Human diseases with impaired mitochondrial protein synthesis

Mitochondrial respiratory chain deficiencies represent one of the major causes of metabolic disorders that are related to genetic defects in mitochondrial or nuclear DNA. The mitochondrial protein synthesis allows the synthesis of the 13 respiratory chain subunits encoded by mtDNA. Altogether, about 100 different proteins are involved in the translation of the 13 proteins encoded by the mitochondrial genome emphasizing the considerable investment required to maintain mitochondrial genetic system. Mitochondrial protein synthesis deficiency can be caused by mutations in any component of the translation apparatus including tRNA, rRNA and proteins. Mutations in mitochondrial rRNA and tRNAs have been first identified in various forms of mitochondrial disorders. Moreover abnormal translation due to mutation in nuclear genes encoding tRNA-modifying enzymes, ribosomal proteins, aminoacyl-tRNA synthetases, elongation and termination factors and translational activators have been successively described. These deficiencies are characterized by a huge clinical and genetic heterogeneity hampering to establish genotype-phenotype correlations and an easy diagnosis. One can hypothesize that a new technique for gene identification, such as exome sequencing will rapidly allow to expand the list of genes involved in abnormal mitochondrial protein synthesis.     

Identification of a molecular component of the mitochondrial acetyltransferase programme: a novel role for GCN5L1

SIRT3 (sirtuin 3) modulates respiration via the deacetylation of lysine residues in electron transport chain proteins. Whether mitochondrial protein acetylation is controlled by a counter-regulatory program has remained elusive. In the present study we identify an essential component of this previously undefined mitochondrial acetyltransferase system. We show that GCN5L1 [GCN5 (general control of amino acid synthesis 5)-like 1; also known as Bloc1s1] counters the acetylation and respiratory effects of SIRT3. GCN5L1 is mitochondrial-enriched and displays significant homology with a prokaryotic acetyltransferase. Genetic knockdown of GCN5L1 blunts mitochondrial protein acetylation, and its reconstitution in intact mitochondria restores protein acetylation. GCN5L1 interacts with and promotes acetylation of SIRT3 respiratory chain targets and reverses global SIRT3 effects on mitochondrial protein acetylation, respiration and bioenergetics. The results of the present study identify GCN5L1 as a critical prokaryote-derived component of the mitochondrial acetyltransferase programme.     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

The diabetes-associated 3243 mutation in the mitochondrial tRNA(Leu(UUR)) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis rate.

Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNA(Leu(UUR)) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNA(Leu(UUR)), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed.     

Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III₂ and I-III₂-IV_1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function.     

Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria

Three mitochondrial DNA–encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1.     

FAST is a BCL-X(L)-associated mitochondrial protein

The TIA-1-interacting protein Fas-activated serine/threonine phosphoprotein (FAST) is a component of a signaling cascade that is initiated by ligation of the Fas receptor. Immunofluorescence microscopy using affinity-purified antibodies raised against recombinant FAST reveals that the endogenous protein associates with mitochondria. Subcellular fractionation confirms that FAST is a component of mitochondria. FAST is tethered to mitochondria by a lysine/arginine-rich domain at its carboxyl terminus that is structurally similar to the mitochondrial tethering motifs of monoamine oxidase B and cytochrome b5. At the mitochondrial membrane, FAST interacts with BCL-X(L). The BCL-X(L) binding domain maps to a BCL-2-homology-3 (BH3)-related domain that is distinct from the mitochondrial-tethering domain (MTD). Although interactions between FAST and BCL-X(L) require both the BH3-related domain and the MTD, the requirement for mitochondrial tethering can be conferred by a heterologous MTD. Our results suggest that FAST-BCL-X(L) interactions are likely to regulate mitochondrial metabolism during Fas-induced apoptosis.     

SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability

Stomatin is a member of a large family of proteins including prohibitins, HflK/C, flotillins, mechanoreceptors and plant defense proteins, that are thought to play a role in protein turnover. Using different proteomic approaches, we and others have identified SLP-2, a member of the stomatin gene family, as a component of the mitochondria. In this study, we show that SLP-2 is strongly associated with the mitochondrial inner membrane and that it interacts with prohibitins. Depleting HeLa cells of SLP-2 lead to increased proteolysis of prohibitins and of subunits of the respiratory chain complexes I and IV. Further supporting the role of SLP-2 in regulating the stability of specific mitochondrial proteins, we found that SLP-2 is up-regulated under conditions of mitochondrial stress leading to increased protein turnover. These data indicate that SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.     

BMCP1: a mitochondrial uncoupling protein in neurons which regulates mitochondrial function and oxidant production

Outside the nervous system, members of the mitochondrial uncoupling protein (UCP) family have been proposed to contribute to control of body temperature and energy metabolism, and regulation of mitochondrial production of reactive oxygen species (ROS). However, the function of brain mitochondrial carrier protein 1 (BMCP1), which is highly expressed in brain, remains to be determined. To study BMCP1 expression and function in the nervous system, a high-affinity antibody to BMCP1 was generated and used to analyze tissue expression of BMCP1 protein in mouse. BMCP1 protein was highly expressed in heart and kidney, but not liver or lung. In the nervous system, BMCP1 was present in cortex, basal ganglia, substantia nigra, cerebellum, and spinal cord. Both BMCP1 mRNA and protein expression was almost exclusively neuronal. To study the effect of BMCP1 expression on mitochondrial function, neuronal (GT1-1) cell lines with stable overexpression of BMCP1 were generated. Transfected cells had higher State 4 respiration and lower mitochondrial membrane potential (psi(m)), consistent with greater mitochondrial uncoupling. BMCP1 expression also decreased mitochondrial production of ROS. These data suggest that BMCP1 can modify mitochondrial respiratory efficiency and mitochondrial oxidant production, and raise the possibility that BMCP1 might alter the vulnerability of brain to both acute injury and to neurodegenerative conditions.     

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F₀F₁-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F₀F₁-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.     

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 micrograms/100 g body wt) of triiodothyronine (T3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T3 increases the numbers of functional respiratory chain units. T3 also induces mitochondrial protein synthesis by 50-100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T3 belong to two subsets, which were either induced or repressed by hormone. Thus, T3 stimulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.     

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene

The A3243G mutation in the human mitochondrial tRNA^Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA^Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA^Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.     

The effect of diabetes on protein synthesis and the respiratory chain of rat skeletal muscle and kidney mitochondria

The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa₃ and a 27% decrease in cytochrome b, both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro, nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b, c + c₁, and aa₃, the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.