Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 February 2021 Upregulation of COX4-2 via HIF-1α in mitochondrial COX4-1deficiency Liza Douiev1*, Chaya Miller1, Shmuel Ruppo2, Hadar Benyamini2, Bassam Abu-Libdeh3, Ann Saada1,4* Affiliations 1 Department of Genetics, Hadassah Medical Center, Jerusalem 9112001, Israel. 2 Info-CORE, I-CORE Bioinformatics Unit of the Hebrew University of Jerusalem and Hadassah Medical Center 3 Department of Pediatrics, Makassed Hospital and Al-Quds University, Jerusalem 91220, Palestinian Authority 4Faculty of Medicine, Hebrew University of Jerusalem, 9112001, Israel *corresponding authors Prof. Ann Saada (Reisch), Department of Genetics, Hadassah Medical Center, Jerusalem 9112001, Israel. [email protected], [email protected] Liza Douiev, Department of Genetics, Hadassah Medical Center, Jerusalem 9112001, Israel [email protected] Abstract Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF), and compared to patient's cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were carried out by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrate elevated COX4-2 levels in the COX4-1-deficient cells with a concomitant HIF-1α stabilization, nuclear localization and upregulated hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α has the are upregulated, also in normoxia as a compensatory mechanism in COX4-1 deficiency. Key Words Mitochondria, cytochrome c oxidase, COX4-1, COX4-2, HIF-1α © 2021 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 February 2021 1. Introduction: The mammalian cytochrome c oxidase (COX, mitochondrial respiratory chain complex IV) is a dimeric multi-subunit complex which is comprised of fourteen mitochondrial and nuclear-encoded subunits. COX is considered the rate-limiting complex of the oxidative phosphorylation system (OXPHOS). The two unique regulatory mechanisms (compared to the other OXPHOS complexes) are; the expression of isoforms and the binding of specific regulatory factors to nuclear-encoded subunits [ 2 Arnold. Mitochondrion. 2012]. One important regulatory mechanism involves the COX4 isoforms and feedback inhibition by ATP. COX4 is the largest nuclear encoded subunit and plays important roles in COX function, assembly and regulation. It is allosterically inhibited at high ATP/ADP ratios, and by phosphorylation, allowing fine tuning of the mitochondrial respiratory capacity. COX4 isoform 1 (COX4-1), which is the main subunit 4 of COX, is ubiquitously expressed in mammalian tissues under normoxic conditions. COX4 isoform 2 (COX4-2) the less common isoform, is primarily expressed in the lung and in lower levels in the placenta, heart, brain and pancreas. COX4-2 is preferentially expressed under hypoxia and oxidative stress and it is suggested that the isoform switch results in a more efficient COX activity. [2 Arnold, 3 Timon]. In 2017, we reported a novel form of COX deficiency caused by a K101N variant in COX4I1 gene encoding COX4-1 in a patient presented with Fanconi anemia-like features, short stature and mild dysmorphism, while all known Fanconi Anemia (FA) gene sequences were intact. We proved the pathogenicity of the homozygous K101N variant by demonstrating 25% residual COX activity in patients’ fibroblasts homogenate, almost undetectable COX4-1 protein in mitochondria, and by performing complementation studies with the wild-type gene [4Abu-Libdeh, 2017]. Additionally, we verified chromosomal instability by Phospho-Histone H2A.X (γH2AX) staining [5 Douiev 2018]. Recently, an additional pathogenic COX4I1 variant (P152T), associated with a much more severe phenotype resembling Leigh syndrome with developmental regression abnormal MRI and 16% residual COX activity in muscle, was reported by Pilali et al. [6 Pilali]. Notably, the phenotype in our patient was different and relatively mild compared to the COX4-1 P152T patient and other patients with isolated COX deficiencies, and nuclear- encoded mitochondrial diseases in general [7 RAK, 8 shoubridge,9Hock 2020]. Accordingly, we hypothesized that a compensatory isoform switching to COX4-2 is able to, at least partially, compensate for the deficient COX4-1. In this work, we demonstrate elevated COX4-2 in the K101N patient's fibroblasts as well as in a human foreskin fibroblasts cell line (HFF) with stable COX4-1 knockdown. This upregulation is linked to HIF-1α stabilization, nuclear localization and upregulation of both hypoxia and glycolysis pathways. 