KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.

New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier.

The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).

Functional characterization and organ distribution of three mitochondrial ATP-Mg/Pi carriers in Arabidopsis thaliana.

The Arabidopsis thaliana genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Three members of this family, here named AtAPC1, AtAPC2, and AtAPC3, exhibit high structural similarities to the human mitochondrial ATP-Mg(2+)/phosphate carriers. Under normal physiological conditions the AtAPC1 gene was expressed at least five times more than the other two AtAPC genes in flower, leaf, stem, root and seedlings. However, in stress conditions the expression levels of AtAPC1 and AtAPC3 change. Direct transport assays with recombinant and reconstituted AtAPC1, AtAPC2 and AtAPC3 showed that they transport phosphate, AMP, ADP, ATP, adenosine 5'-phosphosulfate and, to a lesser extent, other nucleotides. AtAPC2 and AtAPC3 also had the ability to transport sulfate and thiosulfate. All three AtAPCs catalyzed a counter-exchange transport that was saturable and inhibited by pyridoxal-5'-phosphate. The transport activities of AtAPCs were also inhibited by the addition of EDTA or EGTA and stimulated by the addition of Ca(2+). Given that phosphate and sulfate can be recycled via their own specific carriers, these findings indicate that AtAPCs can catalyze net transfer of adenine nucleotides across the inner mitochondrial membrane in exchange for phosphate (or sulfate), and that this transport is regulated both at the transcriptional level and by Ca(2+).

Riboflavin responsive mitochondrial myopathy is a new phenotype of dihydrolipoamide dehydrogenase deficiency. The chaperon-like effect of vitamin B2.

Dihydrolipoamide dehydrogenase (DLD, E3) is a flavoprotein common to pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenases. We found two novel DLD mutations (p.I40Lfs*4; p.G461E) in a 19 year-old patient with lactic acidosis and a complex amino- and organic aciduria consistent with DLD deficiency, manifesting progressive exertional fatigue. Muscle biopsy showed mitochondrial proliferation and lack of DLD cross-reacting material. Riboflavin supplementation determined the complete resolution of exercise intolerance with the partial restoration of the DLD protein and disappearance of mitochondrial proliferation in the muscle. Morphological and functional studies support the riboflavin chaperon-like role in stabilizing DLD protein with rescue of its expression in the muscle.

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

Transcription of the mitochondrial citrate carrier gene: identification of a silencer and its binding protein ZNF224.

In the last few years, we have been functionally characterizing the promoter of the human mitochondrial citrate carrier (CIC). In this study we show that CIC silencer activity extends over 26 bp (-595/-569), which specifically bind a protein present in HepG2 cell nuclear extracts. This transcription factor was purified by DNA affinity and identified as ZNF224. Overexpression of ZNF224 decreases LUC transgene activity in cells transfected with a construct containing the CIC silencer region, whereas ZNF224 silencing activates reporter transcription in cells transfected with the same construct. Moreover, overexpression and silencing of ZNF224 diminishes and enhances, respectively, CIC transcript and protein levels. Finally, ZNF224 is abundantly expressed in fetal tissues contrary to CIC. It is suggested that CIC transcriptional repression by ZNF224 explains, at least in part, the low expression of CIC in fetal tissues in which fatty acid synthesis is low.

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA.

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.