4.4.2. Mutations in ATP Synthase Nuclear Structural Subunits

At present, only a small number of mitochondrial pathologies have been attributed to mutations in nuclear genes coding for subunits of the ATP synthase and for chaperones that function in assembly of the enzyme. Several studies found that patients with lactic acidosis, persisting 3-methylglutaconic aciduria, cardiomyopathy, and early death were correlated with severe deficiencies in ATP synthase, but the genetic lesions in these studies were not identified [209–211]. *ATP5F1E* and *ATP5F1D*, the human homologs of the yeast *ATP15* and *ATP16*, code for the ε and δ subunits, respectively, of the central stalk of F1. A homozygous missense mutation in *ATP5F1E* was the first reported instance of a patient with an ATP synthase defect stemming from a mutation in a nuclear encoded subunit of the enzyme. The mutation caused mild mental retardation and the patient developed peripheral neuropathy [6]. The mitochondrial synthesis of ATP, the oligomycin ATPase activity and the ATP synthase content were reduced by 60–70%. The residual enzyme with the mutated ε subunit had normal activity, indicating that the primary effect of the mutation was on assembly of the synthase. Interestingly, this mutation, when introduced in the ε subunit of yeast ATP synthase, did not elicit any detectable effect on either the activity of assembly of the enzyme [212]. This suggested that the mutation in the human ε subunit, perhaps because of a weaker physical interaction with the c ring, exerted a deleterious effect on assembly of the human but not of the yeast enzyme [212]. In a more recent report, two patients with homozygous mutations in *ATP5F1D* were shown to suffer from metabolic disorders in one case and from encephalopathy in another [213]. *ATP5F1A,* coding for human α subunit of F1, is the third nuclear ATP synthase gene identified in two patients presenting fatal neonatal encephalopathy with intractable seizures [5]. Interestingly, reduced levels of cellular ATP5F1 were also shown to correlate significantly with earlier-onset prostate cancer [214]. Indeed, transitioning from oxidative phosphorylation to anaerobic glycolysis for energy production occurs in many types of tumors, and could explain the pathophysiology of the disease.


**Table 7.** Pathologies resulting from mutations in genes encoding ATP synthase subunits and assembly factors, and their yeast homologs.


**Table 7.** *Cont.*

<sup>1</sup> Review describing many mutations. <sup>2</sup> In different patients. <sup>3</sup> In the same patient.

None of the patients with mutations in the nuclear ATP synthase genes discussed in this and the next sections exhibit the clinical NARP and Leigh syndrome phenotypes characteristic of patients with mutations in the two mitochondrially-encoded genes of the enzyme.

#### 4.4.3. Nuclear ATP Synthase Assembly Gene Mutations

A substantial number of nuclear gene products of *S. cerevisiae* are known to regulate and chaperone different steps of ATP synthase assembly [215]. At present, however, the only known human regulatory factors with identified mutations in a small number of patients are TMEM70, a protein essential for ATP synthase assembly and ATPAF2, the homolog of the yeast Atp12 chaperone that interacts with the α subunit of F<sup>1</sup> during assembly of this ATP synthase module [216]. TMEM70 does not have a yeast homolog. The phenotypes associated with mutations in TMEM70 and the role of its product in ATP synthase assembly have been reviewed elsewhere [217,218].

A study in which two patients, ascertained to have nuclear mutations that affected mitochondrial ATP synthase assembly, were screened by sequencing human homologs of yeast genes previously shown to affect F<sup>1</sup> biogenesis led to the identification in one patient of a mutation in *ATPAF2*. This patient, diagnosed with lactic acidosis, glutaconic aciduria, encephalomyopathy and a range of different developmental problems, was found to have a homozygous W94R amino acid substitution that resulted in severe deficits of ATP synthase in the heart, liver and to a lesser degree in skeletal muscle, resulting in death at 14 months of age [219]. The deleterious effect of the W94R substitution in a highly conserved region of *ATPAF2* on ATP synthase assembly was confirmed by Meulemans et al. [7], who showed that the wild-type, but not the mutant human gene, restored the ATP synthase activity of a yeast *atp12* mutant. The *atp12* mutant also failed to be complemented by the yeast *ATP12* harboring the equivalent W102R mutation expressed from a low copy yeast CEN, but not from a high-copy plasmid. The rescue by the W102R, however, depended on the presence of wild-type *FMC1*, a gene implicated in regulating the activity of yeast Atp12 [220].