2. Materials and Methods: 2.1. Tissue cultures Previously established skin primary fibroblasts cultures from the patient (Informed consent was obtained IRB #0485-09) [4Abu-Libdeh 2017] controls and human foreskin fibroblasts (HFF-1 ATCC) were maintained in high-glucose DMEM supplemented with 15% fetal bovine serum, L-glutamine, pyruvate and 50µg/mL uridine (Biological Industries, Beit Ha'emek, Israel). For immunocytochemistry, cells were seeded on u-slide 8 well- ibiTreat sterile tissue culture slides (NBT; New Biotechnology Ltd., Jerusalem, Israel). For western blot or RNA analysis, cells were seeded, cells were grown in tissue culture bottles. For positive hypoxia controls in (Immunostaining and western blot) cells were pre- incubated with 500µL CoCl2 supplemented within a new fresh media, for 6 or 24 hours, at 37°C, 5% CO2 [10 Shteyer2009]. All cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 February 2021 2.2. RNA interference For the downregulation of COX4I1, we employed the MISSION® shRNA plasmid DNA vector system. The system included five plasmids each is targeted against different site of COX4I1. In addition, we used a non-mammalian shRNA Control Plasmid DNA target as a control vector (Sigma-Aldrich). We introduced each of the DNA plasmids into HEK293FT cells by co-transfection with pLP1, pLP2, and pLP/VSVG plasmids using lipofectamine (ViraPower; Invitrogen, California, USA). Human foreskin fibroblasts were infected with viral supernatant containing polybrene. Stably transfected cells were selected with puromycin (2µg/mL) for three-weeks. Preliminary results showed that shRNA #TRCN0000232554 (COX4-1 shRNA) was the most suitable. 2.3. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) Total RNA was isolated from patient, HFF-shRNA, HFF-CV and healthy control primary fibroblasts with Tri-Reagent (Telron, Isarel) and cDNA from poly(A)+mRNA was generated using Improm II, Promega, Madison, WI, USA. Real time, quantitative PCR for the quantification of COX4I1, COX4I2, PDK1, SLC2A1, HK1, HK2, GUSB and GAPDH transcripts - was performed using Fast SYBR GreenMaster Mix and the ABI PRISM7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The following primer sequences were used fo qPCR: Gene Forward primer Reverse primer COX4I1 (NM_001861.6) 5'-TTTCACCGCGCTCGTTAT-3' 5'-CTTCATGTCCAGCATCCTCTT-3' COX4I2 (NM_032609) 5'-GAAGACGAGGGATGCACAG-3' 5'-GGCTCTTCTGGCATGGG-3' PDK1 (NM_001278549.2) 5'- CAGGACACCATCCGTTCAAT-3' 5'- AGCTTTAGCATCCTCAGCAC-3' GLUT1 (NM_006516.4) 5'-GCTACAACACTGGAGTCATCAA-3' 5'-ACTGAGAGGGACCAGAGC-3' HK1 (NM_000188.3) 5'- CCCTAAATGCTGGGAAACAAAG-3' 5'- CCCTTCTTGGTGAAGTCGATTA-3' HK2 (NM_000189.5) 5'- CATCCTCCTCAAGTGGACAAA-3' 5'- ACCACATCCAGGTCAAACTC-3' GUSB (NM_000181.4) 5'-GAAAATATGTGGTTGGAGAGCTCATT-3' 5'-CCGAGTGAAGATCCCCTTTTTA-3' GAPDH (NM_001357943.2) 5'-CAAGAGCACAAGAGGAAGAGAG-3' 5'-CTACATGGCAACTGTGAGGAG-3' 2.4. COX enzymatic activity COX activity in cells disrupted by sonication and solubilization with 0.5mg/mL -dodecyl- β-D-maltoside (Calbiochem, San-Diego, CA, US) on ice, was determined by spectrophotometry monitoring the oxidation of 50µM reduced cytochrome c at 550nm in 10mM potassium phosphate buffer, pH7.0 at 37°C on a Kontron UVICON xs double beam spectrophotometer (Secomam, France) 2.5. Oxygen Consumption rate Oxygen consumption rate (OCR) was measured using Agilent Seahorse XF Cell Mito Stress Test Kit (#103015-100) (Seahorse Biosciences, North Billeric, MA, USA). 10,000 cells/well were seeded on an XF96-well plate. Following 72 hr, the growth medium was changed to an unbuffered DMEM (Agilent Technologies, Inc. Wilmington, USA) supplemented with 10mM glucose 1mM pyruvate and 2mM glutamine and the plate was equilibrated at 37°C for 1 hr before the measurements. In the absence and in the presence of sequentially added 2.5 µM Oligomycin, 2 µM Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), 0.5 µM Rotenone and Antimycin), according to the manufacturer's instructions. Oxygen consumption rate (OCR) and extra cellular acidification rate ECAR,