposed to apoptogenic treatment, which contributes to the neuroprotective activity of parkin [225]. 3.4.2. ENDOG As pointed out above (Section 3.2), ENDOG is also involved in DDR [ 163 ]. Furthermore, ENDOG elicits DDR in starvation conditions to promote autophagy [ 163 ]. Damage to DNA is an early event following starvation-induced autophagy, and it is mediated by poly-(ADP-ribose) polymerase-1 (PARP1) and AMP-activated protein kinase (AMPK) [ 226 ]. The loss of ENDOG blocks the activation of PARP1 and AMPK, thus repressing starvationinduced DNA damage [ 163 ]. The latter observation proves that ENDOG-mediated DNA- damage response promotes autophagy. In fact, ENDOG without endonuclease activity does not induce the DNA-damage response or autophagy [ 163 ]. Cell treatment with the DNA-damage inducer etoposide—a DNA topoisomerase II inhibitor—enhances the ENDOG-induced DDR (measured as increments in the DNA-damage sensors γ -H2AX and phosphorylated ataxia-telangiectasia mutated or p-ATM), and also increases autophagosome formation [ 163 ]. Furthermore, mitochondrial ENDOG is released and translocated to nuclei to sustain nuclear DNA damage in autophagy-defective cells. This suggests that DNA-damage-induced autophagy is not needed for recovery from nuclear DNA damage [ 227 ]. Although DNA damage induces ENDOG’s nuclear translocation, the mechanism underlying this translocation and the specific role of ENDOG in DNA repair are still under investigation. 3.4.3. Fumarase In response to DSBs, yeast fumarase is recruited from cytosol to the nucleus, where it seems to play a key role in the protection of cells from DNA damage [ 228 ]. The enzyme fumarase, or fumarate hydratase (FH), takes part in the tricarboxylic acid (TCA) cycle in the mitochondrial matrix. It catalyses the reversible conversion of fumaric acid to malic acid. Fumarase redistribution between the mitochondria and cytosol has been observed in all the eukaryotes studied, including yeast and humans [ 228 ]. In yeast, it has been proposed, cytosolic fumarase participates in the catabolism of amino acids [ 229 ], and as a scavenger of fumarate from the urea cycle [ 230 ]. Nevertheless, this does not explain the high levels of FH in cytosol [ 228 ]. Apparently, fumarase is first targeted to mitochondria, but a subpopulation of the enzyme is located in cytosol [ 231 ]. If, during import, the nascent chain begins to fold in the mitochondrial matrix, it is localised in the mitochondria, but if the import slows and the polypeptide chain begins to fold in the cytosol, the chain withdraws from the import machinery, and it is localised in the cytosol [232]. Upon the induction of DSBs, a lack of nuclear fumarase prevents histone H2AX phosphorylation and cell-cycle-checkpoint activation, making yeast and human cells more sensitive to DNA damage. This suggests that fumarase is related to the early detection or signalling of DSBs. This protective nuclear effect depends on the enzymatic production of fumaric acid by fumarase. High concentrations of this metabolite can indeed overcome the absence of the enzyme [ 228 ]. To perform its DDR-related role, FH does not accumulate at the sites of breaks in separate nuclear foci, as is the case with many members of the DDR. Int. J. Mol. Sci. 2023,24, 13656 18 of 38 Recent evidence shows that nuclear fumarase promotes DNA repair in response to ionizing irradiation through the hindering of histone demethylation [ 233 , 234 ]. Thus, exposure to ionizing radiation induces the phosphorylation of nuclear fumarase—at Thr 236—leading to an interaction between fumarase and the histone variant, H2A.Z, adjacent to DSB regions [ 233 ]. This FH recruitment results in the local accumulation of fumarate in DSBs, which inhibits the activity of lysine-specific demethylase 2B (KDM2B). This results in the enhanced dimethylation of histone H3—at Lys 36—which, in turn, favours the accumulation of DNA-dependent protein kinase (DNA-PK) at DSB foci [ 233 ]. In NHEJ, DSBs are recognised by the DNA–PK complex—consisting of the heterodimer, Ku70- Ku80, and the catalytic subunit, DNA-PKcs—which then recruits end-processing enzymes, polymerases, and DNA ligase IV [ 208 ]. Altogether, this evidence proves that nuclear fumarase actively participates in DNA repair by NHEJ in mammalian cells. On the other hand, a tumour-suppressing role has also been proposed for fumarase in human cells, owing to FH mutations, which predispose individuals to common types of tumour [ 235 ]. Accordingly, FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylase, further stabilizing HIF by preventing its proteasomal degradation [ 236 ]. Subsequently, the transcription factor HIF promotes tumour growth and survival by inducing glycolysis and blood-vessel growth—facilitating the adaptation of tumoral cells to low-oxygen conditions [ 237 ]. However, in some cases of FH deficiency, there is no accumulation of fumarate, nor is there stabilization of HIF [ 238 ]. The latter observation can be explained by the above-mentioned role of fumarase in the DDR, suggesting that the tumorigenesis function of FH is due to its role in the cellular response to DSBs. Finally, high fumarate levels in FH-defective cells have been shown to increase genetic instability by impairing cell-cycle G2 arrest and causing early mitotic start in response to DNA damage [239]. 3.4.4. Hexokinase 2 Hexokinase 2 (HK2)—another mitochondrial enzyme—has been reported to modulate the DDR. Hexokinases are a family of ubiquitous enzymes, primary found in cytosol, which participates in the first and rate-limiting step in glycolysis by catalysing the phosphorylation of glucose to glucose 6-phosphate. Hexokinase 2 is the second most predominant isozyme in the family, and it is mainly expressed in insulin-sensitive tissues—the heart, skeletal muscles, adipose tissues, and a wide range of tumours [ 240 ]. It can dock to mitochondria, where it performs additional functions in autophagy and cell-death inhibition, which are independent of its enzymatic activity [ 241 ]. Mitochondrial HK2 contributes to the metabolic reprogramming of tumoral cells by enhancing glucose consumption and lactate production, as well as decreasing mitochondrial respiration in parallel [ 241 ]. Interestingly, the anchoring of HK2 to the outer mitochondrial membrane is key to its oncogenic activity, and it is regulated by SUMOylation. Furthermore, HK2 can localise to the nuclei of acute myeloid leukaemia (AML) and normal hematopoietic stem cells, and it is involved in the maintenance of stemness [ 242 ]. Importantly, nuclear HK2 alters stem-cell function and differentiation independently of its enzymatic function. The nuclear import of HK2 is mediated by importin IPO5, whereas HK2 nuclear export requires exportin 1 (XPO1) [ 242 ]. In addition, HK2 can localise to the nuclei of acute myeloid leukaemia (AML) and normal hematopoietic stem cells, and it is involved in the maintenance of the stemness [242]. Remarkably, nuclear HK2 interacts with several proteins associated with the DDR pathways in stem cells. Accordingly, the overexpression of nuclear HK2 in stem cells treated with a chemotherapeutic agent that causes DSBs, daunorubicin, results in a decrease in the number of DSBs—measured by the levels of the DSBs marker g-H2AX—and in increased expression levels of tumour-suppressor p53 binding protein 1 (53BP1) and RAD51 recombinase, which are essential factors for NHEJ and HR, respectively [ 242 ]. Thus, nuclear HK2 seems to mediate the enhanced DNA-damage responses of AML stem cells. Int. J. Mol. Sci. 2023,24, 13656 19 of 38 On the other hand, nuclear HK2 modulates chromatin accessibility and maintains DNA integrity by interacting with proteins involved in chromatin organisation and regulation, such as the RNA-polymerase-associated protein CTR9, myc-associated factor X (MAX), the histone lysine demethylase plant homeodomain finger (PHF) protein 8 (PHF8), protein 10 (PHF10), and spindlin1 (SPIN1) [ 242 ]. In addition, MAX recognises basic helix–loop–helix (bHLH) E-box motifs, which are implicated in cell proliferation, stem-cell maintenance and differentiation, and the DNA-damage response [ 243 ]. The overexpression of nuclear HK2 increases chromatin accessibility, whereas its downregulation decreases chromatin accessibility, demonstrating that stem cells have more accessible chromatin [ 242 ]. It can be inferred that the opening of the chromatin structure enhances DNA-damage repair by increasing access to DNA-repair proteins. Additionally, nuclear HK2 also interacts with proteins related to the DNA-damage response, e.g., the NAD-dependent protein deacetylase Sirtuin 1 (Sirt1), tyrosyl-DNA phosphodiesterase 2 (TDP2), and ubiquitin-protein ligase N-recognin 5 (UBR5, a regulator of transcription in damaged chromatin) [242]. 