Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress.

BACKGROUND: Tissue hypoxia is a key feature of several endemic hepatic diseases, including alcoholic and non-alcoholic fatty liver disease, and organ failure. Hypoxia imposes a severe metabolic challenge on the liver, potentially disrupting its capacity to carry out essential functions including fuel storage and the integration of lipid metabolism at the whole-body level. Mitochondrial respiratory function is understood to be critical in mediating the hepatic hypoxic response, yet the time-dependent nature of this response and the role of the respiratory chain in this remain unclear. RESULTS: Here, we report that hepatic respiratory capacity is enhanced following short-term exposure to hypoxia (2 days, 10% O2) and is associated with increased abundance of the respiratory chain supercomplex III2+IV and increased cardiolipin levels. Suppression of this enhanced respiratory capacity, achieved via mild inhibition of mitochondrial complex III, disrupted metabolic homeostasis. Hypoxic exposure for 2 days led to accumulation of plasma and hepatic long chain acyl-carnitines. This was observed alongside depletion of hepatic triacylglycerol species with total chain lengths of 39-53 carbons, containing palmitic, palmitoleic, stearic, and oleic acids, which are associated with de novo lipogenesis. The changes to hepatic respiratory capacity and lipid metabolism following 2 days hypoxic exposure were transient, becoming resolved after 14 days in line with systemic acclimation to hypoxia and elevated circulating haemoglobin concentrations. CONCLUSIONS: The liver maintains metabolic homeostasis in response to shorter term hypoxic exposure through transient enhancement of respiratory chain capacity and alterations to lipid metabolism. These findings may have implications in understanding and treating hepatic pathologies associated with hypoxia.

Exploring the molecular interface between hypoxia-inducible factor signalling and mitochondria.

Oxygen is required for the survival of the majority of eukaryotic organisms, as it is important for many cellular processes. Eukaryotic cells utilize oxygen for the production of biochemical energy in the form of adenosine triphosphate (ATP) generated from the catabolism of carbon-rich fuels such as glucose, lipids and glutamine. The intracellular sites of oxygen consumption-coupled ATP production are the mitochondria, double-membraned organelles that provide a dynamic and multifaceted role in cell signalling and metabolism. Highly evolutionarily conserved molecular mechanisms exist to sense and respond to changes in cellular oxygen levels. The primary transcriptional regulators of the response to decreased oxygen levels (hypoxia) are the hypoxia-inducible factors (HIFs), which play important roles in both physiological and pathophysiological contexts. In this review we explore the relationship between HIF-regulated signalling pathways and the mitochondria, including the regulation of mitochondrial metabolism, biogenesis and distribution.

CHCHD4 regulates the expression of mitochondrial genes that are essential for tumour cell growth.

CHCHD4 (MIA40) is central to the functions of the mitochondrial disulfide relay system (DRS). CHCHD4 is essential and evolutionarily conserved. Previously, we have shown CHCHD4 to be a critical regulator of tumour cell growth. Here, we use integrated analysis of our genome-wide CRISPR/Cas9 and SILAC proteomic screening data to delineate mechanisms of CHCHD4 essentiality in cancer. We identify a shortlist of common essential genes/proteins regulated by CHCHD4, including subunits of complex I that are known DRS substrates, and genes/proteins involved in key metabolic pathways. Our study highlights a range of CHCHD4-regulated nuclear encoded mitochondrial genes/proteins essential for tumour cell growth.

Integrative transcriptomic and metabolic analyses of the mammalian hibernating brain identifies a key role for succinate dehydrogenase in ischemic tolerance.

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites 1 . Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus , provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage 2 . Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and α-ketoglutarate-αKG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo . Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

The Contextual Essentiality of Mitochondrial Genes in Cancer.

Mitochondria are key organelles in eukaryotic evolution that perform crucial roles as metabolic and cellular signaling hubs. Mitochondrial function and dysfunction are associated with a range of diseases, including cancer. Mitochondria support cancer cell proliferation through biosynthetic reactions and their role in signaling, and can also promote tumorigenesis via processes such as the production of reactive oxygen species (ROS). The advent of (nuclear) genome-wide CRISPR-Cas9 deletion screens has provided gene-level resolution of the requirement of nuclear-encoded mitochondrial genes (NEMGs) for cancer cell viability (essentiality). More recently, it has become apparent that the essentiality of NEMGs is highly dependent on the cancer cell context. In particular, key tumor microenvironmental factors such as hypoxia, and changes in nutrient (e.g., glucose) availability, significantly influence the essentiality of NEMGs. In this mini-review we will discuss recent advances in our understanding of the contribution of NEMGs to cancer from CRISPR-Cas9 deletion screens, and discuss emerging concepts surrounding the context-dependent nature of mitochondrial gene essentiality.

Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia.

Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.

Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

[This corrects the article DOI: 10.3389/fonc.2018.00388.].

Corrigendum: CHCHD4 Regulates Intracellular Oxygenation and Perinuclear Distribution of Mitochondria.

[This corrects the article DOI: 10.3389/fonc.2017.00071.].

CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain.

Background: Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus, mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is critical for regulating intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers correlates with increased tumour progression and poor patient survival. Results: Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I-V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). Conclusions: Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology.