#### *4.5. Coenzyme Q*

Coenzyme Q (ubiquinone, CoQ or CoQ<sup>10</sup> in humans) is a lipophilic redox molecule found in virtually all eukaryotic organisms and most bacteria. It is composed of a quinone ring connected to a polyisoprenoid side chain of variable length. Coenzyme Q serves several crucial functions in mitochondria, including transfer of electrons from Complexes I and II to Complex III, acting as an essential cofactor in the uncoupler protein mediated transfer of protons to the matrix, prevention of lipid peroxidation, biosynthesis of uridine, beta-oxidation of fatty acids, and binding to and regulating the permeability transition of the voltage-dependent anion channel [221].

The CoQ biosynthetic pathway in eukaryotes has been studied in yeast *coq* mutants arrested at different steps of the pathway [222,223]. Like human cells, yeast relies on de novo synthesis of CoQ; and any deficiency in the biosynthetic pathway results in a growth arrest on media containing non-fermentable carbon sources [3,224]. Most enzymes of the CoQ biosynthetic pathway are organized in a multi-subunit complex known as the CoQ synthome [222,225]. The CoQ synthome is spatially linked to the endoplasmic reticulum–mitochondria contact sites, providing optimal CoQ production with an efficient intracellular distribution as well as minimizing the escape of toxic intermediates [226,227]. Expression of functional CoQ in yeast depends on at least 14 nuclear gene products (Coq1-Coq11, Yah1, Arh1, and Hfd1), all located in mitochondria [227].

CoQ biosynthesis is achieved by three separate and highly conserved pathways:

(1) Synthesis of the quinone ring from 4- hydroxybenzoate (4HB), derived from tyrosine [228], or from p-aminobenzoic acid (pABA), in yeast but not in humans [225,227,229]. The early steps of 4HB formation are still to be determined but deamination of tyrosine starts with Aro8 or Aro9 catalyzed transamination [230]. The formation of the final intermediate 4-hydroxybenzaldehyde (4 HBz) is catalyzed by the aldehyde dehydrogenase Hfd1 [230,231].

(2) Synthesis of isopentenyl pyrophosphate (IPP) and dimethylally pyrophosphate (DMAPP), catalyzed by Coq1 in yeast [232] and PDSS1 and PDSS2 in humans.

(3) Prenylation of parahydroxybenzoate by the polisoprenyl transferase Coq2 [233] and further modifications of the benzoquinone ring by hydroxylases and methyl transferases. The isoprenoid side chain is important for proper CoQ localization at the mid-plane of phospholipid bilayers. Yeast coenzyme Q contains 6 isopentenyl units (CoQ6) while in humans the major coenzyme Q isoform contains 10 isopentenyl units (CoQ10).

