Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer

Abstract

Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of 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.

Keywords: Poldip2; hypoxia; lipoylation; metabolism; mitochondria.

Fig. 1.

Mitochondrial protein Poldip2 is required for oxidative metabolism. (A) HASMC fractions showing Poldip2 mitochondrial localization. (B) Profile of mitochondrial function over time in siControl- and siPoldip2-treated HASMCs. C, cytosol; M, mitochondria; N, nucleus. Four basal OCR and ECAR measurements were made, and 1 μg/mL oligomycin (O), 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (F), and 10 μM antimycin-A (A) were then sequentially injected. (C) Box plot summarizing the data obtained from the analysis of OCR/ECAR profiles (basal corresponds to antimycin-A–inhibitable, ATP-linked corresponds to oligomycin-inhibitable, and maximal capacity corresponds to the increase induced by the uncoupler FCCP). (D) Energy map showing the metabolic reprograming induced by Poldip2 deficiency. All data are presented as mean ± SE from three to six independent experiments. Seahorse experiments were performed three independent times with similar results.

Fig. 2.

Poldip2 deficiency induces the expression of metabolic shift markers. (A, Left) Western blot of metabolic shift markers in HASMCs treated with siControl or siPoldip2. (A, Right) Box plots represent mean ± SE from five independent experiments. The P value indicated was calculated by an independent t test. (B) Total intracellular α-KG concentrations (C) HIF-1α stabilization induced by Poldip2 deficiency is reversed by incubation with a cell-permeable α-KG analog (octyl-α-KG, 1 mM). The blot is representative of four independent results.

Fig. 3.

Poldip2 deficiency induces loss of DLAT and DLST lipoylation and degradation of ACSM1. (A, Left) Western blot showing the expression of proteins of the lipoylation pathway in siControl- and siPoldip2-treated cells. (A, Right) Box plots represent mean ± SE from six independent experiments. (B and C) PDH and αKGDH activities in isolated mitochondria from Poldip2-deficient cells. All data are presented as mean ± SE from three to six independent experiments.

Fig. 4.

Forced expression of ACSM1 reverses the phenotype of Poldip2-deficient cells. (A, Left) Western blot showing the effect of ACSM1 forced expression in siControl- and siPoldip2-treated cells on the lipoylation of DLAT and DLST. (A, Right) Box plots represent mean ± SE from four to six independent experiments. (B, Left) Western blot showing the effect of ACSM1 forced expression in siControl- and siPoldip2-treated cells on the lipoylation of DLAT and DLST. (B, Right) Box plots represent mean ± SE from four to six independent experiments. Symbols above boxes indicate statistical significance. Pairs of bars that do not share any symbol are significantly different from each other.

Fig. 5.

Poldip2 regulates DLAT and DLST lipoylation by Clp-protease complex–mediated degradation of ACSM1. (A) LC-MS/MS’s peptide-spectrum match scores for CLPX binding to myc-tagged Poldip2. IP, immunoprecipitation; PSM, peptide-spectrum match. (B) Coimmunoprecipitation of overexpressed tagged Poldip2 and endogenous Poldip2 shows specific binding to CLPX. IB, immunoblot. (C, Left) Western blot showing the expression of proteins of the salvage lipoylation pathway in siControl-, siPoldip2-, siCLPP-, and siPoldip2 and siCLPP combined-treated HASMCs. (A, Right) Box plots represent mean ± SE from four to six independent experiments. Symbols above boxes indicate statistical significance. Pairs of bars that do not share any symbol are significantly different from each other.

Fig. 6.

Model. Decreased Poldip2 expression and binding to CLPX permit activation of the Clp-protease and degradation of the lipoic acid-activating enzyme ACSM1. As a result, no lipoyl-AMP is synthetized and LIPT1 has no substrate for lipoylation of DLAT and DLST. Lipoylation deficiency inhibits the TCA cycle, inducing PHD2 metabolic inhibition, HIF-1α stabilization, and signaling. PP, pyrophosphate.

Fig. 7.

Poldip2 is an oxygen-sensitive protein that controls protein lipoylation under hypoxia. (A, Left) Poldip2 in HASMCs cultured under normoxia or hypoxia for 48 h. (A, Right) Box plots represent the mean ± SE from four to six independent experiments. (B, Left) Western blot of ACSM1 expression and protein lipoylation in cells infected with control adenovirus or AdPoldip2 after being maintained under normoxia or hypoxia for 48 h. (B, Right) Box plots represent the mean ± SE from four to six independent experiments. Symbols above boxes indicate statistical significance. Pairs of bars that do not share any symbol are significantly different from each other.

Fig. 8.

Poldip2 down-regulation participates in BT549 metabolic reprograming. (A) Highly glycolytic TNBCs exhibit low Poldip2 expression and inhibition of DLAT and DLST lipoylation. (B) Triple-negative cancer cell line BT549 was stably transfected with empty or Poldip2-expressing vectors. Poldip2 expression increases ACSM1 stabilization, increases DLAT and DLST lipoylation, and reduces HIF-1α. Western blots are representative of two experiments from cell lines; samples after were taken 4 wk and 8 wk after transfection. (C) Basal mitochondrial respiration in BT549 compared with BT549 expressing Poldip2. (D) Growth curves of BT549 compared with BT549 expressing Poldip2. Experiments were done in triplicate. The seahorse experiment was performed once with 10 replicates. Data and statistics shown were calculated by the wave software from one 96-well plate.