Inhibition of ATPIF1 ameliorates severe mitochondrial respiratory chain dysfunction in mammalian cells - PubMed (original) (raw)

. 2014 Apr 10;7(1):27-34.

doi: 10.1016/j.celrep.2014.02.046. Epub 2014 Mar 27.

Walter W Chen # 1 2 3 4, Kivanc Birsoy # 1 2 3 4, Maria M Mihaylova 1 2 3 4, Iwona Stasinski 5, Burcu Yucel 1 2 3 4, Erol C Bayraktar 1 2 3 4, Jan E Carette 6, Clary B Clish 3, Thijn R Brummelkamp 7, David D Sabatini 5, David M Sabatini 1 2 3 4 8

Affiliations

Inhibition of ATPIF1 ameliorates severe mitochondrial respiratory chain dysfunction in mammalian cells

Walter W Chen et al. Cell Rep. 2014.

Abstract

Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human ρ° cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Haploid genetic screen identifies loss of ATPIF1 as protective against complex III inhibition

(A) Mutagenized KBM7 cells were treated with antimycin and resistant cells were pooled. Gene-trap insertions were identified by massively parallel sequencing and mapped to the human genome. The y-axis represents the statistical significance of a given gene, while the x-axis represents the collection of genes with insertions. The red line indicates the cut-off of statistical significance chosen to determine whether a gene scored as a hit in the screen. For ATPIF1, WT1, and TP53, the number of unique insertions per gene is given in parentheses. (B) Map of unique insertions in ATPIF1 in the resistant cell population. The arrow denotes 5′-3′ directionality, boxes represent exons, and black bars indicate insertions. (C) Immunoblots for indicated proteins in WT and ATPIF1_KO KBM7 cells. (D) Micrographs (left) and viability (right) of WT and ATPIF1_KO KBM7 cells treated with antimycin for 4 days. Error bars are ± s.e.m. (n = 3). Scale bars, 20 μm. (E) Immunoblots for indicated proteins in WT, ATPIF1_KO, and ATPIF1_KO KBM7 cells with restored ATPIF1 expression (left) and viability of cells treated with antimycin (135 μM) for 2 days (right). Error bars are ± s.e.m. (n = 3). ***P < 0.001. (F) Cellular ATP (left) and ΔΨm (right) in WT and ATPIF1_KO KBM7 cells treated with antimycin (135 μM) and oligomycin (1 μM). Error bars are ± s.e.m. (n = 3). (G) Viability of WT and ATPIF1_KO KBM7 cells treated with antimycin (135 μM) and oligomycin (1 μM) for 2 days. Error bars are ± s.e.m. (n = 3). ***P < 0.001. See also Figures S1, S2, S3, and Table S1.

Figure 2

Figure 2. Loss of ATPIF1 is beneficial in both pharmacological and genetic models of ETC dysfunction

(A) Immunoblots for indicated proteins in SH-SY5Y cells expressing a control shRNA against Luciferase (shLuc) or an shRNA against ATPIF1 (shATPIF1_3) (left). Viability of SH-SY5Y (middle) and HeLa (right) cells treated with antimycin for 4 days. Error bars are ± s.e.m. (n = 3). (B) Immunoblots for indicated proteins in Malme-3M cells overexpressing control RAP2A or ATPIF1 (left). Viability of Malme-3M cells treated with antimycin for 4 days (right). Error bars are ± s.e.m. (n = 3). (C) Viability of WT and ATPIF1_KO KBM7 cells treated with piericidin (top) or tigecycline (bottom) for 4 days. Error bars are ± s.e.m. (n = 3). (D) Relative ATPIF1 mRNA levels (top) and immunoblots for indicated proteins (bottom) in HeLa WT and ρ0 cells. Error bars are ± s.e.m. (n = 3). **P < 0.01. (E) Immunoblots (left) and relative proliferation (right) of HeLa WT and ρ0 cells transduced with control vector, ATPIF1 (WT), or ATPIF1 (E55A) constructs. Error bars are ± s.e.m. (n = 3).

Figure 3

Figure 3. Loss of ATPIF1 improves cell viability during progressive mtDNA depletion

(A) Schematic depicting experimental paradigm of long-term treatment of WT and ATPIF1_KO KBM7 cells with ddI. Early (days 0 – 10), middle (days 10 – 40), and late (days 40 +) periods of ddI treatment are indicated and demarcated by dotted lines. During each period, mtDNA copy number, cell proliferation over four days, and ATPIF1 expression were analyzed. (B – D) mtDNA copy number (left), cell proliferation (middle), and immunoblots for indicated proteins (right) of WT and ATPIF1_KO KBM7 cells treated with ddI during the early (B), middle (C), and late (D) periods. Error bars are ± s.e.m. (n = 3). ***P < 0.001.

Figure 4

Figure 4. Inhibition of ATPIF1 ameliorates the effects of complex III blockade in primary hepatocytes

(A) Immunoblots for indicated proteins of primary hepatocytes derived from WT and ATPIF1 -/- mice. (B) Cellular ATP of WT and ATPIF1 -/- primary hepatocytes treated with antimycin (0.625 μM) for 1.5 hours. Error bars are ± s.e.m. (n = 3). ***P < 0.001. (C) ΔΨm of WT and ATPIF1 -/- primary hepatocytes treated with antimycin (10 μM) for 1.5 hours. Error bars are ± s.e.m. (n = 3). **P < 0.01. (D) Viability of WT and ATPIF1 -/- primary hepatocytes treated with antimycin (1.25 μM) for 2 days. Error bars are ± s.e.m. (n = 3). **P < 0.01. (E) Schematic diagramming the behavior of cells with ETC dysfunction under conditions where ATPIF1 is active or inhibited. Inhibition of ATPIF1 is depicted by absence of the protein but represents any strategy to block ATPIF1 activity on the F1-F0 ATP synthase. See also Figure S4.

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