The benzoquinone head group is modified by hydroxylations catalyzed by Coq6 and Coq7 [234–236], methylation of the resultant hydroxyls by the Coq3 methyl transferase [237], methylation of the ring by Coq5 [238] and a decarboxylation step catalyzed by a still unidentified enzyme of this pathway. Other gene products linked to coenzyme Q biosynthesis and utilization include Coq4 and Coq9, that have been assigned a role in assembly and stability of the CoQ synthome, and Coq8, a member of a protein family that includes kinases and ATP-dependent ligases. Coq8 has been implicated in the phosphorylation state of Coq3, Coq5 and Coq7 [239–241]. The steady-state concentrations of Coq4, 6, 7, and 9 are markedly decreased in a *coq8* null mutant, as a result of which assembly of the CoQ synthome is abrogated [242]. The *coq8* null mutant, however, contains a complex of Coq6 and Coq7 thought to be an early intermediate of the synthome [243]. Coq10 is a low molecular weight member of the START protein family that binds coenzyme Q. Although Coq10 is required for respiration, its synthesis is only partially affected in log but not in stationary phase yeast cells, suggesting that its function is related to the delivery of coenzyme Q from the synthome located at the endoplasmic-mitochondrial contact sites to the regions of the inner membrane containing the respiratory chain complexes [226,244]. The respiratory deficiency of *coq10* mutants is partially rescued in a *coq11* mutant, which codes for a component of the synthome that has been proposed to down- regulate the synthesis of coenzyme Q [244]. Coq11 (human NDUFA9), a separate protein in yeast, is a component of the CoQ synthome [245] and appears to regulate its formation and stability [244]. CoQ yeast and human genes are listed in Table 8.


**Table 8.** Coenzyme Q genes required for functional expression of CoQ and their yeast homologs.


**Table 8.** *Cont.*

#### Mutations in COQ Genes

CoQ<sup>10</sup> deficiency, a biochemical lesion first described over three decades ago by Ogasahara et al. [246], is subdivided into primary CoQ<sup>10</sup> deficiency, when caused by a pathogenic mutation in one of the genes required for the coenzyme's biosynthesis, and secondary CoQ<sup>10</sup> deficiency, when the mutated gene is not directly related to the biosynthetic pathway [247]. Of the two, the latter has been reported more frequently in patients, with phenotypes including mitochondrial myopathies, mitochondrial DNA depletion syndrome and multiple acyl-CoA dehydrogenase deficiency (MADD) [248]. However, the pathogenic mechanisms linking these disorders with the observed CoQ<sup>10</sup> deficiency have yet to be elucidated.

Primary CoQ<sup>10</sup> deficiency is far rarer [248]. Emmanuele et al. [247] first classified the clinical manifestations of primary CoQ<sup>10</sup> deficiency into five distinct phenotypes: encephalomyopathy, isolated myopathy, nephropathy, infantile multisystemic disease, and cerebellar ataxia. However, it has been argued that this subdivision should be updated, as new cases have been discovered with novel mutations presenting a wide range of other clinical phenotypes, as well as different combinations of the previously described symptoms [248]. To date, mutations in ten genes have been associated with primary CoQ<sup>10</sup> deficiency (Table 9).

With the exception of Coq3, patients have been reported with mutations in all other components of the CoQ multi-subunit complex [249,250]. These patients can be treated with CoQ<sup>10</sup> supplementation with partial success. Early treatment based on early diagnosis is critical for the best outcome [251]. Because of its poor solubility, CoQ<sup>10</sup> is only administrated in oral formulations, despite its destitute bioavailability [252,253]. Similarly, uptake of CoQ6 in yeast *coq* mutants is inefficient [254,255].

Due to the striking homology between human and yeast COQ genes [227], studies of CoQ proteins in *S. cerevisiae* may provide insight into human homologs, leading to the identification of residues critical for protein function and, therefore, with higher pathogenic potential [256]. Indeed, yeast *coq3*, *coq8*, *coq9*, and *coq10* mutants are complemented by the human counterparts [239,257–259], while yeast *coq5* null mutants are complemented by human *COQ5* combined with overexpression of *COQ8* [260]. Furthermore, studies of pathogenic mutations in *COQ* genes have been validated in yeast *coq1* [261], *coq2* [261,262], *coq4* [263], *coq6* [264], *coq8* [241], and *coq9* mutants [265].

