Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER

Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER

Effect of 'binary mitochondrial heteroplasmy' on respiration and ATP synthesis: implications for mitochondrial diseases.

Respiratory-chain-complex subunits in mitochondria are encoded by nuclear or mitochondrial DNA. This property might have profound implications for the phenotypic expression of mutations affecting oxidative phosphorylation complexes. The aim of this paper is to study the importance of the origin of the mutation (nuclear or mitochondrial) on the expression of mitochondrial defects. We have therefore developed theoretical models illustrating three mechanisms of nuclear or mitochondrial DNA mutation giving rise to a deficiency in the respiratory-chain complex: (1) a partial deficiency, homogeneously distributed in all of the mitochondria; (2) a complete deficiency, only affecting some of the mitochondria ('binary mitochondrial heteroplasmy'); and (3) a partial deficiency, affecting only some of the mitochondria. We show that mutations affecting oxidative phosphorylation complexes will be expressed in different ways depending on their origins. Although the expression of nuclear or mitochondrial mutations is evidence of a biochemical threshold, we demonstrate that the threshold value depends on the origin and distribution of the mutation (homogeneous or not) and also on the energy demand of the tissue. This last prediction has been confirmed in an experimental model using hexokinase for the simulation of the energy demand and a variation in mitochondrial concentration. We also emphasize the possible role of 'binary mitochondrial heteroplasmy' in the expression of mitochondrial DNA mutations and thus the importance of the origin of the deficit (mutation) for the diagnosis or therapy of mitochondrial diseases

Influence of mitochondrial DNA level on cellular energy metabolism: implications for mitochondrial diseases

The total amount of cellular mitochondrial DNA (mtDNA) varies widely and seems to be related to the nature and metabolic state of tissues and cells in culture. It is not known, however, whether this variation has any significance in vivo, and to which extent it regulates energy production. To better understand the importance of the cellular mtDNA level, we studied the influence of a gradual reduction of mtDNA copy number on oxidative phosphorylation in two models: (a) a control human cell line treated with different concentrations of 2', 3'-dideoxycytidine, a nucleoside analogue that inhibits mtDNA replication by interfering with mitochondrial DNA polymerase gamma, and (b) a cell line derived from a patient presenting mtDNA depletion. The two models were used to construct biochemical and phenotypic threshold curves. Our results show that oxidative phosphorylation activities are under a tight control by the amount of mtDNA in the cell, and that the full complement of mtDNA molecules are necessary to maintain a normal energy production level

Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis.

International audienceDiversity of respiratory chain spectrophotometric assays may lead to difficult comparison of results between centers. The French network of mitochondrial diseases diagnostic centers undertook comparison of the results obtained with different protocols in the French diagnostic centers. The diversity of protocols was shown to have striking consequences, which prompted the network to undertake standardization and optimization of the protocols with respect to clinical diagnosis, i.e. high velocity while maintaining linear kinetics relative to time and enzyme concentration. Assays were set up on animal tissues and verified on control human muscle and fibroblasts. Influence of homogenization buffer and narrow range of optimal concentration of phosphate, substrate and tissue were shown. Experimental data and proposed protocols have been posted on a free access website. Their subsequent use in several diagnostic centers has improved consistency for all assays

Statistical Analysis of Mitochondrial Pathologies in Childhood: Identification of Deficiencies using Principal Component Analysis

International audienceMitochondrial pathologies are a heterogeneous group of metabolic disorders that are frequently characterized by anomalies of oxidative phosphorylation, especially in the respiratory chain. The identification of these anomalies may involve many investigations, and biochemistry is a main tool. However, considering the whole set of biochemical data, the interpretation of the results by the traditionally used statistical methods remains complex and does not always lead to an unequivocal conclusion about the presence or absence of a respiratory chain defect. This arises from three main problems: (a) the absence of an a priori-defined control population, because the determination of the control values are derived from the whole set of investigated patients, (b) the small size of the population studied, (c) the large number of variables collected, each of which creates a wide variability. To cope with these problems, the principal component analysis (PCA) has been applied to the biochemical data obtained from 35 muscle biopsies of children suspected of having a mitochondrial disease. This analysis makes it possible for each respiratory chain complex to distinguish between different subsets within the whole population (normal, deficient, and, in between, borderline subgroups of patients) and to detect the most discriminating variables. PCA of the data of all complexes together showed that mitochondrial diseases in this population were mainly caused by multiple deficits in respiratory chain complexes. This analysis allows the definition of a new subgroup of newborns, which have high respiratory chain complex activity values. Our results show that the PCA method, which simultaneously takes into account all of the concerned variables, allows the separation of patients into subgroups, which may help clinicians make their diagnoses