3.4.5. HIGD1A and DNA Damage In cells, responses often result from crosstalk between pathways that are initially unconnected. Thus, HIGD1A was found to be expressed during hypoxia. Some metabolic stress— e.g., glucose starvation coupled with hypoxia or DNA damage induced by etoposide— triggers its nuclear accumulation, as well as its interaction with nuclear AIF [ 244 ]. A more recent study found that HIGD1A moves into the nucleus in response to DSBs, thereby participating in the regulation of the HR pathway of DDR by interacting with replication protein A (RPA) [ 245 ]. Consequently, RPA binds and stabilises single-stranded DNA intermediates, thus playing an essential role both in DNA replication and in the cellular response to DNA damage [ 246 ]. In the early stages of HR, nuclear HIGD1A promotes RPA loading at DSB sites and enhances the chromatin binding of the 9-1-1 complex, thereby activating the ataxia–telangiectasia and Rad3-related (ATR)-mediated phosphorylation of checkpoint kinase 1 (CHK1)—a G2/M-cycle-damage checkpoint, resulting in a high survival rate [ 245 ]. By contrast, in the later stages of HR and after promoting RPA binding, HIGD1A inhibits the aberrant persistence of RPA by enhancing RPA ubiquitination and its subsequent proteasomal degradation, which enables the accomplishment of HR [ 245 ]. Furthermore, the absence of nuclear HIGD1A has been linked to the impairment of the DDR displayed by replication stress in cells deficient in COX4-1 (a common isoform of COX subunit 4) [247]. The mechanism underlying the nuclear translocation of HIGD1A observed in response to ionizing radiation involves the recognition of HIGD1A by the nucleoporin NUP93 through a conserved sequence (residues 46 to 60) of HIGD1A [ 245 ]. Intriguingly, the nuclear localization of HIGD1A is also dependent on the presence of BCL-2-related proteins, specifically BAX and BCL-2 homologous antagonist/killer (BAK), positive regulators of the intrinsic pathway of apoptosis, which suggests that HIGD1A requires apoptosis-related molecules to be translocated into the nucleus [244,248]. 3.4.6. B-Cell Lymphoma 2 Proteins and DDR The members of the BCL-2 family act on DDR by means beyond the modulation of the translocation of mitochondrial factors. The BCL-2 family of proteins consists of 25 pro- and anti-apoptotic members that maintain a balance between newly forming cells and old dying cells [ 249 ]. The members of the family localise to endomembranes (of the mitochondria and the endoplasmic reticulum (ER)) through a transmembrane (TM) domain and govern the permeability of transition pores by interacting with each other [ 249 , 250 ]. The BCL-2 family members share their homology in the BCL-2 homology (BH) domains [ 251 ]. When antiapoptotic members are overexpressed, the ratio of pro- and anti-apoptotic BCL-2 family members is disturbed, and apoptosis is avoided [ 252 ]. Consequently, the targeting of the anti-apoptotic members of the BCL-2 family triggers apoptosis and, thus, overcomes drug resistance to cancer chemotherapy [ 253 ]. In addition to their accepted role in apoptosis Int. J. Mol. Sci. 2023,24, 13656 20 of 38 regulation, several studies suggest roles for BCL-2 members in DNA repair, specifically for the BCL-2 protein. The BCL-2 protein is an anti-apoptotic factor anchored to the external mitochondrial membrane, the ER, as well as in the outer nuclear envelope [ 254 ]. Pro-survival members of the BCL-2 family—including BCL-2—possess four BH domains, i.e., BH1, BH2, BH3, and BH4 [ 251 ]. The BCL-2 protein performs its anti-apoptotic activity by impairing the release of Cyt-cfrom the mitochondrial IMS, thus preventing caspase activation [ 252 ]. This anti-apoptogenic activity makes the BCL-2 protein behave as an oncogene and a potent tumour promoter [255]. Some studies highlight the links between BCL-2-dependent carcinogenesis and genetic instability due to impaired DNA repair [ 256 , 257 ]. Indeed, in addition to anti-apoptotic activity, BCL-2 negatively regulates the NHEJ pathway. The BCL-2 protein suppresses DSB repair by inhibiting the heterodimer, Ku70-Ku80—part of the DNA-PK complex functioning in NHEJ—and, thus, increasing genetic instability [ 258 ]. The Ku70-Ku80 heterodimer recognises and binds to DSBs ends. Subsequently, the catalytic subunit (DNA-PKcs) joins to form the active DNA–PK complex, which recruits end-processing enzymes and other factors to DSBs [ 259 , 260 ]. Ionizing radiation stimulates the accumulation of BCL-2 in the nucleus, which then interacts with the Ku70-Ku80 heterodimer and inhibits its binding to DSB ends [ 258 ]. Remarkably, BCL-2 interacts with Ku70 and Ku80 via its BH1 and BH4 domains, which are the regions responsible for BCL-2 nuclear accumulation in response to ionizing radiation [ 258 ]. It is unclear whether such effects of BCL-2 in the nucleus are caused by an increase in its expression or by its translocation from the mitochondria to the nucleus following DNA damage. The BCL-2 protein also regulates DDR by interacting with breast cancer tumour suppressor 1 (BRCA1), which plays