*PDSS1* and *PDSS2*, both human homologs of yeast *COQ1*, encode two proteins that form a heterotetramer that catalyzes the elongation of the isoprenoid side chain. *PDSS1* does not complement the yeast *coq1* null mutant [261]. Mutations in both of these genes lead to infantile multisystemic disease, a heterogeneous disorder characterized by psychomotor regression, encephalopathy, optic atrophy, retinopathy, hearing loss, renal dysfunction and heart valvulopathy [247,261]. Mutations in *PDSS2* have been associated with additional phenotypes, including Leigh syndrome, steroid resistant nephrotic syndrome (SRNS)—an atypical manifestation for other mitochondrial disorders but quite common for CoQ<sup>10</sup> deficiencies; and hepatocellular carcinoma [247,266,267]. It has been shown that the downregulation of *PDSS2* can induce a shift from aerobic metabolism to anaerobic glycolysis, as well as increased chromosomal instability—a possible pathogenic mechanism for hepatocellular carcinoma [267].

Pathogenic mutations in *COQ2*, encoding a parahydroxybenzoate-polyprenyltransferase that catalyzes the addition of the isoprenoid chain to the benzoquinone ring, were the first to be associated with primary CoQ<sup>10</sup> deficiency [268]. Pathogenic mutations in human *COQ2* have been confirmed in yeast by complementation studies of the yeast *coq2* null mutant [261,262]. Clinical manifestations of these mutations include isolated SRNS, SRNS with encephalomyopathy resembling MELAS, fatal infantile multisystemic disease, and late-onset multiple-system atrophy and retinopathy [247,248].

The first pathogenic mutation in *COQ4*, required for the stability of the CoQ synthome, was found as a haploinsufficiency, with a phenotype similar to that of a heterozygous yeast mutant [263]. The patient presented facial dysmorphism and muscle hypotonia, which improved significantly with CoQ<sup>10</sup> supplementation [263]. Since then, a total of 19 patients, all infants, have been identified with mutations in *COQ4*, with clinical phenotypes that included cerebellar atrophy, lactic acidosis, seizures, muscle weakness, cardiomyopathy, ataxia, and Leigh syndrome [269,270]. None of the patients suffered from nephropathy, typically found in primary CoQ<sup>10</sup> deficiency.

To date, only three cases of pathogenic mutations in the methyltransferase encoded by *COQ5*, have been recorded. The affected individuals were three female siblings presenting with non-progressive cerebellar ataxia, dysarthria, and mild to moderate cognitive disability [250]. Two of the three siblings exhibited myoclonic jerks and generalized tonic-clonic seizures in adolescence and early 20s. Next-generation sequencing identified a tandem duplication of the last four exons of *COQ5*, while biochemical studies showed a 33% reduction of CoQ<sup>10</sup> in skeletal muscle—sufficient to sustain a basal rate but insufficient to reach maximal efficiency of respiration [250].

Mutations in *COQ6*, encoding an enzyme involved in hydroxylation and deamination reactions during CoQ biosynthesis, have been primarily associated with SRNS [264], characterized by significant proteinuria with resulting hypoalbuminemia and edema and presenting with focal segmental glomerulosclerosis [248]. The pathogenicity of the first six *COQ6* mutations in human patients was confirmed by complementation studies using the yeast *coq6* null mutant [264]. Three patients harboring a *COQ6* mutation also suffered from infantile multisystemic disease [247]. Based on in vitro and in vivo studies of renal podocyte cell lines, it has been hypothesized that the pathogenicity of *COQ6* mutations relates to respiratory chain deficiency, ROS generation, disruption of podocyte cytoskeleton and induction of cellular apoptosis, ultimately resulting in SRNS [271].

Three cases of primary CoQ<sup>10</sup> deficiency caused by mutations in *COQ7*, responsible for the penultimate step of CoQ biosynthesis, have been reported in two children with similar phenotypes of spasticity, sensorineural hearing loss and muscle hypotonia; and a third more severe case of fatal mitochondrial encephalo-myo-nephro-cardiopathy, persistent lactic acidosis, and basal ganglia lesions [272–274]. In one of the patients, treatment with the unnatural biosynthesis precursor 2,4-dihydroxybenzoate (DHB), a hydroxylated variant of the native 4-hydroxybenzoic acid (4-HB) normally modified by COQ7, increased CoQ<sup>10</sup> levels and partially restored mitochondrial respiration [273].

