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Poldip2 Is an Oxygen-Sensitive Protein That Controls PDH and Αkgdh Lipoylation and Activation to Support Metabolic Adaptation in Hypoxia and Cancer

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Poldip2 Is an Oxygen-Sensitive Protein That Controls PDH and Αkgdh Lipoylation and Activation to Support Metabolic Adaptation in Hypoxia and Cancer Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer Felipe Paredesa, Kely Sheldona, Bernard Lassèguea, Holly C. Williamsa, Elizabeth A. Faidleya, Gloria A. Benavidesb, Gloria Torresa, Fernanda Sanhueza-Olivaresa, Samantha M. Yeligarc,d, Kathy K. Griendlinga, Victor Darley-Usmarb, and Alejandra San Martina,1 aDivision of Cardiology, Department of Medicine, Emory University, Atlanta, GA 30322; bDepartment of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294; cDivision of Pulmonary, Allergy, Critical Care, and Sleep, Department of Medicine, Emory University, Atlanta, GA 30322; and dAtlanta Veterans Affairs Medical Center, Decatur, GA 30033 Edited by Hening Lin, Cornell University, Ithaca, NY, and accepted by Editorial Board Member Michael A. Marletta January 5, 2018 (received for review December 3, 2017) Although the addition of the prosthetic group lipoate is essential to the prosthetic group. It is required for the activity of two key catabolic activity of critical mitochondrial catabolic enzymes, its regulation is enzymes: the pyruvate dehydrogenase (PDH) and the α-ketoglu- unknown. Here, we show that lipoylation of the pyruvate dehydroge- tarate dehydrogenase (αKGDH) complexes. Specifically, α-lipoic nase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dy- acid is covalently attached to dihydrolipoamide S-acetyltransferase namically regulated process that is inhibited under hypoxia and in (DLAT) and dihydrolipoamide S-succinyltransferase (DLST), the cancer cells to restrain mitochondrial respiration. Mechanistically, we dihydrolipoyl transacetylase (or E2) subunits of the PDH and the α found that the polymerase-δ interacting protein 2 (Poldip2), a KGDH, respectively. nuclear-encoded mitochondrial protein of unknown function, controls Lipoic acid synthesis and the mechanisms leading to protein the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyl- lipoylation have been investigated most thoroughly in Escher- transferase subunits by a mechanism that involves regulation of ichia coli and, recently, in Saccharomyces cerevisiae, but they are the caseinolytic peptidase (Clp)-protease complex and degradation still poorly understood in mammals. Currently, two mammalian BIOCHEMISTRY of the lipoate-activating enzyme Ac-CoA synthetase medium-chain pathways are proposed. In the first, which is evolutionarily con- family member 1 (ACSM1). ACSM1 is required for the utilization of served, lipoic acid is synthesized in the mitochondria from an octanoyl-acyl carrier protein provided by the fatty acid synthesis lipoic acid derived from a salvage pathway, an unacknowledged pathway. The octanoyl moiety is attached to lipoate-dependent lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation enzymes by the ligase LIPT2. Next, the [Fe-S] cluster-containing represses mitochondrial function and induces the stabilization of enzyme LIAS catalyzes the radical-mediated insertion of two hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition α– sulfur atoms into the C-6 and C-8 positions of the octanoyl of prolyl-4-hydroxylases (PHDs). HIF-1 mediated retrograde signal- moiety bound to the lipoyl domains of lipoate-dependent en- ing results in a metabolic reprogramming that resembles hypoxic and zymes. The second and less characterized is the salvage pathway, cancer cell adaptation. Indeed, we observe that Poldip2 expression is which uses exogenous lipoate that is taken up via the sodium- down-regulated by hypoxia in a variety of cell types and basally re- dependent multivitamin transporter (7, 8). Exogenously scavenged pressed in triple-negative cancer cells, leading to inhibition of lipoic acid is then activated by addition of AMP and transferred lipoylation of the pyruvate and α-KDH complexes and mitochondrial to lipoate-containing enzymes. In contrast to bacteria and yeast, dysfunction. Increasing mitochondrial lipoylation by forced expres- sion of Poldip2 increases respiration and reduces the growth rate of Significance cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation. The present work establishes that the addition of the pros- thetic group lipoic acid to catabolic enzymes is a dynamically regulated posttranslational modification that increases meta- Poldip2 | lipoylation | mitochondria | hypoxia | metabolism bolic plasticity under hypoxia and in cancer cells. We show that δ δ that the polymerase- interacting protein 2 (Poldip2) is an oxygen- he polymerase- interacting