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Upregulation of COX4-2 Via HIF-1 in Mitochondrial COX4-1 Deficiency

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Upregulation of COX4-2 Via HIF-1 in Mitochondrial COX4-1 Deficiency cells Article Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency Liza Douiev 1,*, Chaya Miller 1, Shmuel Ruppo 2 , Hadar Benyamini 2, Bassam Abu-Libdeh 3 and Ann Saada 1,4,* 1 Department of Genetics, Hadassah Medical Center, Jerusalem 9112001, Israel; [email protected] 2 Info-CORE, I-CORE Bioinformatics Unit of the Hebrew, University of Jerusalem and Hadassah Medical Center, Jerusalem 91120011, Israel; [email protected] (S.R.); [email protected] (H.B.) 3 Department of Pediatrics and Genetics, Makassed Hospital and Al-Quds University, East Jerusalem 91220, Palestine; [email protected] 4 Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 9112001, Israel * Correspondence: [email protected] (L.D.); [email protected] or [email protected] (A.S.) 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 a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the 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 quantified by RT-qPCR. COX activity and OXPHOS function were Citation: Douiev, L.; Miller, C.; Ruppo, S.; Benyamini, H.; measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by Abu-Libdeh, B.; Saada, A. CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, Upregulation of COX4-2 via HIF-1α with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and in Mitochondrial COX4-1 Deficiency. glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a Cells 2021, 10, 452. https://doi.org/ compensatory mechanism in COX4-1 deficiency. 10.3390/cells10020452 Keywords: mitochondria; cytochrome c oxidase; COX4-1; COX4-2; HIF-1α Academic Editor: Yidong Bai Received: 9 February 2021 Accepted: 18 February 2021 1. Introduction Published: 20 February 2021 The mammalian cytochrome c oxidase (COX, mitochondrial respiratory chain complex IV) is a dimeric multisubunit complex that is comprised of fourteen mitochondrial and Publisher’s Note: MDPI stays neutral nuclear-encoded subunits. COX is considered the rate-limiting complex of the oxidative with regard to jurisdictional claims in phosphorylation system (OXPHOS). The two unique regulatory mechanisms (compared to published maps and institutional affil- iations. the other OXPHOS complexes) are the expression of isoforms and the binding of specific regulatory factors to nuclear-encoded subunits [1,2]. 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 the fine Copyright: © 2021 by the authors. tuning of the mitochondrial respiratory capacity. COX4 isoform 1 (COX4-1), which is the Licensee MDPI, Basel, Switzerland. main subunit 4 of COX, is ubiquitously expressed in mammalian tissues under normoxic This article is an open access article conditions. COX4 isoform 2 (COX4-2), the less common isoform, is primarily expressed distributed under the terms and conditions of the Creative Commons in the lung and at lower levels in the placenta, heart, brain and pancreas. COX4-2 is Attribution (CC BY) license (https:// preferentially expressed under hypoxia and oxidative stress, and it is suggested that the creativecommons.org/licenses/by/ isoform switch results in a more efficient COX activity. [2,3]. 4.0/). Cells 2021, 10, 452. https://doi.org/10.3390/cells10020452 https://www.mdpi.com/journal/cells Cells 2021, 10, 452 2 of 15 In 2017, we reported a novel form of isolated COX deficiency caused by a K101N variant of the COX4I1 gene encoding COX4-1 in a patient presenting with Fanconi anemia- like features, short stature and mild dysmorphism, while all the known Fanconi anemia (FA) gene sequences were intact. We proved the pathogenicity of the homozygous K101N variant by demonstrating 25% residual COX activity in the patient fibroblast homogenate and almost undetectable COX4-1 protein in mitochondria, and by performing complementation studies with the wild-type gene [4]. Additionally, we verified chromosomal instability by phospho-histone H2A.X (γH2AX) staining [5]. 