an essential role in HR [ 261 ]. The BCL-2 protein and BRCA1 co-localise to mitochondria and the ER through the TM domain of BCL-2, leading to a decrease in nuclear BRCA1 and, subsequently, the inhibition of HR [ 261 ]. Importantly, targeting BRCA1 through BCL-2 not only affects HR, but also other DDR pathways, such as NHEJ or BER, as well as several cellular processes, e.g., cell-cycle regulation, cell death, or the inactivation of the X-chromosome [250]. Furthermore, BCL-2 attenuates DNA repair by the NER and BER pathways. It has been reported that BCL-2 avoids the NER-mediated restoration of cyclobutane pyrimidine dimers (CPD), kinds of photo-induced DNA lesion, although the underlying molecular mechanisms have not been explored [ 262 ]. On the other hand, BCL-2 inhibits the BER- dependent correction of the chemical changes produced in the DNA bases [ 257 ]. First, BER is started by the elimination of the damaged base to generate an abasic site (AP). Next, apurinic/apyrimidinic endonuclease (APE1) cleaves the DNA to the 50end of the AP site, thus creating a space for the filling with one nucleotide [ 250 ]. To become active, APE1 forms a complex with X-ray repair cross-complementing protein 1 (XRCC1) [ 250 ]. Scholars have proposed two mechanisms involving APE1 to explain the inhibition of BER exerted by BCL-2: (i) BCL-2 increases the expression of c-MYC, resulting in a decrease in the gene expression of APE1 [ 263 ]; and (ii) BCL-2 and APE1 establish a direct interaction, which destabilises the APE1–XRCC1 complex, resulting in the reduced endonuclease activity of APE1, thereby hampering BER [264]. Researchers have reported a direct interaction between BCL-2 and the enzyme PARP1 in the nuclei of B-cell lymphoma cells, which suppresses PARP1 enzymatic activity, thereby hampering DNA repair [ 265 ]. One of the earliest events in the DDR is the recruitment of PARP1 to diverse types of DNA lesion. The PARP1 is rapidly recruited to sites of DNA damage through its DNA-binding domains [ 266 ], where it catalyses the attachment of a negatively charged polymer—poly(ADP-ribose) or PAR—to itself and to multiple target proteins [ 267 ]. Subsequently, DDR-related proteins can be recruited to sites of DNA injury by binding to PAR through non-covalent interactions [ 268 ]. This PARylation activity not only contributes to the repair of single-strand breaks (SSBs) and DSBs—through HR and NHEJ—but also seems to be involved in BER and NER DNA-repair pathways [ 267 ]. Int. J. Mol. Sci. 2023,24, 13656 21 of 38 Interestingly, BCL-2 is overexpressed and located within the nuclei of several tumour-cell lines [ 258 , 263 , 269 ], and then colocalises and inhibits PARP1 to attenuate DNA repair [ 265 ]. Lastly, it has been hypothesised that the inhibition of PARP1 by BCL-2 also suppresses a form of non-apoptotic cell death named parthanatos (from PARylation and θανατ o ζ , or death, in Greek) [ 265 ], distinct from apoptosis, necrosis or autophagy, in which PARP1 triggers the nuclear translocation of mitochondrial AIF and cell death [ 270 ], and then colocalises and inhibits PARP1 to attenuate DNA repair [265]. In addition, BCL-2 plays a role in the MMR pathway. When initiating MMR, a protein named mutS homolog 2 (MSH2) forms several complexes that recognise base– base mismatches and insertion/deletion mispairs [ 271 ]. Scholars have proposed two different mechanisms to explain the BCL-2 inhibition of MMR. According to the first, BCL-2 decreases MSH2 gene expression [ 256 ], whereas in the second, BCL-2 is translocated to the nucleus to sequester MSH2 through a direct interaction [ 269 ]. Both mechanisms reduce the effectiveness of MMR [ 250 ]. Intriguingly, the second mechanism is dependent on the BH4 domain of BCL-2, which is also needed for BCL-2 nuclear accumulation under genotoxic stresses [250]. 3.4.7. Cytochrome c The Cyt-cis a heme-containing protein that resides in the intermembrane spaces of mitochondria as a component of oxidative phosphorylation, and transports electrons between cytochrome bc 1 (complex III) and COX (complex IV) [ 272 ]. In mammalian cells undergoing apoptosis, Cyt-cis released to the cytosol, where it binds to the apoptosis-protease- activating factor 1 (APAF-1), as well as to procaspase-9, to form a huge macromolecular complex, the so-called apoptosome [ 12 ]. This results in the activation of caspase-9, which, in turn, triggers the activation of effector caspases—i.e., caspase-3 and caspase-7, which are responsible for the morphologic and biochemical changes associated with apoptosis, which execute cell death [ 12 ]. Certainly, Cyt-cis released from