Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression
Jun Yang, Oliver Staples, Luke W. Thomas, Thomas Briston, Mathew Robson, Evon Poon, Maria L. Simões, Ethaar El-Emir, Francesca M. Buffa, Afshan Ahmed, Nicholas P. Annear, Deepa Shukla, Barbara R. Pedley, Patrick H. Maxwell, Adrian L. Harris, Margaret Ashcroft
Jun Yang, Oliver Staples, Luke W. Thomas, Thomas Briston, Mathew Robson, Evon Poon, Maria L. Simões, Ethaar El-Emir, Francesca M. Buffa, Afshan Ahmed, Nicholas P. Annear, Deepa Shukla, Barbara R. Pedley, Patrick H. Maxwell, Adrian L. Harris, Margaret Ashcroft
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Research Article Oncology

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression

Abstract

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.

Authors

Jun Yang, Oliver Staples, Luke W. Thomas, Thomas Briston, Mathew Robson, Evon Poon, Maria L. Simões, Ethaar El-Emir, Francesca M. Buffa, Afshan Ahmed, Nicholas P. Annear, Deepa Shukla, Barbara R. Pedley, Patrick H. Maxwell, Adrian L. Harris, Margaret Ashcroft

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Figure 2

CHCHD4 proteins contain an evolutionarily conserved CHCH domain and localize to mitochondria.

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CHCHD4 proteins contain an evolutionarily conserved CHCH domain and loca...
(A) Sequence alignment of the CHCH domain of CHCHD4 and orthologs showing identical (red, asterisk), conserved (blue, double dot), and partially conserved (green, single dot) amino acid residues. The conserved 6 cysteine residues (red) are highlighted in yellow. The GEO accession numbers ( http://www.ncbi.nlm.nih.gov/protein) are as follows: H. sapiens (Hm, NP_653237), M. musculus (Mm, NP_598689), X. laevis (Xl, AAH59772), D. melanogaster (Dm, AAN14214), A. mellifera (Am, XP_392382), C. elegans 1 (Ce1, NP_500803), C. elegans 2 (Ce2, NP_510159), C. elegans 3 (Ce3, NP_500804), and C. elegans 4 (Ce4, NP_498876). (B) Immunostaining analysis of HCT116 cells transfected with myc-tagged CHCHD4.1 or CHCHD4.2 expression vectors. Cells were fixed and stained with an anti-myc antibody (CHCHD4, green) and imaged by confocal microscopy. The mitochondria were visualized using an antibody to cytochrome c (red). The nuclei were stained with DAPI (blue). The overlay shows colocalized proteins in yellow. (C) Western blot showing endogenous and exogenously expressed CHCHD4 proteins in cytosolic (Cyto) and mitochondrial (Mito) fractions. Cytochrome c was used to confirm efficient mitochondrial fractionation. (D) Western blot analysis showing endogenous CHCHD4 proteins in HCT116 cells from cytosolic (C) and mitochondrial (M) fractions. Cytochrome c was used to confirm efficient mitochondrial fractionation. (E and F) Immunostaining analysis of HCT116 cells transfected with myc-tagged CHCHD4.2 cysteine mutants as indicated. Cells were fixed, stained, and imaged as described in B. (F) The ER was visualized using an antibody to PDI. The partial yellow staining in the overlay image indicates colocalization of CHCHD4.2 with PDI. Original magnification, ×60 (B); ×100 (E and F).