GLTSCR2/PICT1 links mitochondrial stress and Myc signaling - PubMed (original) (raw)

GLTSCR2/PICT1 links mitochondrial stress and Myc signaling

John C Yoon et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Mitochondrial defects underlie a multitude of human diseases. Genetic manipulation of mitochondrial regulatory pathways represents a potential therapeutic approach. We have carried out a high-throughput overexpression screen for genes that affect mitochondrial abundance or activity using flow-cytometry-based enrichment of a cell population expressing a high-complexity, concentration-normalized pool of human ORFs. The screen identified 94 candidate mitochondrial regulators including the nuclear protein GLTSCR2, also known as PICT1. GLTSCR2 enhances mitochondrial function and is required for the maintenance of oxygen consumption, consistent with a pivotal role in the control of cellular respiration. RNAi inactivation of the Caenorhabditis elegans ortholog of GLTSCR2 reduces respiration in worms, indicating functional conservation across species. GLTSCR2 controls cellular proliferation and metabolism via the transcription factor Myc, and is induced by mitochondrial stress, suggesting it may constitute a significant component of the mitochondrial signaling pathway.

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Conflict of interest statement

D.A.S. is a scientific consultant for GlaxoSmithKline, Cohbar, Ovascience, and Metrobiotech.

Figures

Fig. 1.

Schematic diagram of the genome-scale overexpression screen. The human ORFeome collection was cloned into the lentiviral vector pHAGE T-Rex by Gateway cloning and packaged into viruses. C2C12 mouse myoblast cells were transduced with the viruses, selected, and stained with NAO and MitoTracker deep red dyes for FACS sorting. Microarray hybridization was used to compare the genomic DNA samples collected from high NAO, high MT-DR cells (shown as grayed out areas in the FACS profiles) vs. the unsorted cells. Similarly, the low NAO, low MT-DR cells and the unsorted cells were compared.

Fig. 2.

Further analysis of the confirmed hits from the screen using secondary assays. (A) Distribution of TMRM signals among the 94 confirmed hits expressed in C2C12 cells. The genes that produce less than a 20% change in the TMRM signal relative to the control cells are colored in a lighter shade. (B) Distribution of the NAO vs. TMRM signals among the 94 confirmed hits. (C) Distribution of NAO signals among the 94 confirmed hits. The genes whose overexpression produce less than a 20% change in the NAO signal relative to the control cells are colored in a lighter shade. (D) Distribution of MitoTracker deep red signals among the 94 confirmed hits. The genes that produce less than a 20% change in the MT-DR signal relative to the control cells are colored in a lighter shade. (E) Distribution of the NAO versus MitoTracker deep red signals among the 94 confirmed hits. (F) NAO signal values for the 11 genes that scored in the mini-RNAi screen in IMR90 cells with at least two of three independent siRNAs. (G) MitoTracker deep red signal values for the 11 genes that scored in the mini-RNAi screen in IMR90 cells with at least two of three independent siRNAs indicated by different colored bars.

Fig. 3.

GLTSCR2 regulates mitochondrial respiration in primary human fibroblasts and in the nematode C. elegans. *P < 0.05 by unpaired t test; **P < 0.01. (A) Stable expression of GLTSCR2 cDNA in C2C12 cells increases both NAO and MitoTracker deep red signals. (B) Stable expression of GLTSCR2 cDNA in IMR90 cells increases oxygen consumption. (C) Depletion of GLTSCR2 in IMR90 cells by siRNA transfection reduces oxygen consumption. Cells were examined 4 d after transfection with the Seahorse XF24 flux analyzer. (D) Depletion of GLTSCR2 in IMR90 cells by stable shRNA expression reduces oxygen consumption, which can be rescued by expressing an RNAi-resistant GLTSCR2 cDNA. (E) Stable expression of GLTSCR2 cDNA in IMR90 cells increases cellular ATP content. (F) Depletion of GLTSCR2 in IMR90 cells by stable shRNA expression reduces cellular ATP content. (G) RNAi inactivation of the GLTSCR2 ortholog Y39B6A.33 in C. elegans reduces oxygen consumption.

Fig. 4.

GLTSCR2 responds to mitochondrial stress and controls cell proliferation and respiration via Myc. (A) Depletion of GLTSCR2 in IMR90 cells by siRNA transfection (Left) or by stable shRNA expression (Right) induces Myc and p53 levels. Protein samples were isolated 72 h after siRNA transfection. (B) Stable expression of GLTSCR2 in IMR90 cells induces Myc and p53 levels. (C) GLTSCR2 increases cell proliferation in IMR90 cells. Cells were counted in triplicates every day for 5 consecutive days. (D) GLTSCR2 interacts with RPL11. The 293T-Rex cells stably carrying HA-GLTSCR2 under a tetracycline-inducible promoter were treated with doxycycline for 24 h and the lysates were immunoprecipitated with anti-HA antibody or IgG. (E) Depletion of Myc in IMR90 cells by stable shRNA expression eliminates GLTSCR2-mediated increases in oxygen consumption. (F) Depletion of Myc in IMR90 cells by stable shRNA expression abolishes GLTSCR2-mediated increases in cell proliferation. Cells were counted in triplicates every day for 5 consecutive days. (G) Expression of mutant OTC in IMR90 cells induces GLTSCR2 and Myc expression. The wild-type OTC protein runs at 39 kDa, whereas the deletion mutant lacking amino acids 30–114 runs at 30 kDa. (H) Model of mitochondrial stress signaling involving GLTSCR2 and Myc.

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GLTSCR2/PICT1 links mitochondrial stress and Myc signaling - PubMed (original) (raw)