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Proteomic approach to the identification of voltage-dependent anion channel protein isoforms in guinea pig brain synaptosomes
Abstract
Voltage-dependent anion channel (VDAC) proteins are small, abundant, pore-forming proteins belonging to the eukaryotic mitochondrial porins. At least three different VDAC genes have been identified in vertebrates. VDAC proteins are known to play an essential role in cellular metabolism and in the early stages of apoptosis. A proteomic approach, consisting of two-dimensional gel electrophoresis followed by two-dimensional immunoblotting with anti-VDAC and anti-phosphotyrosine antibodies and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, was exploited to define the expression pattern of VDAC isoforms in guinea pig brain synaptosomes, both in normoxic and hypoxic conditions. In this way a total of five different VDAC isoforms were identified, as both VDAC1 and VDAC2 were detected in more than one electrophoretic spot. Moreover, VDAC isoforms selectively undergo hypoxia-induced tyrosine phosphorylation, suggesting that tyrosine phosphorylation may contribute to the modulation of VDAC protein function/conformation or interaction with other proteins in hypoxic conditions.
... All amino acid residues are similarly frequent in VDAC2 and the other VDAC isoforms except cysteine (C), which is exclusively higher in mammalian VDAC2 (9 in hVDAC2 vs 2 in hVDAC1 and 6 in hVDAC3). Four of the C′s (47, 76,103,133) in VDAC2 are in the linkers between the membrane spanning domains, seemingly at the same side of the membrane. They might play a role in redox regulation, protein-protein interactions or might have an effect on the stability of β-barrels and on VDAC2 interaction with the lipid environment [46][47][48]. ...
... For instance in adipogenesis [130], iron deprivation [131] and aging [132] VDAC2 level increased and in hypoxia it decreased. Not only changes in the level of the protein but also post-translational modification like phosphorylation can be affected under Naghdi various pathological conditions [133]. Interestingly VDAC seems to be associated to neurodegenerative diseases as well. ...
... Adipogenesis VDAC2↑ 3T3-L1 fibroblast (mouse) [130] Exercise after myocardial infarction VDAC2↓ Heart (rat) [144] Hyperglycemia VDAC2↓,VDAC1↑ INS1E pancreatic β cells (rat) [145] Iron deprivation VDAC2↑ K562 erythroleukemia cell (human) [131] Aging VDAC2↑ Skeletal muscle mitochondria (aged rat) [132] Hypoxia Phosphorylation of VDAC1 and 2 Brain fraction (guinea pig) [133] Hypoxia Vdac2 gene↑ ...
Voltage Dependent Anion-selective Channel 2 (VDAC2) contributes to oxidative metabolism by sharing a role in solute transport across the outer mitochondrial membrane (OMM) with other isoforms of the VDAC family, VDAC1 and VDAC3. Recent studies revealed that VDAC2 also has a distinctive role in mediating sarcoplasmic reticulum to mitochondria local Ca2 + transport at least in cardiomyocytes, which is unlikely to be explained simply by the expression level of VDAC2. Furthermore, a strictly isoform-dependent VDAC2 function was revealed in the mitochondrial import and OMM-permeabilizing function of pro-apoptotic Bcl-2 family proteins, primarily Bak in many cell types. In addition, emerging evidence indicates a variety of other isoform-specific engagements for VDAC2. Since VDAC isoforms display 75% sequence similarity, the distinctive structure underlying VDAC2-specific functions is an intriguing problem. In this paper we summarize studies of VDAC2 structure and functions, which suggest a fundamental and exclusive role for VDAC2 in health and disease.
... The use of purification protocols allowed the isolation of mitochondrial porins from mammalian tissues and from cells 292 of lower eukaryotes with high yield (Colombini, 1983; De Pinto et al., 1987b, 1989b, 1985 Freitag et al., 1982; Linden et al., 293 1982b; Ludwig et al., 1989 Ludwig et al., , 1988 Roos et al., 1982; Troll et al., 1992; Zalman et al., 1980)Yamamoto et al., 2006), as previously noted (De Pinto et al., 1985 ), whereas VDAC2, despite a high- 310 er molecular weight, has the same mobility as VDAC1, while VDAC3 has the highest mobility in SDS–PAGE (Yamamoto et al., 311 2006). These results were confirmed upon purification of VDAC isoforms from bovine spermatozoa (Menzel et al., 2009) that the purified protein is found in more spots (Liberatori et al., 2004; Linden et al., 1982b; Menzel et al., 317 2009). This mobility pattern can result from post-translational modifications of VDAC isoforms (seeTable 1). ...