CHCHD4 regulates tumour proliferation and EMT-related phenotypes, through respiratory chain-mediated metabolism.

BACKGROUND: Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. RESULTS: Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. CONCLUSIONS: CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.

The mitochondrial oxidoreductase CHCHD4 is present in a semi-oxidized state in vivo.

Disulfide formation in the mitochondrial intermembrane space is an essential process catalyzed by a disulfide relay machinery. In mammalian cells, the key enzyme in this machinery is the oxidoreductase CHCHD4/Mia40. Here, we determined the in vivo CHCHD4 redox state, which is the major determinant of its cellular activity. We found that under basal conditions, endogenous CHCHD4 redox state in cultured cells and mouse tissues was predominantly oxidized, however, degrees of oxidation in different tissues varied from 70% to 90% oxidized. To test whether differences in the ratio between CHCHD4 and ALR might explain tissue-specific differences in the CHCHD4 redox state, we determined the molar ratio of both proteins in different mouse tissues. Surprisingly, ALR is superstoichiometric over CHCHD4 in most tissues. However, the levels of CHCHD4 and the ratio of ALR over CHCHD4 appear to correlate only weakly with the redox state, and although ALR is present in superstoichiometric amounts, it does not lead to fully oxidized CHCHD4.

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

CHCHD4 Regulates Intracellular Oxygenation and Perinuclear Distribution of Mitochondria.

Hypoxia is a characteristic of the tumor microenvironment and is known to contribute to tumor progression and treatment resistance. Hypoxia-inducible factor (HIF) dimeric transcription factors control the cellular response to reduced oxygenation by regulating the expression of genes involved in metabolic adaptation, cell motility, and survival. Alterations in mitochondrial metabolism are not only a downstream consequence of HIF-signaling but mitochondria reciprocally regulate HIF signaling through multiple means, including oxygen consumption, metabolic intermediates, and reactive oxygen species generation. CHCHD4 is a redox-sensitive mitochondrial protein, which we previously identified and showed to be a novel regulator of HIF and hypoxia responses in tumors. Elevated expression of CHCHD4 in human tumors correlates with the hypoxia gene signature, disease progression, and poor patient survival. Here, we show that either long-term (72 h) exposure to hypoxia (1% O2) or elevated expression of CHCHD4 in tumor cells in normoxia leads to perinuclear accumulation of mitochondria, which is dependent on the expression of HIF-1α. Furthermore, we show that CHCHD4 is required for perinuclear localization of mitochondria and HIF activation in response to long-term hypoxia. Mutation of the functionally important highly conserved cysteines within the Cys-Pro-Cys motif of CHCHD4 or inhibition of complex IV activity (by sodium azide) redistributes mitochondria from the perinuclear region toward the periphery of the cell and blocks HIF activation. Finally, we show that CHCHD4-mediated perinuclear localization of mitochondria is associated with increased intracellular hypoxia within the perinuclear region and constitutive basal HIF activation in normoxia. Our study demonstrates that the intracellular distribution of the mitochondrial network is an important feature of the cellular response to hypoxia, contributing to hypoxic signaling via HIF activation and regulated by way of the cross talk between CHCHD4 and HIF-1α.

Serine 162, an essential residue for the mitochondrial localization, stability and anti-apoptotic function of Mcl-1.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family that plays a key role in normal development, but also in pathologies such as cancer. It has some unusual properties compared to other anti-apoptotic members of the Bcl-2 family, and its expression and function are dynamically regulated by a variety of post-transcriptional and post-translational processes. Of note, Mcl-1 protein has a very short half life, and its stability and function may be regulated by reversible phosphorylation. There is also evidence to suggest that it may be localized to different subcellular compartments. The aim of this work was to determine whether residues within the PEST region of Mcl-1 that may undergo reversible phosphorylation, also regulate its subcellular distribution. We show that EGFP:Mcl-1 localizes mainly to the mitochondria of HeLa cells, with some additional cytoplasmic and nuclear localization. The mutations, S64A, S64E, S121A, S159A, T163A and T163E did not significantly affect the localization of Mcl-1. However, mutation of Ser162 to the phospho-null residue, Alanine resulted in an essentially nuclear localization, with some cytoplasmic but no mitochondrial localization. This mutant Mcl-1 protein, S162A, showed significantly decreased stability and it decreased the ability to protect against Bak-induced apoptosis. These data identify a new molecular determinant of Mcl-1 function, localization and stability that may be important for understanding the role of this protein in disease.

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

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.

Mcl-1; the molecular regulation of protein function.

Apoptosis, an essential and basic biological phenomenon, is regulated in a complex manner by a multitude of factors. Myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, exemplifies a number of the mechanisms by which a protein's contribution to cell fate may be modified. The N-terminus of Mcl-1 is unique amongst the Bcl-2 family, in that it is rich in experimentally confirmed and putative regulatory residues and motifs. These include sites for ubiquitination, cleavage and phosphorylation, which influence the protein's stability, localisation, dimerization and function. Here we review what is known about the regulation of Mcl-1 expression and function, with particular focus on post-translational modifications and how phosphorylation interconnects the complex molecular control of Mcl-1 with cellular state.