The human genes *ADCK3* and *ADCK4*, also known as *COQ8A* and *COQ8B*, are both homologs of yeast *COQ8*. Mutations in *COQ8A* have mostly been associated with autosomal recessive progressive cerebellar ataxia (ARCA), often accompanied by childhood onset cerebellar atrophy, with and without seizures, and exercise intolerance [241,248,275]. The pathogenic nature of the *ADCK3* mutations was corroborated using the yeast counterpart system [241]. However, isolated cases of psychiatric disorders, seizures, migraines, and dysarthria have been reported [248]. Studies of fibroblast cell lines isolated from ARCA patients showed an increased sensitivity to oxidative stress induced by hydrogen peroxide, high levels of oxidative stress and changes in mitochondrial homeostasis as a result of loss-of-function mutations in *COQ8A* [275]. Interestingly, concomitant with the upregulation of ROS production, increased respiratory supercomplex stability and basal respiratory rate were observed, suggesting that the loss of ADCK3 could result in a compensatory elevation of respirasome formation [275]. The relationship between the two human paralogs *COQ8A* and *COQ8B* is unclear. However, pathogenic mutations in the two genes lead to completely different clinical phenotypes. Indeed, all patients with *COQ8B* mutations suffered from SNRS, with only a single case of neurological involvement reported [248,276].


**Table 9.** Pathologies resulting from mutations in Coenzyme Q genes and their yeast homologs.

<sup>1</sup> Review describing many mutations. <sup>2</sup> In different patients.

Primary CoQ<sup>10</sup> deficiency caused by mutations in *COQ9* is extremely rare, with only seven cases from four families having been reported. The *COQ9* gene product binds to the polyprenyl tail of CoQ intermediates with high specificity, allowing the modification of the benzene ring by Coq7 and other components of the CoQ synthome [259,277]. The reported cases include an infant suffering from lethal lactic acidosis, seizures, cerebral atrophy, HCM, and renal dysfunction [248,265], a boy diagnosed with neonatal Leigh-like syndrome who died at 18 days of age from cardio-respiratory failure [278], four siblings with an unknown and ultimately lethal condition characterized by dilated cardiomyopathy, anemia, abnormal appearing kidney, and suspected Leigh syndrome [279]; and a nine-month old girl presenting with microcephaly, truncal hypotonia, and dysmorphic features [280].

It has been shown that the pathogenicity of primary and secondary CoQ<sup>10</sup> deficiencies is linked to the impairment of electron transfer to Complex III, ultimately resulting in decreased mitochondrial respiration and ATP synthesis [248,281]. It has also been noted that the neurological presentation of CoQ<sup>10</sup> deficiency is likely associated with oxidative damage and caspase-independent apoptotic cell death in the brain, as a result of mitochondrial impairment [281]. While renal dysfunction is not uncharacteristic of mitochondrial deficiencies, the glomerular involvement, as opposed to the tubular damage seen in other mitochondrial cytopathies, is perplexing and could be a result of impaired CoQ<sup>10</sup> antioxidant function [248]. Equally puzzling is the remarkable diversity of clinical phenotypes resulting from mutations in different COQ genes. Based on in vivo studies of mouse models harboring *COQ9* mutations, Luna-Sánchez et al. [281] have hypothesized that a key factor in determining the degree of severity and particular clinical phenotypes of CoQ<sup>10</sup> deficiencies is the stability of the CoQ synthome. Lastly, decreased CoQ<sup>10</sup> levels impair the activity of sulfide:quinone oxidoreductase, an enzyme involved in the catabolism of H2S [282]. While at physiological conditions H2S serves as an electron donor to the mitochondrial respiratory chain, at elevated levels it inhibits Complex IV activity, resulting in reduced cellular respiration. Over-physiological levels of H2S could therefore participate in the pathogenic mechanism of CoQ<sup>10</sup> deficiency [282].