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]. 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 [7–9]. 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 demonstrated elevated COX4-2 in the K101N patient’s fibroblasts as well as in a human foreskin fibroblast cell line (HFF) with stable COX4-1 knockdown. This upregulation is linked to HIF-1α stabilization, nuclear localization and the upregulation of both hypoxia and glycolysis pathways. 2. Materials and Methods 2.1. Tissue Cultures Previously established skin primary fibroblast cultures from the patient (informed consent was obtained, IRB #0485-09) [4], controls and human foreskin fibroblasts (HFF-1 ATCC) were maintained in high-glucose DMEM supplemented with 15% fetal bovine serum, L-glutamine, pyruvate and 50 µg/mL uridine (Biological Industries, Beit Ha’emek, Israel). For immunocytochemistry, cells were seeded on µ-slide 8 well ibiTreat sterile tissue culture slides (NBT; New Biotechnology Ltd., Jerusalem, Israel). For Western blotting or RNA analysis, cells were seeded, and grown in tissue culture bottles. For positive hypoxia controls in the immunostaining and Western blotting, cells were preincubated with 500 µL ◦ of CoCl2 supplemented with a new fresh medium, for 6 or 24 h, at 37 C and 5% CO2 [10]. ◦ All the cells were incubated at 37 C in a humidified 5% CO2 atmosphere. 2.2. RNA Interference For the downregulation of COX4I1, we employed the MISSION® shRNA plasmid DNA vector system. The system included five plasmids; each targeted against a different site of COX4I1. In addition, we used a nonmammalian shRNA Control Plasmid DNA target as a control vector (Sigma-Aldrich, St.Louis, MO, USA)). We introduced each of the DNA plasmids into HEK293FT cells by co-transfection with pLP1, pLP2, and pLP/VSVG plas- mids using Lipofectamine (ViraPower; Invitrogen, Carlsbad, CA, USA). Human foreskin fibroblasts were infected with viral supernatant containing polybrene. Stably transfected cells were selected with puromycin (2 µg/mL) for three weeks. Preliminary results showed that shRNA #TRCN0000232554 (COX4-1 shRNA) was the most suitable for further study. 2.3. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) Total RNA was isolated from patient, HFF-shRNA, HFF-CV and healthy control pri- mary fibroblasts with Tri-Reagent (Telron, Israel), 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 primer se- quences were used for qPCR are detailed in Table1. Cells 2021, 10, 452 3 of 15 Table 1. Primers qPCR Gene Forward Primer Reverse Primer COX4I1 (NM_001861.6) 50-TTTCACCGCGCTCGTTAT-30 50-CTTCATGTCCAGCATCCTCTT-30 COX4I2 (NM_032609) 50-GAAGACGAGGGATGCACAG-30 50-GGCTCTTCTGGCATGGG-30 PDK1 (NM_001278549.2) 50- CAGGACACCATCCGTTCAAT-30 50- AGCTTTAGCATCCTCAGCAC-30 GLUT1 (NM_006516.4) 50-GCTACAACACTGGAGTCATCAA-30 50-ACTGAGAGGGACCAGAGC-30 HK1 (NM_000188.3) 50- CCCTAAATGCTGGGAAACAAAG-30 50- CCCTTCTTGGTGAAGTCGATTA-30 HK2 (NM_000189.5) 50- CATCCTCCTCAAGTGGACAAA-30 50- ACCACATCCAGGTCAAACTC-30 GUSB (NM_000181.4) 50-GAAAATATGTGGTTGGAGAGCTCATT-30 50-CCGAGTGAAGATCCCCTTTTTA-30 GAPDH (NM_001357943.2) 50-CAAGAGCACAAGAGGAAGAGAG-30 50-CTACATGGCAACTGTGAGGAG-30 2.4. COX Enzymatic Activity COX activity in cells disrupted by sonication and solubilization with 0.5 mg/mL dodecyl-β-D-maltoside (Calbiochem, San-Diego, CA, US) on ice was determined by spec- trophotometry, monitoring the oxidation of 50 µM reduced cytochrome c at 550 nm in 10 mM potassium phosphate buffer, pH 7.0, at 37 ◦C on a Kontron UVICON xs double beam spectrophotometer (Secomam, Ales, France). 2.5. Oxygen Consumption Rate The oxygen consumption rate (OCR) was measured using an Agilent Seahorse XF Cell Mito Stress Test Kit (#103015-100) (Seahorse Biosciences, North Billeric, MA, USA). A total of 10,000 cells/well were seeded on an XF96-well plate.
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