RAS signalling in energy metabolism and rare human diseases.

The RAS pathway is a highly conserved cascade of protein-protein interactions and phosphorylation that is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Recent findings indicate that the RAS pathway plays a role in the regulation of energy metabolism via the control of mitochondrial form and function but little is known on the participation of this effect in RAS-related rare human genetic diseases. Germline mutations that hyperactivate the RAS pathway have been discovered and linked to human developmental disorders that are known as RASopathies. Individuals with RASopathies, which are estimated to affect approximately 1/1000 human birth, share many overlapping characteristics, including cardiac malformations, short stature, neurocognitive impairment, craniofacial dysmorphy, cutaneous, musculoskeletal, and ocular abnormalities, hypotonia and a predisposition to developing cancer. Since the identification of the first RASopathy, type 1 neurofibromatosis (NF1), which is caused by the inactivation of neurofibromin 1, several other syndromes have been associated with mutations in the core components of the RAS-MAPK pathway. These syndromes include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), which was formerly called LEOPARD syndrome, Costello syndrome (CS), cardio-facio-cutaneous syndrome (CFC), Legius syndrome (LS) and capillary malformation-arteriovenous malformation syndrome (CM-AVM). Here, we review current knowledge about the bioenergetics of the RASopathies and discuss the molecular control of energy homeostasis and mitochondrial physiology by the RAS pathway.

Multi-site control and regulation of mitochondrial energy production.

With the extraordinary progress of mitochondrial science and cell biology, novel biochemical pathways have emerged as strategic points of bioenergetic regulation and control. They include mitochondrial fusion, fission and organellar motility along microtubules and microfilaments (mitochondrial dynamics), mitochondrial turnover (biogenesis and degradation), and mitochondrial phospholipids synthesis. Yet, much is still unknown about the mutual interaction between mitochondrial energy state, biogenesis, dynamics and degradation. Meanwhile, clinical research into metabolic abnormalities in tumors as diverse as renal carcinoma, glioblastomas, paragangliomas or skin leiomyomata, has designated new genes, oncogenes and oncometabolites involved in the regulation of cellular and mitochondrial energy production. Furthermore, the examination of rare neurological diseases such as Charcot-Marie Tooth type 2a, Autosomal Dominant Optic Atrophy, Lethal Defect of Mitochondrial and Peroxisomal Fission, or Spastic Paraplegia suggested involvement of MFN2, OPA1/3, DRP1 or Paraplegin, in the auxiliary control of mitochondrial energy production. Lastly, advances in the understanding of mitochondrial apoptosis have suggested a supplementary role for Bcl2 or Bax in the regulation of mitochondrial respiration and dynamics, which has fostered the investigation of alternative mechanisms of energy regulation. In this review, we discuss the regulatory mechanisms of cellular and mitochondrial energy production, and we emphasize the importance of the study of rare neurological diseases in addition to more common disorders such as cancer, for the fundamental understanding of cellular and mitochondrial energy production.

Bioenergetics of lung tumors: alteration of mitochondrial biogenesis and respiratory capacity.

Little is known on the metabolic profile of lung tumors and the reminiscence of embryonic features. Herein, we determined the bioenergetic profiles of human fibroblasts taken from lung epidermoid carcinoma (HLF-a) and fetal lung (MRC5). We also analysed human lung tumors and their surrounding healthy tissue from four patients with adenocarcinoma. On these different models, we measured functional parameters (cell growth rates in oxidative and glycolytic media, respiration, ATP synthesis and PDH activity) as well as compositional features (expression level of various energy proteins and upstream transcription factors). The results demonstrate that both the lung fetal and cancer cell lines produced their ATP predominantly by glycolysis, while oxidative phosphorylation was only capable of poor ATP delivery. This was explained by a decreased mitochondrial biogenesis caused by a lowered expression of PGC1alpha (as shown by RT-PCR and Western blot) and mtTFA. Consequently, the relative expression of glycolytic versus OXPHOS markers was high in these cells. Moreover, the re-activation of mitochondrial biogenesis with resveratrol induced cell death specifically in cancer cells. A consistent reduction of mitochondrial biogenesis and the subsequent alteration of respiratory capacity was also observed in lung tumors, associated with a lower expression level of bcl2. Our data give a better characterization of lung cancer cells' metabolic alterations which are essential for growth and survival. They designate mitochondrial biogenesis as a possible target for anti-cancer therapy.

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases.

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

Physiological diversity of mitochondrial oxidative phosphorylation.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1-1.5]:[30-135]:[3]:[9-35]:[6.5-7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.