mitochondria in numerous organisms, including plants, yeasts, and flies, in addition to mammals [273–275]. Beyond the cell-death-related functions of Cyt-cin cytosol as a key element in the activation of mammalian caspases [ 12 , 276 ] or plant-caspase-like enzymes [ 277 ], numerous studies have shown that the heme protein migrates to the cell nucleus under non-apoptotic conditions [ 278 – 283 ]. Following DNA DSBs, Cyt-cgradually collects in the nucleus and displaces the acetylated histone H2A from the DNA, thereby producing the chromatin condensation and nuclear pyknosis observed during apoptosis [279]. However, the implications of such chromatin changes in the DNA-repair response have not been clarified. When genotoxic stress leads to DNA breaks, Cyt-cshuttles to the nuclei of mammalian cells to interact with the suppressor of variegation, the enhancer of zeste, and trithorax (SET)/template-activating factor (TAF)-I β , a.k.a., SET/TAF-I β [ 281 ]. The SET/TAF-I β is a histone chaperone primarily located in the cell nucleus, where it mediates histone incorporation or the removal of nucleosomes during chromatin remodelling [ 284 ]. This chaperone is also recruited to the DSBs and, in fact, it is a key modulator of DNA repair in the chromatin surrounding those sites [ 285 ]. Accordingly, in response to the damage to DNA caused by the use of radiomimetic drugs, the downregulation of SET/TAF-I β increases DDR, and, by contrast, the upregulation of the chaperone impairs DNA repair. Under such conditions, SET/TAF-I β interacts with the Kruppel-associated box (KRAB)-associated co-repressor (KAP1), resulting in the retention of KAP1 and heterochromatin protein 1 (HP1) on chromatin, thereby hampering DNA repair by HR [ 285 ]. Therefore, SET/TAF-I β is recruited to DNA breaks to moderate uncontrolled DDR. In addition, upon the triggering of DNA breaks, nuclear Cyt-cbinds to SET/TAF-I β and to its plant orthologue—nucleosome assembly protein 1 (NAP1)-related protein 1 (NRP1)—in mammalian and plant cells, respectively, thereby inhibiting their histone-chaperone abilities [ 281 , 286 ]. Although these observations suggest a role for nuclear Cyt-cin the DDR, further research is needed to clarify whether such Cyt-c-mediated chaperone inhibition results in increased or decreased DNA repair. Int. J. Mol. Sci. 2023,24, 13656 22 of 38 In addition to the chromatin-remodelling and DDR-modulation activities of SET/TAF- I β , this chaperone also inhibits protein phosphatase 2A (PP2A) [ 287 ], which dephosphorylates a number of DDR effectors, including the phosphorylated variant of the histone H2AX ( γ -H2AX) [ 288 ]. The γ -H2AX histone signals and recruits DDR factors on chromatin surrounding DSBs, so that γ -H2AX foci must be removed to proceed with the repair [ 288 ]. Using structural and computational approaches, it has been reported that Cyt-cforms a dynamic ensemble with SET/TAF-I β able to activate PP2A [ 289 ]. It is noteworthy that another histone chaperone and PP2A inhibitor—acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B)—is also targeted by the haemoprotein in the cell nucleus upon DSBs, yielding Cyt-c-mediated PP2A activation. In addition to SET/TAF-I β and ANP32B, Cyt-cforms complexes with other histone chaperones in the nucleus under DNA-damage conditions, as is the case of nucleolin (NCL) [ 290 ]. One of the most abundant proteins in the nucleolus of mammalian cells, NCL moves to the nucleoplasm after genotoxic stress, where it binds to histones H2A and H2B [ 291 ]. Under these conditions, NCL is recruited to DSB foci by binding to γ -H2AX [ 292 ]. Furthermore, this histone chaperone promotes cell-cycle arrest and DNA repair by binding to the mediator of DNA damage checkpoint protein 1 (MDC1) [ 292 ]. Moreover, NCL interacts with the DDR factors RAD50/51 and replication protein A (RPA) [ 291 ]. Remarkably, another abundant nucleolar protein, a histone chaperone named nucleophosmin (NPM), is specifically recognised by Cyt-cupon DNA breaks [ 293 ]. The metalloprotein binds to nucleolar NPM to drive the release of alternative reading frame (ARF)—a tumour-suppressor protein sequestered by NPM in the nucleoli [ 293 ]. In this way, mitochondrial Cyt-cmay control the trafficking and availability of nucleolar proteins under stress conditions. Other histone chaperones recognised by Cyt-cin the cell nucleus are the heterogeneous ribonuclear proteins C1 and C2 (hnRNP C1/C2), which bind to damaged chromatin sites near DSBs [ 294 ]. In addition to chromatin remodelling, hnRNP C1/C2 participates in mRNA splicing, transport, stability, and translation [ 295 ], as well as in the NHEJ-repair pathway, by means of its interaction with the heterodimer Ku70-Ku80 [296]. 