Voltage-dependent anion channels: the wizard of the mitochondrial outer membrane.

Voltage dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane. Although they are essential in metabolite exchange, cell defense and apoptosis, the molecular mechanism of these VDAC-mediated processes remains elusive. Here we review recent progress in terms of VDACs' structure and regulation, with a special focus on the molecular aspects of gating and the interaction with effector proteins.

Expression and localization of voltage-dependent anion channels (VDAC) in human spermatozoa.

Voltage-dependent anion channels (VDAC), also known as mitochondrial porins, are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures. VDAC allow the passage of the metabolites across the mitochondrial outer membrane, and are involved in metabolite transport and signal transduction. Several recent studies have indicated the important roles of VDAC in maintaining normal structure and motility of mammalian spermatozoa. To study the expression and localization of VDAC in human spermatozoa, different experimental approaches were applied: (1) specific primers were designed and VDAC gene sequences were cloned by PCR amplification from human testis cDNA library; (2) recombinant VDAC proteins were produced in the expression vector Escherichia coli BL21 (DE3); (3) human sperm VDAC proteins were extracted, separated and analyzed by Western blotting; (4) the localization of VDAC in human spermatozoa were detected using immunofluorescence. The three gene sequences and recombinant VDAC proteins were obtained, respectively. VDAC proteins were detected to be located in human spermatozoa, especially in sperm flagella. Our study elucidated for the first time that VDAC were synthesized and secreted at the testis level and eventually became an integral part of sperm proteins.

Mitochondrial modulation is involved in the hepatoprotection of Limonium sinense extract against liver damage in mice.

AIM OF THE STUDY: Limonium sinense (Girard) Ktze is a Chinese folk medicine used to treat fever, hemorrhage, hepatitis, and other disorders. The present research focused on the protective effects of L. sinense extracts (LSE) against liver damage. MATERIALS AND METHODS: In this study the extract from the root of Limonium sinense was used. Aminotransferase activity detection, electron microscopy, mitochondrial function evaluation, RT-PCR and western blot were used to evaluate the hepatoprotection of LSE in LPS/d-GalN-intoxicated mice. RESULTS: Pretreatment with 100, 200 or 400mg/kg LSE significantly blocked the increase in both serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT) levels induced by treatment with LPS plus d-GalN (LPS/d-GalN). Ultrastructural observation by electron microscopy showed reduced hepatocyte nuclear condensation and less lipid deposition. The decrease in both the mitochondrial membrane potential (14.6%) and sensitivity to mitochondrial swelling induced by Ca(2+) (45.9%) observed in the liver of LPS/d-GalN-treated mice were prevented by pretreatment with LSE. In addition, different doses of LSE increased both the transcription and the translation of voltage-dependent anion channels (VDAC), which was down-regulated by LPS/d-GalN treatment. CONCLUSIONS: In summary, LSE protects livers against LPS/d-GalN-induced damage, possibly by mitochondrial mechanisms related to increased expression of VDAC.

Ontogeny and nutritional programming of mitochondrial proteins in the ovine kidney, liver and lung.

This study investigated the developmental and nutritional programming of two important mitochondrial proteins, namely voltage-dependent anion channel (VDAC) and cytochrome c, in the sheep kidney, liver and lung. The effect of maternal nutrient restriction between early and mid-gestation (i.e. 28- to 80-day gestation, the period of maximal placental growth) on the abundance of these proteins was also examined in fetal and juvenile offspring. Fetuses were sampled at 80 and 140 days of gestation (term approximately 147 days), and postnatal animals at 1 and 30 days and 6 months of age. The abundance of VDAC peaked at 140 days of gestation in the lung, compared with 1 day after birth in the kidney and liver, whereas cytochrome c abundance was greatest at 140 days of gestation in the liver, 1 day after birth in the kidney and 6 months of age in lungs. This differential ontogeny in mitochondrial protein abundance between tissues was accompanied with very different tissue-specific responses to changes in maternal food intake. In the liver, maternal nutrient restriction only increased mitochondrial protein abundance at 80 days of gestation, compared with no effect in the kidney. In contrast, in the lung mitochondrial protein, abundance was raised near to term, whereas VDAC abundance was decreased by 6 months of age. These findings demonstrate the tissue-specific nature of mitochondrial protein development that reflects differences in functional adaptation after birth. The divergence in mitochondrial response between tissues to maternal nutrient restriction early in pregnancy further reflects these differential ontogenies.

