A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics

Abstract

Background: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed.

Results: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology.

Conclusions: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

Keywords: Alternative open reading frame; Homo sapiens; Mitochondria; Mitochondrial bioenergetics; Mitochondrial proteome; Mitogenome; Protein coding potential; nd4.

Fig. 1

Small ORFs and alternative ORFs in the human mitochondrial genome. In dark gray in the external circle: the typical 13 protein-coding genes. In gray in the external circle: the two rRNA genes. In white in the external circle: the D-loop. Arrows positioned inside the circle indicate smORFs and altORFs on the main coding 5′–3′ (frames 1, 2, and 3: black arrows) and complementary 3′–5′ (frames − 1, − 2, and − 3: white arrows) strands. In blue: the ORFs coding for the micropeptides Humanin, SHLP1-6, and MOTs-c found in the 16S and 12S rRNA genes and SHMOOSE found in the tRNA-Ser overlapping with a small portion of nad5. The putative gau gene is also shown. In pink: the eight chosen candidates for antibody production. In red: MTALTND4

Fig. 2

Identification of an alternative protein in the mtDNA-encoded nd4 gene. A The MTALTND4 ORF and its DNA and protein sequences (antigenic sequence underlined), as well as its protein structure prediction. This ORF is frameshifted by 2 nucleotides relative to ND4 reading frame. B MTALTND4 detected by western blotting in HeLa and HEK-293 T cells—a protein lysate of HeLa cells transfected with the coding sequence of the corresponding MTALTND4 protein with a FLAG tag was used as a positive control for MTALTND4. C MTALTND4, mtDNA-encoded cytochrome c oxidase subunit I (CO1), nuclear-encoded Actin and nuclear-encoded mitochondrial ATP5 in HeLa (control) and HeLa cells treated with chloramphenicol (cam) or actinonin (act), and in HeLa Rho0 cells. D Detection of MTALTND4 and nuclear-encoded mitochondrial ATP5 by immunofluorescence in HeLa cells treated with chloramphenicol. E Multiple peptide sequence alignment in five primate species. Black background for residues conserved in all species and gray for residues conserved in 4 out of 5 species. Asterisk indicates a stop codon. F Detection of MTALTND4 and nuclear-encoded mitochondrial ATP5 by immunofluorescence in HeLa cells. G Detection of MTALTND4 and nuclear-encoded mitochondrial ATP5 by western blotting in human plasma

Fig. 3

Impact of MTALTND4 upon mitochondrial and cell physiology. A–B Dose-dependent effect on intact (ce) HeLa and HEK-293 T cells routine respiration (n = 3 and n = 4, respectively). Respirometry data were normalized for the routine respiration in the absence of the peptide. For each titration point, data are represented as fraction of their own simultaneous control (blue dotted line). C–D Mitochondrial respiration in intact (ce) HeLa and HEK-293 T cells in the presence of 10 µM MTALTND4 (n = 6–9 and n = 6, respectively). Respirometry data were expressed as flux control ratios (FCR), normalized for the routine respiration before treatment (ce_R). ce-pept_R routine respiration with MTALTND4, ce-pept_L leak respiration with MTALTND4, ce-pept_E maximal uncoupled respiration with MTALTND4. E Mitochondrial spare reserve capacity (SRC) in HeLa and HEK-293 T cells in the presence of 10 µM MTALTND4 (n = 6 and n = 6, respectively). F Mitochondrial respiration in permeabilized (pce) HEK-293 T cells in the presence of 10 µM MTALTND4 (n = 6). Respirometry data were normalized for the CI + II-sustained coupled respiration before treatment (CI + II_P). CI + II-pept_P CI + II-linked coupled respiration with MTALTND4, CI + II-pept_E CI + II-linked uncoupled respiration with MTALTND4, CIV-pept_E cytochrome c oxidase standalone capacity with MTALTND4. G Hydrogen peroxide efflux rate (pmol H₂O₂ ∙ mg proteins⁻¹ ∙ min⁻¹) in intact HEK-293 T cells in the presence of 30 µM MTALTND4 (n = 5). H Catalase activity (U ∙ mg proteins⁻¹) in intact HEK-293 T cells incubated 4 h with 30 µM MTALTND4 (n = 5). I Lactate dehydrogenase activity (U ∙ mg proteins⁻¹) in intact HEK-293 T cells incubated 4 h with 30 µM MTALTND4 (n = 5). L ATP content (nmol ATP ∙ mg proteins.⁻¹) in intact HEK-293 T cells incubated 4 h with 30 µM MTALTND4 (n = 5). M–N Proliferation (Mx cells) and viability (%) of HeLa cells measured at different time points (24, 48, and 72 h) and different MTALTND4 concentrations (0, 0.1, 10, and 30 µM) (n = 3–5). Statistical analyses: A–B one sample t test, C, D, F, G, H, I, L paired t test, E linear mixed model. Factors cell type (2 levels) and treatment (2 levels) plus interaction. M, N linear mixed model. Factor treatment (4 levels), with letters indicating statistical difference following a post hoc multi comparison test. Data shown as mean ± sem. 0.05 > p ≤ 0.09; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. A detailed summary (individual values) is reported in Additional file 1: Tables S6-S10