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

Identification of a lysosomal pathway regulating degradation of the bone morphogenetic protein receptor type II.

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease

Follow-Up in Carriers of the ‘MELAS’ Mutation without Strokes

Eight carriers of the A3243G mutation of mitochondrial DNA without stroke-like episodes were monitored for up to 7 years in clinical and metabolic studies, by magnetic resonance imaging (MRI) and positron emission tomography (PET). None developed mitochondrial encephalopathy (MELAS), but 2 developed diabetes mellitus, 1 terminal kidney failure and 2 cardiomyopathy. One patient improved markedly under ubiquinone. Electroencephalography showed progressive slowing in 2 cases, but electrophysiological tests and MRI were otherwise noncontributary. PET showed widespread cortical and basal ganglion metabolic deficits in 6 cases. We conclude that internal medical complications are more common than MELAS in adult carriers of the mutation. PET findings, firstly reported in such patients, suggest that chronic subclinical encephalopathy is very frequent, and PET may play a role in monitoring in the future.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

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

Abstract 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

Complement C1q is hydroxylated by collagen prolyl 4 hydroxylase and is sensitive to off-target inhibition by prolyl hydroxylase domain inhibitors that stabilize hypoxia-inducible factor

Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. C1q is composed of A, B, and C chains that require post-translational prolyl 4-hydroxylation of their N-terminal collagen-like domain to enable the formation of the functional triple helical multimers. The prolyl 4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognized prolyl 4-hydroxylases include collagen prolyl-4-hydroxylases (CP4H) and the more recently described prolyl hydroxylase domain (PHD) enzymes that act as oxygen sensors regulating hypoxia-inducible factor (HIF). We show that several small-molecule prolyl hydroxylase inhibitors that activate HIF also potently suppress C1q secretion by human macrophages. However, reducing oxygenation to a level that activates HIF does not compromise C1q hydroxylation. $\textit{In vitro}$ studies showed that a C1q A chain peptide is not a substrate for PHD2 but is a substrate for CP4H1. Circulating levels of C1q did not differ between wild-type mice or mice with genetic deficits in PHD enzymes, but were reduced by prolyl hydroxylase inhibitors. Thus, C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. Hence, reduction of C1q levels may be an important off-target side effect of small molecule PHD inhibitors developed as treatments for renal anemia.This work was supported by the Wellcome Trust and the NIHR Cambridge Biomedical Research Centre Senior Investigator Awards (to PHM, supporting SSH and NB), the Wellcome Trust Scientific strategic award [100140]. SK was supported by a British Heart Foundation grant awarded to PHM as well as a Kennedy Institute of Rheumatology trustees fund

Reduced expression of mitochondrial heavy strand transcripts in m.547A>T patient-derived cells.

<p>(A, C) RNA from patient- and control-derived cells (n = 4 each) was analysed for expression of a panel of mitochondrial tRNAs by quantitative Northern blot. The graphs show mean expression of heavy strand tRNAs relative to light strand tRNA Gln(Q). Values are expressed relative to the mean for the control cell lines in each case. Error bars indicate the standard deviation. Heavy strand tRNAPhe and tRNALeu expression is reduced relative to light strand tRNA Gln in m.547A>T fibroblasts (A, p<0.001) and cybrids (C, p<0.001 for tRNA Phe(F), Val (V) and Leu (L)). Experiments were repeated three times with equivalent results. Labelling for 5S rRNA was used to confirm equal loading. Heavy chain transcripts RNR1 (B) and CO1 (D) were measured by quantitative RT-PCR and were reduced in m.547A>T cybrids relative to the light strand transcript ND6. Each point represents the mean of several independent cybrid cell lines derived from a single donor.</p

Mitochondrial function is impaired in m.547A>T fibroblasts and cybrids.

<p>Oxygen consumption rate (OCR) was measured to assess mitochondrial function in fibroblasts (A, C) and cybrids (B, D), normalised for cell number. Representative comparison of fibroblasts (A) and cybrids (B) from control individuals (red) with fibroblasts or cybrids from patients with the m.547A>T substitution (blue) showing a substantial change in respiratory profile. There was a significant decrease in baseline oxygen consumption, and in maximal oxygen consumption following addition of FCCP in both fibroblasts (C) and cybrids (D). Asterisks *p < 0.05, **p < 0.005, and ***p < 0.001 for control versus patient groups, represented as the mean ± SD of separate experiments performed in triplicate with four patient and four control cell lines.</p

mt.616T>C cybrids have a defect in respiration.

<p>(A) Two pedigrees showing potential maternal inheritance of renal disease. Individuals with kidney disease are represented by filled shapes as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006620#pgen.1006620.g001" target="_blank">Fig 1</a>. Family 8 from ref 13 forms part of pedigree II, and is indicated by the dotted box. Pedigree III is Family 6 from ref 13. Individuals from whom DNA was sequenced in the current study are marked with asterisks. All affected individuals who were sequenced were found to have homoplasmic levels of the m.616T>C substitution. The mitochondrial haplotype is T1a1. (B) Measurement of oxygen consumption in patient-derived and control cybrids showing a reduction in basal (before addition of oligomycin) and maximal respiration (after addition of FCCP) in patient-derived cybrids. (C) Expression levels of tRNAs quantified by Northern blot of three control and patient-derived cybrids show reduced mitochondrial tRNAPhe levels relative to the light strand encoded tRNAGln in mt.616T>C cells.(p = 0.006) The tRNA levels for valine and leucine were unaffected. (D) Conservation of mt tRNA Phe within vertebrates. The anticodon (GAA) is highlighted in bold, the nucleotides forming the last pair of the anticodon stem are underlined. Sequences were aligned with clustal omega and manually adjusted. The stem (s) and loop (l) secondary structure of the tRNA Phe of humans is indicated at the bottom.</p