Protein biosynthesis in mitochondria

Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.     

Supernatant protein factor in complex with RRR-alpha-tocopherylquinone: a link between oxidized Vitamin E and cholesterol biosynthesis

The vast majority of monomeric lipid transport in nature is performed by lipid-specific protein carriers. This class of proteins can enclose cognate lipid molecules in a hydrophobic cavity and transport them across the aqueous environment. Supernatant protein factor (SPF) is an enigmatic representative of monomeric lipid transporters belonging to the SEC14 family. SPF stimulates squalene epoxidation, a downstream step of the cholesterol biosynthetic pathway, by an unknown mechanism. Here, we present the three-dimensional crystal structure of human SPF in complex with RRR-alpha-tocopherylquinone, the major physiological oxidation product of RRR-alpha-tocopherol, at a resolution of 1.95A. The structure of the complex reveals how SPF sequesters RRR-alpha-tocopherylquinone (RRR-alpha-TQ) in its protein body and permits a comparison with the recently solved structure of human alpha-tocopherol transfer protein (alpha-TTP) in complex with RRR-alpha-tocopherol. Recent findings have shown that RRR-alpha-TQ is reduced in vivo to RRR-alpha-TQH(2), the latter has been suggested to protect low-density lipoprotein (LDL) particles from oxidation. Hence, the antioxidant function of the redox couple RRR-alpha-TQ/RRR-alpha-TQH(2) in blocking LDL oxidation may reduce cellular cholesterol uptake and thus explain how SPF upregulates cholesterol synthesis.     

Therapy of Cushing''s syndrome with steroid biosynthesis inhibitors

Several substances with different inhibitory effects on adrenal steroid biosynthesis were investigated in patients with Cushing's syndrome. It has been shown that trilostane, a 3β-hydroxysteroid-dehydrogenase inhibitor, is not potent enough to block cortisol biosynthesis in patients with hypercortisolism. Aminoglutethimide inhibits side chain cleavage of cortisol synthesis, but it has been demonstrated that the blocking effect on cortisol secretion is not strong enough to normalize urinary cortisol excretion in patients with Cushing's disease. For metyrapone, an inhibitor of adrenal 11β-hydroxylase, promising results were reported for the treatment of Cushing's syndrome. However, the drug has several side effects and depending on the definition of the desired reduction of cortisol secretion a true remission was only found in a minority of patients. The antifungal drug ketoconazole in vitro predominantly blocks 17,20-desmolase (IC₅₀ 1 μM) and to a lesser extent 17-hydroxylase (IC₅₀ 10 μM) and 11β-hydroxylase (IC₅₀ 15–40 μM). Therefore, ketoconazole in vivo most potently suppresses androgen secretion and only to a lesser extent cortisol biosynthesis. Several therapeutic trials with ketoconazole treatment in patients with pituitary Cushing's disease showed various remission rates between 30 and 90%. In contrast, in almost all patients with benign, primary adrenal Cushing's syndrome cortisol levels were normalized. In patients with ectopic ACTH syndrome ketoconazole was effective in about 50% of all reported cases, while cortisol hypersecretion due to adrenocortical carcinoma was only rarely inhibited by ketoconazole. The main side effect of ketoconazole treatment was liver toxicity which occurred in 12% of all treated patients. In contrast to ketoconazole, the narcotic drug etomidate shows a strong inhibitory effect on 11β-hydroxylase (IC₅₀ 0.03–0.15 μM) but only a weak inhibition of 17,20 desmolase (IC₅₀ 380 μM). This correlates with in vivo studies where even low, non-hypnotic doses of etomidate induced a pronounced fall in serum cortisol levels in normals and in patients with Cushing's syndrome. However, its clinical use is limited by its mandatory intravenous application and its sedative effects. In conclusion, ketoconazole remains the only available steroid-inhibitory drug for a therapeutic trial in patients with Cushing's syndrome who cannot be treated definitively by surgery.     

The missing link between hydrogenosomes and mitochondria

Mitochondria typically respire oxygen and possess a small DNA genome. But among various groups of oxygen-shunning eukaryotes, typical mitochondria are often lacking, organelles called hydrogenosomes being found instead. Like mitochondria, hydrogenosomes are surrounded by a double-membrane, produce ATP and sometimes even have cristae. In contrast to mitochondria, hydrogenosomes produce molecular hydrogen through fermentations, lack cytochromes and usually lack DNA. Hydrogenosomes do not fit into the conceptual mold cast by the classical endosymbiont hypothesis about the nature of mitochondria. Accordingly, ideas about their evolutionary origins have focussed on the differences between the two organelles instead of their commonalities. Are hydrogenosomes fundamentally different from mitochondria, the result of a different endosymbiosis? Or are our concepts about the mitochondrial archetype simply too narrow? A new report has uncovered DNA in the hydrogenosomes of anaerobic ciliates. The sequences show that these hydrogenosomes are, without a doubt, mitochondria in the evolutionary sense, even though they differ from typical mitochondria in various biochemical properties. The new findings are a benchmark for our understanding of hydrogenosome origins.     