3.4.8. Dynamin-Related Protein 1 Dynamin-related protein 1 (DRP1) is a large GTPase belonging to the dynamin superfamily, and it is the key component of the mitochondrial and peroxisomal fission machinery [ 297 ]. Mitochondria constantly change their shape, keeping a balance between fusion and fission [ 298 ]. Mitochondrial fission requires the translocation of DRP1 from cytosol to the mitochondria, after which DRP1 presumably docks an adaptor protein on the outer membrane and uses the energy from GTP to self-assemble as helical- or spiralshaped superstructures that constrict and cleave mitochondria [ 299 , 300 ]. Several studies link excessive mitochondrial fission and DRP1 with apoptosis, neuronal dysfunction, and cell death [ 301 , 302 ]. Recent investigations show that ionizing radiation triggers DRP1- dependent mitochondrial fission, and that DRP1 inhibition attenuates a form of cell death associated with aberrant mitosis—the so-called mitotic catastrophe [298]. Furthermore, DRP1-mediated mitochondrial fragmentation has also been associated with the cytotoxicity exerted by some commonly-used anticancer drugs—e.g., cisplatin. This links DRP1 with cancer progression and sensitivity to cancer treatments [ 303 ]. The expression of nuclear DRP1 is often found in lung adenocarcinomas, and it is correlated with poor prognoses, i.e., there is a significant difference in survival between patients with high levels of nuclear DRP1 and those with the cytosolic protein [ 303 ]. The nuclear transportation of DRP1 is mediated by its interaction with the human homologue of the yeast RAD23 protein A (hHR23A) [ 303 ]. In vitro , hypoxia raises nuclear DRP1 levels and cisplatin resistance in lung adenocarcinoma cells. How DRP1 accumulation in the nucleus causes resistance to antitumour drug treatments is still unknown. Conversely, DRP1 inhibition triggers replication stress, which then induces G2/M cell-cycle arrest and starts the DNA-damage response [ 304 ]. In addition, DRP1 loss causes centrosome overduplication and chromosomal instability. Thus, deficient mitochondrial fission—due Int. J. Mol. Sci. 2023,24, 13656 23 of 38 to the loss of DRP1—causes genome instability [ 304 ]. Further, the disruption of DRP1 induces lethality and cellular senescence [ 305 , 306 ]. Thus, it is tempting to speculate that the genomic instability resulting from DRP1 deficiency is related to the ability of DRP1 to confer resistance to anticancer drugs. 3.4.9. CR6-Interacting Factor 1 The CR6-interacting factor 1 (CRIF1) is a mitochondrial protein involved in the assembly of the oxidative phosphorylation complexes into the inner mitochondrial membranes of mammalian cells [ 307 ]. The expression of CRIF1 expression is high in osteosarcoma tissues, and it has been associated with cancer radio-resistance [308]. In response to ionizing radiation, CRIF1 is translocated to the nucleus—it has a nuclear localization signal—where it negatively regulates cell-cycle progression and cell growth by means of its interaction with growth arrest and DNA-damage-inducible protein 45 (GADD45) [ 309 ]. A recent study states that CRIF1 prompts the nuclear transport of the DNA-damage-checkpoint regulator cyclin-dependent kinase 2 (CDK2) when DNA is damaged by ionizing irradiation [ 310 ]. Thus, CRIF1 promotes the phosphorylation of CDK2 at Thr14 and Thr160 in the cell nucleus, which ultimately facilitates G1/S-checkpoint activation and DNA-damage repair [310]. 3.5. Mitochondrial Metabolites and Proteins Affecting Chromatin Remodelling As pointed out in Section 2, mitochondrial functioning can affect chromatin remodelling to adapt response to changes in organelle performance, among other purposes. Thus, metabolites derived from and the activity of mitochondrial enzymes—including those in the TCA cycle—can influence cellular physiology through epigenetic modifications. In addition, diverse mitochondrial proteins can also take part in the governance of the chromatin state. 