2.3.2. NDUFA3

The NDUFA3 gene is located on chromosome 19. The NDUFA3 protein is a 9.3 kDa protein that consists of 83 amino acids. It is a single-pass protein located in the inner mitochondrial membrane. It lies in the junction between the membrane and matrix arms of complex I and contains a kinked transmembrane alpha-helix domain that spans residues 17–35 and 37–49. During processing, the initiator methionine is cleaved, and Ala at position 2 is N-acetylated by analogy with the bovine enzyme [48]. NDUFA3 contacts core subunits ND1, ND3, and NDUFS8. Additionally, it contacts supernumerary subunits NDUFA8 and NDUFA13 (see Figure 7). In a knockout strain of cultured human cells, the loss of NDUFA3 resulted in a reduced expression of complex I and migrated as a smaller-than-normal-sized complex in BN gel electrophoresis [11]. An earlier study demonstrated the role of NDUFA3 in the assembly of the Q-module of complex I [49].

Though no clinical mutations in NDUFA3 have been identified yet, it has been shown that the full deletion of the NDUFA3 gene, along with other genes on chromosome 19, results in retinitis pigmentosa (RP), a disorder caused by photoreceptor cell degeneration. It is characterized by vision loss following night-blindness. In 2006, a family was identified as carrying a 30 kb deletion in chromosome 19 [50]. The family members afflicted with RP contained at least one copy of chromosome 19 that lacked a region containing genes NDUFA3, TFPT, PRP31, and the OSCAR promoter. The ages of onset of the RP symptoms varied from 3 to 30 years of age. Surprisingly, the patients were not afflicted by any disease other than RP. From this study, the absence of symptoms relating to complex I deficiencies suggests that only one copy of the gene for NDUFA3 may be sufficient for complex I activity.

Similarly, in 2011, another family was discovered to harbor a 112 kb deletion that encompassed the PRP31 gene and five of its upstream genes: TFPT, OSCAR, NDUFA3, TARM-1, and VSTM-1 [51]. Only one family member, a 33-year-old female, was diagnosed with RP. Once again, the RP patient showed no signs of complex I deficiency, and the authors suggested that one copy of NDUFA3 was sufficient for complex I activity.

#### 2.3.3. NDUFA13

The NDUFA13 gene is located on chromosome 19. NDUFA13 is a unique complex I subunit because it functions as a cell-death regulatory protein, GRIM-19, as well. It has been suggested that GRIM-19 regulates STAT3, a signal transducer and transcription activator, and is involved in interferon-β- and retinoic acid-induced cancer cell death [52]. For this reason, although NDUFA13 is mainly found in the inner mitochondrial membrane as a single-pass membrane protein, it can also be translocated to the nucleus to serve its apoptotic purpose. NDUFA13 is a 16 kDa protein synthesized as a 144-residue polypeptide, but the initiator methionine is removed during processing. It has a transmembrane alpha helix that spans residues 30–51 (see Figure 7). NDUFA13 has a longer alternate isoform that exists as a 222-residue polypeptide. NDUFA13 shares a large contact surface area with core subunits ND1, ND6, and NDUFS2, NDUFS5, NDUFA3, and NDUFA8. It weakly contacts ND3, NDUFS8, and supernumerary subunits NDUFA1 and NDUFA7. In a knockout line of cultured human cells, the loss of NDUFA13 resulted in a reduced expression of complex I, and it migrated as a smaller-than-normal-sized complex in BN gel electrophoresis [11]. Furthermore, when homozygous GRIM-19-deficient mice were generated, embryonic lethality resulted [53].

At least two diseases have been associated with mutations in NDUFA13. In 2015, a pair of sisters were discovered to harbor the first pathogenic germinal mutation identified in the short isoform one of NDUFA13, p.Arg57His (c.170 G > A in exon 2) [54]. Both sisters were homozygous for the mutation and presented with early onset hypotonia, dyskinesia, auditory neuropathy, and severe optic neuropathy. The patients were siblings who had been born from a consanguineous marriage. Their parents and elder sister were heterozygous for the p.Arg57His mutation and asymptomatic. The sisters were still living in 2015, the elder sister being 12 years old at the time. Though the sisters experienced early-onset neurological symptoms, the progression of neurological symptoms has been slow. Arg57 is a highly conserved residue across species. Upon biochemical analysis, NDUFA13 expression was reduced by a mean of 70% in the two sisters. Additionally, NDUFA9 and NDUFB8 expression was reduced by 95% and 90%, respectively. Due to the absence of intermediary complex I structures in the BN gel electrophoresis, the p.Arg57His mutation was concluded to cause major instability in NDUFA13 and prevent complex I assembly. It was suggested that the varied clinical symptoms in these sisters could be attributed to the dual function of NDUFA13/GRIM-19.

Somatic mutations in the long isoform two of NDUFA13 have been implicated in Hurthle cell thyroid tumors. Isoform two of NDUFA13 has 222 amino acids instead of 144 amino acids like isoform one. In 2005, four unrelated patients with Hurthle cell thyroid carcinomas were found to harbor four different heterozygous missense mutations that affected the N-terminal sequence of NDUFA13 [55]. Because the patients' transcripts arose from an alternate reading frame and coded for isoform two, a structural analysis of these mutations cannot be performed.