Different effects of maternal parity, cold exposure and nutrient restriction in late pregnancy on the abundance of mitochondrial proteins in the kidney, liver and lung of postnatal sheep.

Adaptation to the extrauterine environment at birth relies upon the onset of postnatal function and increased metabolism in the lungs, liver and kidney, mediated partly by activation of mitochondrial proteins such as the voltage-dependent anion channel (VDAC), cytochrome c and, in the lung only, uncoupling protein (UCP)2. The magnitude of adaptation is dependent on the maternal metabolic and endocrine environment. We, therefore, examined the influence of maternal cold exposure (MCE) induced by winter shearing of pregnant sheep in conjunction with nutrient restriction (NR; 50% reduction in maternal food intake from 110 days gestation up to term). The effect of parity was also examined, as the offspring of nulliparous mothers are growth restricted compared with multiparous offspring. All sheep were twin bearing. One twin was sampled after birth and its sibling at 30 days. In the lung, both MCE and maternal nulliparity enhanced UCP2 abundance. However, whilst VDAC abundance was decreased in both the offspring of nulliparous mothers and by NR, it was transiently raised by MCE. Kidney VDAC abundance was reduced by MCE and nulliparity, adaptations only influenced by NR in multiparous mothers. Cytochrome c abundance was raised by MCE and by NR in multiparous controls and raised in offspring of nulliparous mothers. Liver VDAC and cytochrome c abundance were transiently reduced by MCE and persistently lower in offspring of nulliparous mothers. In conclusion, changes in the maternal metabolic environment have marked tissue-specific effects on mitochondrial protein abundance in the lungs, liver and kidney that may be important in enabling the newborn to effectively adapt to the extrauterine environment.

A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage.

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.

Detection of the mitochondrial apoptosis-induced channel (MAC) and its regulation by Bcl-2 family proteins.

Apoptosis is a phenomenon fundamental to higher eukaryotes that is integral to such diverse cellular processes as tissue homeostasis, organogenesis, and response to toxins. The release from mitochondria of apoptotic factors such as cytochrome c is a key step during apoptosis of most cells. Cytochrome c release occurs through the MAC (mitochondrial apoptosis-induced channel), a pore which forms in the mitochondrial outer membrane during early apoptosis and is exquisitely regulated by the Bcl-2 family of proteins. This unit presents basic and advanced tools for detecting MAC and defining its regulation by Bcl-2 family proteins and pharmacological agents. Protocols include the use of time-lapse video-microscopy to monitor the onset of apoptosis in living cells and patch-clamp techniques for mitochondria or proteoliposomes containing mitochondrial proteins, which allow direct detection of MAC. These approaches enable an evaluation of the role of MAC and mitochondria in apoptosis of a variety of cell types by many inducers.

Tissue-specific effects of leptin administration on the abundance of mitochondrial proteins during neonatal development.

Many tissues undergo a rapid transition after birth, accompanied by dramatic changes in mitochondrial protein function. In particular, uncoupling protein (UCP) abundance increases at birth in the lung and adipose tissue, to then gradually decline, an adaptation that is important in enabling normal tissue function. Leptin potentially mediates some of these changes and is known to promote the loss of UCP1 from brown fat but its effects on UCP2 and related mitochondrial proteins (i.e. voltage-dependent anion channel (VDAC) and cytochrome c) in other tissues are unknown. We therefore determined the effects of once-daily jugular venous administration of ovine recombinant leptin on mitochondrial protein abundance as determined by immunoblotting in tissues that do (i.e. the brain and pancreas) and do not (i.e. liver and skeletal muscle) express UCP2. Eight pairs of 1-day-old lambs received either 100 mug leptin or vehicle daily for 6 days, before tissue sampling on day 7. Administration of leptin diminished UCP2 abundance in the pancreas, but not the brain. Leptin administration had no affect on the abundance of VDAC or cytochrome c in any tissue examined. In leptin-administered animals, but not controls, UCP2 abundance in the pancreas was positively correlated with VDAC and cytochrome c content, and UCP2 abundance in the brain with colonic temperature. In conclusion, leptin administration to neonatal lambs causes a tissue-specific loss of UCP2 from the pancreas. These effects may be important in the regulation of neonatal tissue development and potentially for optimising metabolic control mechanisms in later life.