3.5.1. Mitochondrial Metabolites and Chromatin Condensation State α-Ketoglutarate, Succinate, and Fumarateple Some mutations affecting enzymes in the TCA cycle, namely, isocitrate dehydrogenase, succinate dehydrogenase, or fumarase, leads to the accumulation of a-ketoglutarate, succinate, and fumarate in the mitochondria and cytosol [311,312]. The accumulation of α -KG leads to its conversion to 2-hydroxyglutarate, a potent inhibitor of α -KG-dependent dioxygenases. The most notable dioxygenases that are inhibited by excess α -KG are HIF prolyl hydroxylases [ 313 ] and the Jumonji-domain-family histone lysine demethylases (JMJ-KDMs) [ 314 ]. The inhibition of these dioxygenases leads to significant epigenetic and transcriptional changes [ 315 ]. Thus, the α -KG-mediated inhibition of HIF prolyl hydroxylases stabilises HIF, thereby activating hypoxic signalling, so 2-hydroxyglutarate is considered an oncometabolite, and it accumulates in many cancers [ 316 ]. It is believed that succinate accumulation has a similar impact on HIF prolyl hydroxylases, so this has also been linked to tumorigenesis [ 315 , 317 ]. Fumarate accumulation results in the succinylation of the cysteine residues of Kelch-like ECH associated protein 1 (KEAP1), which leads to the stabilization of the transcriptional factor nuclear factor erythroid-derived 2-like (NRF2), driving the activation of the NRF2 antioxidant transcriptional response [ 318 ]. Moreover, glutathione (GSH) can be succinylated, leading to the depletion of the cellular pools of GSH, which also results in activation of the NRF2- mediated antioxidant response [ 319 ]. Although it is seemingly beneficial, the persistent activation of the NRF2 response can be detrimental, and it has been linked to oncogenesis [ 319 ]. Strikingly, fumarate accumulation also results in increased trimethylation at Lys4 and acetylation at Lys27 of histone H3, processes that are related to the epigenetic changes observed in trained immunity [ 320 ]. Thus, fumarate acts as an inhibitor of the lysine demethylase 5 (KDM5) family of proteins responsible for H3K4 demethylation [ 321 ]. Int. J. Mol. Sci. 2023,24, 13656 24 of 38 Flavin Adenine Dinucleotide Flavin adenine dinucleotide (FAD) is a coenzyme derived from the vitamin riboflavin, which is produced in the mitochondria and acts as an electron carrier during catabolism. Importantly, FAD is also a cofactor for lysine demethylases [ 322 ]. Lysine-specific demethylase 1 (LSD1) is a flavin-containing protein endowed with FAD as a prosthetic factor [ 323 ]. Furthermore, LSD1 is a transcriptional corepressor, so it may catalyse the removal of methyl groups from lysine residues of histones whose methylation is linked to active transcription [ 322 ]. Remarkably, it is likely that alterations in the FAD/FADH 2 ratio, which fluctuates with metabolic activities such as fatty-acid oxidation or the TCA cycle, affect LSD1-mediated demethylation [324]. 3.5.2. Mitochondrial Proteins and Chromatin Remodelling Although most investigations are focused on mitochondrial metabolites as epigenetic regulators, some studies have also shown mitochondrial proteins that shuttle to the nucleus to control chromatin remodelling and, consequently, gene expression, through either their enzymatic or their transcriptional activities. Mitochondrial Matrix Dehydrogenases Pyruvate dehydrogenase (PDH) is an essential mitochondrial protein complex consisting of several copies of three enzymes, the pyruvate dehydrogenase (E1), dihydrolipoamide transacetylase (E2), and dihydrolipoamide dehydrogenase (E3) [ 325 ]. In addition, PDH is needed for connecting cytosolic glycolysis and the TCA cycle, through the oxidation of pyruvate to acetyl-CoA. In response to mitochondrial dysfunction—upon the inhibition of respiratory-chain complexes or the depletion of the mitochondrial outer-membrane protein, called mitochondrial carrier homolog 2 (MTCH2)—all three subunits of the PDH complex have been detected in cell nuclei, and it has been proposed that they shuttle there from mitochondria in a cell-cycle-dependent manner [ 326 , 327 ]. To the best of our knowledge, there are no clear data regarding the translocation mechanism of such a large complex. The nuclear PDH complex produces acetyl-CoA from pyruvate, leading to increased histone acetylation, which regulates the cell cycle. This is required for S-phase entry [ 326 , 327 ]. Remarkably, the nuclear-PDH-complex pool can also increase in response to growth signals, including the epidermal growth factor and serum, suggesting an important role in cell-cycle progression [ 326 , 327 ]. Recently, other mitochondrial matrix dehydrogenases have been shown to translocate during the reprogramming of somatic cells, as well as influencing histone acetylation [ 328 ]. Again, the translocation mechanisms are unknown. Thus, further research is needed to confirm this point. Transcription Factor A Mitochondrial transcription factor A (TFAM) is a nuclear-encoded member of a highmobility group (HMG) family of proteins, which is essential for the transcription and replication of mitochondrial DNA [ 329 ]. It was recently reported that TFAM may also be located in the nucleus