Mechanism of protein biosynthesis in mammalian mitochondria

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 g ml^–1 CdCl₂ in the presence or absence of 0.1–1000 ng ml^–1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17-estradiol (E₂) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 g ml^–1 CdCl₂ suppressed P accumulation to 65 and 10%, respectively; accumulation of E₂ (at 5 g ml^–1 CdCl₂) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 g ml^–1 CdCl₂ was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 g ml^–1 CdCl₂, whereas basal E₂ production of granulosa cells was markedly diminished. It is concluded that CdCl₂ suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.     

Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis

Cholesterol transport from the outer to the inner mitochondrial membrane is the rate-determining step in steroid and bile acid biosyntheses. Biochemical, pharmacological and molecular studies have demonstrated that the peripheral-type benzodiazepine receptor (PBR) is a five transmembrane domain mitochondrial protein involved in the regulation of cholesterol transport. PBR gene disruption in Leydig cells completely blocked cholesterol transport into mitochondria and steroid formation, while PBR expression in bacteria, devoid of endogenous PBR and cholesterol, induced cholesterol uptake and transport. Molecular modeling of PBR suggested that cholesterol might cross the membrane through the five helices of the receptor and that synthetic and endogenous ligands might bind to common sites in the cytoplasmic loops. A cholesterol recognition/interaction amino acid consensus (CRAC) sequence in the cytoplasmic carboxy-terminus of the PBR was identified by mutagenesis studies. In vitro reconstitution of PBR into proteoliposomes demonstrated that PBR binds both drug ligands and cholesterol with high affinity. In vivo polymeric forms of PBR were observed and polymer formation was reproduced in vitro, using recombinant PBR protein reconstituted into proteoliposomes, associated with an increase in drug ligand binding and reduction of cholesterol-binding capacity. This suggests that the various polymeric states of PBR might be part of a cycle mediating cholesterol uptake and release into the mitochondria, with PBR functioning as a cholesterol exchanger against steroid product(s) arising from cytochrome P450 action. Taking into account the widespread presence of PBR in many tissues, a more general role of PBR in intracellular cholesterol transport and compartmentalization might be considered.     

The use of bioelectrochemical systems in steroid biosynthesis

The feasibility of electrosynthesis involving the use of biocatalysts has been demonstrated in a model reaction of hydrocortisone oxidation to prednisolone catalyzed by Arthrobacter globiformis cells. Electrochemical regeneration of phenazine methosulfate, an artificial electron acceptor, was performed, using electrodes made of different materials. Transformation products were analyzed by high performance liquid chromatography.     

Heat shock factor 1 in brain tumors: a link with transient receptor potential channels TRPV1 and TRPA1

Journal of Molecular Histology - Novel data report a “cross-talk” between Heat-Shock Factor 1 (HSF1) and the transient receptor potential vanilloid 1 cation channel (TRPV1) located in...     

Galactosyltransferase activities in mitochondria outer membrane: biosynthesis of galactosylated proteins

1. Mitochondria outer membranes prepared from mouse livers were purified on a discontinuous sucrose gradient. Control in electron microscopy and marker enzymes assays confirmed purity and homogeneity of this fraction. 2. Purified mitochondria outer membranes exhibited significant UDP-galactose: glycoprotein galactosyltransferase activities when incubated with endogenous or exogenous glycoprotein acceptors in presence of detergent (Nonidet P40). 3. Some properties of two distinct mitochondrial galactosyltransferases, acting respectively on ovomucoid and ovine asialo-mucin were investigated. 4. Transfer of galactose on ovomucoid was maximal for a pH of 7.6 at 33 degrees C whereas asialo-mucin galactosyltransferase exhibited an optimum pH of 5.6 for an optimal temperature of 46 degrees C. 5. These two distinct membrane-bound enzymes were both inhibited by diacylglycerophospholipids whereas lysophospholipids modulated both enzymes in a different way: at 5 mM lysophosphatidylcholine, asialo-mucin galactosyltransferase was slightly stimulated while ovomucoid galactosyltransferase was markedly activated. 6. The most important activating effect on ovomucoid galactosyltransferase was obtained with a phospholipid containing a long aliphatic side chain linked by an ester bond in sn-1 of glycerol, an hydroxyl group or hydrogen atoms in sn-2 and a phosphorylcholine head group in sn-3.