protein 2 (Poldip2; also known as sensitive protein that regulates the lipoylation and activation TPDIP38 and mitogenin 1), a ubiquitously expressed protein of the pyruvate and α-ketoglutarate dehydrogenase com- δ (1), was initially identified as a DNA polymerase- interacting plexes. Additionally, our work reveals that mitochondrial pep- protein and a binding partner for the proliferating cell nuclear tidases participate in an integrated response needed for metabolic antigen (2). Later studies showed that homozygous deletion of adaptation. This study positions Poldip2 as a key regulator of Poldip2 in mice results in reduced fetal weight and perinatal le- mitochondrial function and cell metabolism. thality of unknown cause (3). Adult heterozygous mice have no evident physiological phenotype, but more detailed characteriza- Author contributions: F.P. and A.S.M. conceived the project; F.P., B.L., K.K.G., V.D.-U., and tion has shown that they display disrupted extracellular matrix A.S.M. designed research; K.K.G. and V.D.-U. conceptualized experiments; F.P., K.S., organization with excessive and disorganized collagen deposition H.C.W., E.A.F., G.A.B., G.T., F.S.-O., S.M.Y., and A.S.M. performed research; A.S.M. con- tributed new reagents/analytic tools; F.P., B.L., G.A.B., G.T., V.D.-U., and A.S.M. analyzed (4) and an impaired response to ischemia-induced collateral data; and A.S.M. wrote the paper. vessel formation (5). Interestingly, Poldip2 contains an N-terminal The authors declare no conflict of interest. mitochondrial localization sequence and has been found to in- ThisarticleisaPNASDirectSubmission.H.L.isaguesteditorinvitedbythe teract with components of the mitochondrial nucleoid (6), sug- Editorial Board. gesting a mitochondrial role that remains unexplored. One of the This open access article is distributed under Creative Commons Attribution-NonCommercial- aims of this study was to understand the function of Poldip2 in NoDerivatives License 4.0 (CC BY-NC-ND). the mitochondria. 1To whom correspondence should be addressed. Email: [email protected]. α The -(R)-lipoic acid (5-[(3R)-1,2-dithiolan-3-yl] pentanoic acid) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is a universally conserved fundamental metabolite that serves as a 1073/pnas.1720693115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1720693115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 A Results 1.5 Poldip2 contains an N-terminal mitochondrial localization sequence ABC M N O that predicts it will be localized to the mitochondrion (15). Indeed, 1.0 37 - Poldip2 to begin to define its function, we investigated the subcellular dis- F 37 - GAPDH tribution of Poldip2. As predicted from the primary sequence, we 60 - Hsp60 0.5 found that endogenous Poldip2 localizes almost exclusively to the 17 - Histone3 mitochondria in a variety of cell types, including human aortic OCR/ECAR(pmol /mpH) 0.0 01530456075smooth muscle cells (HASMCs) (Fig. 1A), human mammary epi- C P<0.0001 D Time (min) thelial cells (HMECs), and mouse embryonic fibroblasts (MEFs) siControl 110 1.4 siPoldip2 (Fig. S1). Poldip2 was detected at an apparent molecular mass of 1.2 100 37 kDa, which corresponds to the predicted molecular mass of the 1.0 P<0.0001 mitochondrial protease-processed form (16, 17). 0.8 P<0.0001 90 The role of Poldip2 in the mitochondria is unknown. There- 0.6 80 fore, to investigate its distinct contribution to mitochondrial 0.4 0.2 function, we performed a series of experiments manipulating OCR (pmol /min) 70 OCR/ECAR (pmol /mpH) 0.0 25 30 35 40 45 Poldip2 expression. Using extracellular flux analysis to determine Basal ATP linked Maximal cellular bioenergetics as a function of time (Fig. 1B), we found ECAR (mpH/min) that Poldip2-deficient cells display a lower ratio of basal oxygen consumption rate to extracellular acidification rate (OCR/ Fig. 1. Mitochondrial protein Poldip2 is required for oxidative metabolism. (A) ECAR), lower OCAR/ECAR associated with ATP synthesis, HASMC fractions showing Poldip2 mitochondrial localization. (B) Profile of mito- and lower OCR/ECAR maximal capacity (Fig. 1C). Despite the chondrial function over time in siControl- and siPoldip2-treated HASMCs. C, cy- fact that Poldip2 deficiency represses mitochondrial function, we tosol; M, mitochondria; N, nucleus. Four basal OCR and ECAR measurements were did not observe a reduction in mitochondrial biogenesis (102 ± made, and 1 μg/mL oligomycin (O), 1 μM carbonyl cyanide-4-(trifluoromethoxy) = ± = μ 14%, P 0.9 at 24 h; 94 7%, P 0.5 at 72 h), and even though phenylhydrazone (FCCP) (F), and 10 M antimycin-A (A) were then sequentially Poldip2-deficient cells produce less ATP by oxidative respiration, injected. (C) Box plot summarizing the data obtained from the analysis of OCR/
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