and, specifically, that it may be anchored to chromatin after the treatment of tumour cells with anticancer drugs [ 330 ]. This nuclear localization of TFAM is likely to be due to the presence of two nuclear localization signals within its two HMG domains [ 331 ]. However, the question of whether nuclear TFAM molecules are translocated from the mitochondria is a subject of debate. The TFAM binds tightly to the nuclear chromatin and regulates the expression of nuclear genes to promote cell growth [ 332 ]. The overexpression of TFAM enhances cell growth in several cancer-cell lines, while its downregulation inhibited their growth [ 332 ]. Thus, predictably, nuclear TFAM confers protection against the genotoxic drugs etoposide, camptothecin, and cisplatin [ 330 ]. Nuclear TFAM is also observed in neuronal cells, where the binding of nuclear TFAM to its promoter results in the suppression of its own expression [333]. Int. J. Mol. Sci. 2023,24, 13656 25 of 38 Clock 1/Coenzyme Q7 Homolog Clock 1 (CLK-1)—and its human homolog, coenzyme Q7 homolog (CoQ7)—is a mitochondrial enzyme involved in the biosynthesis of ubiquinone, a component of the electron transport chain [ 334 ]. In response to the mitochondrial accumulation of ROS, CLK-1/CoQ7 translocate to the nucleus to regulate gene expression in both C. elegans and human cells [ 176 ]. The nuclear fraction of CLK-1/CoQ7 binds to chromatin to regulate the gene transcription of the ROS-mediated response, triggering the adaptive oxidative stress response in order to decrease cellular ROS levels [ 176 ]. When CLK-1/CoQ7 are restricted to the nucleus, ubiquinone biosynthesis is inhibited, suggesting that CLK-1/CoQ7 exhibits different functions in the two compartments. The CLK-1 is an iron-binding protein endowed with hydroxylase activity [ 335 ]. However, it is unknown whether this activity is required for its nuclear role. In this regard, other iron-binding oxygenases have been shown to regulate gene expression through the demethylation of histones and cytosine bases [336]. 4. Concluding Remarks The key role of mitochondria in cell metabolism makes it a target for tight regulation and, conversely, a key governance agent for cells and tissues. This crosstalk between the control mechanisms evolved in the early stages and has grown in complexity over the course of evolution, to the point where it is fully integrated into major regulatory routes. In this work, we supplied an overview of the mechanisms involving mitochondrial proteins and soluble metabolites targeting cell nuclei, although we did not address the role of the plethora of mtRNA species that have recently emerged nor the complex signalling network involving the cytoplasm and other organelles, such as the ER and lysosomes. Several key features of the mitochondrial proteins targeting the nucleus are worth highlighting. Many of them may include an NLS element, enabling them to target the cell nucleus. Alternatively, they can also migrate by piggybacking on other proteins. Nevertheless, the mitochondrial import system is emerging as a key sorter of many of the regulatory proteins described here. The dependence of the import system on redox activity and its coupling to mitochondrial protein folding makes it an ideal mitochondrial-stress- signal transducer, in addition to membrane-pore transition. The mitochondrial factors addressed in this work exert their actions in different ways and in a surprising variety of scenes, most often acting through moonlighting and in a pleiotropic manner. Moonlighting involves the participation of these proteins and metabolites in different processes. The pleiotropic manner involves the mitochondrial factors targeting a variety of genes in diverse ways, either directly or following a cascade of transcriptional events, post-translational modifications, or just shifting the accessibility of genes to processing machineries for repair or transcription. Under unbearable stress, some factors also aid DNA degradation. Finally, it is worth noting that the functional multiplicity of all these factors provides additional links between responses to different sources of stress, enabling them to either aid in the general stress responses of cells or elicit programmed cell death. We speculate that this may help to dampen the deleterious effects of acute stress and favour survival, raising the threshold at which cell death needs to be triggered. Author Contributions: Conceptualization, A.D.-Q. and K.G.-A.; writing, A.D.-Q. and K.G.-A. All authors have read and agreed to the published version of the manuscript. 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