... These and similar cytosolic kinases and phosphatases 1739 may also modulate VDAC activity. 1740 VDAC possesses several potential phosphorylatable residues and several of them were indeed shown to undergo phos- 1741 phorylation (Baines et al., 2003; Bera et al., 1995; Liberatori et al., 2004). VDAC is phosphorylated by protein kinase A 1742 (PKA) (Bera et al., 1995), by protein kinase Ce (PKCe) (Baines et al., 2003 ), while VDAC1 and VDAC2 in guinea pig brain syn- 1743 aptosomes were found to be phosphorylated at a Tyr residue under hypoxic conditions (Liberatori et al., 2004). ...
... 1740 VDAC possesses several potential phosphorylatable residues and several of them were indeed shown to undergo phos- 1741 phorylation (Baines et al., 2003; Bera et al., 1995; Liberatori et al., 2004). VDAC is phosphorylated by protein kinase A 1742 (PKA) (Bera et al., 1995), by protein kinase Ce (PKCe) (Baines et al., 2003 ), while VDAC1 and VDAC2 in guinea pig brain syn- 1743 aptosomes were found to be phosphorylated at a Tyr residue under hypoxic conditions (Liberatori et al., 2004). Recently, the 1744 phosphorylation sites in VDAC were identified by MALDI-TOF mass spectrometry, showing that rat liver VDAC1 is phosphor- 1745 ylated under physiological conditions at Ser-12 and Ser-136, and VDAC2 at Tyr-237 and VDAC3 at Ser-241 and Thr-33 1746 (Distler et al., 2007). ...
Research over the past decade has extended the prevailing view of the mitochondrion to include functions well beyond the generation of cellular energy. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organellar communication, aging, cell proliferation, diseases and cell death. Thus, mitochondria play a central role in the regulation of apoptosis (programmed cell death) and serve as the venue for cellular decisions leading to cell life or death. One of the mitochondrial proteins controlling cell life and death is the voltage-dependent anion channel (VDAC), also known as mitochondrial porin. VDAC, located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, thereby controlling cross-talk between mitochondria and the rest of the cell. VDAC is also a key player in mitochondria-mediated apoptosis. Thus, in addition to regulating the metabolic and energetic functions of mitochondria, VDAC appears to be a convergence point for a variety of cell survival and cell death signals mediated by its association with various ligands and proteins. In this article, we review what is known about the VDAC channel in terms of its structure, relevance to ATP rationing, Ca(2+) homeostasis, protection against oxidative stress, regulation of apoptosis, involvement in several diseases and its role in the action of different drugs. In light of our recent findings and the recently solved NMR- and crystallography-based 3D structures of VDAC1, the focus of this review will be on the central role of VDAC in cell life and death, addressing VDAC function in the regulation of mitochondria-mediated apoptosis with an emphasis on structure-function relations. Understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of functions, all important for cell life and death. This review also provides insight into the potential of VDAC1 as a rational target for new therapeutics.
... In particular VDAC2 originated five distinct spots with a well distinct pattern: they were focussed at two different molecular weights and at three different pIs, giving raise to a characteristic checkerboard distribution. Liberatori et al [38] found in guinea pig brain synaptosomes three spots corresponding to VDAC2, all with the same molecular mass but different pIs. Yamamoto et al [39] identified in rat liver mitochondria two spots as VDAC2, again with the same molecular masses. ...
... The second issue was also justified by the observation that VDAC2 can be phosphorylated in various situations: changes in the isoelectric point of a protein indeed are often due to post-translational modifications. VDAC2 was found to be phosphorylated in guinea pig synaptosomes after hypoxia [38]. Distler et al showed that in VDAC2 Tyr-237 is phosphorylated [INSR site, 28]. ...
... This is because VDAC3 is essentially not active in planar bilayers [12] and VDAC1 is present in a very little amount and has distinguishable, not fully overlapping, features [12]. VDAC2, with a longer N-terminus, has the same electrophoretic mobility than VDAC1 [38] with the exception of the spermatozoa where it is specifically present with two electrophoretic mobilities. In addition its specific activity is much lower than VDAC1 [12]. ...
VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.
... TVDAC1 contains two cysteines; Cys 232 is found in the carboxyamidomethylated form, while Cys 127 is in the oxidized form of sulfonic acid [131]. VDAC1 possesses several potentially phosphorylatable serine and threonine residues, many of which have indeed been shown to undergo phosphorylation [132][133][134] by protein kinase A (PKA) [133], protein kinase C (PKC)ε [132], and GSK3b [135]. Both VDAC1 and VDAC2 are phosphorylated at a specific Tyr residue under hypoxic conditions [134]. ...
... VDAC1 possesses several potentially phosphorylatable serine and threonine residues, many of which have indeed been shown to undergo phosphorylation [132][133][134] by protein kinase A (PKA) [133], protein kinase C (PKC)ε [132], and GSK3b [135]. Both VDAC1 and VDAC2 are phosphorylated at a specific Tyr residue under hypoxic conditions [134]. ...
The voltage-dependent anion channel 1 (VDAC1) protein, is an important regulator of mitochondrial function, and serves as a mitochondrial gatekeeper, with responsibility for cellular fate. In addition to control over energy sources and metabolism, the protein also regulates epigenomic elements and apoptosis via mediating the release of apoptotic proteins from the mitochondria. Apoptotic and pathological conditions, as well as certain viruses, induce cell death by inducing VDAC1 overexpression leading to oligomerization, and the formation of a large channel within the VDAC1 homo-oligomer. This then permits the release of pro-apoptotic proteins from the mitochondria and subsequent apoptosis. Mitochondrial DNA can also be released through this channel, which triggers type-Ι interferon responses. VDAC1 also participates in endoplasmic reticulum (ER)-mitochondria cross-talk, and in the regulation of autophagy, and inflammation. Its location in the outer mitochondrial membrane, makes VDAC1 ideally placed to interact with over 100 proteins, and to orchestrate the interaction of mitochondrial and cellular activities through a number of signaling pathways. Here, we provide insights into the multiple functions of VDAC1 and describe its involvement in several diseases, which demonstrate the potential of this protein as a druggable target in a wide variety of pathologies, including cancer.
... MPT is thought to result from the formation of a mitochondrial pore at contact sites between the inner and outer mitochondrial membranes, between the adenine nucleotide translocator and the voltage-dependent ion channel (VDAC). The selective tyrosine phosphorylation of VDAC1 and VDAC2 isoforms as a consequence of hypoxic stimulus has been reported [30]. It has been suggested that the observed hypoxia-induced tyrosine phosphorylation of VDAC1 and VDAC2 might be crucial for the modulation of protein interactions both in metabolic and proapoptotic/ antiapoptotic equilibrium, leading to activation/inactivation of cellular processes related to cellular apoptosis, neuronal degeneration or survival [30]. ...
... The selective tyrosine phosphorylation of VDAC1 and VDAC2 isoforms as a consequence of hypoxic stimulus has been reported [30]. It has been suggested that the observed hypoxia-induced tyrosine phosphorylation of VDAC1 and VDAC2 might be crucial for the modulation of protein interactions both in metabolic and proapoptotic/ antiapoptotic equilibrium, leading to activation/inactivation of cellular processes related to cellular apoptosis, neuronal degeneration or survival [30]. ...
Introduction: Perinatal hypoxia–ischemia (HI) continues to be a common underlying cause of brain damage. The outcomes of HI encephalopathy (HIE) are devastating and permanent, making it a major burden for the patient, the family and society. The understanding of the pathways that lead to the injury is vital for finding potential therapies. HI triggers a cascade of several detrimental insults: glutamate-mediated excitotoxicity, nitric oxide (NO) and calcium overload, oxidative stress (OS), stress signaling, inflammation and cell death.
Areas covered: The reader will be introduced to the complex biological processes involved in HI injury and how these mechanisms are exploited for current and emerging therapies. Current knowledge on pathways of damage, as well as ongoing experimental therapies are reviewed, based on a comprehensive literature search employing review articles, book chapters and Medline searches.
Expert opinion: Recent studies have demonstrated a protective effect of antioxidant drugs against apoptosis after cerebral ischemia and reperfusion. Iminobiotin provides long- and short-term neuroprotection hindering apoptotic pathways. As an antioxidant it acts as NO synthetase inhibitor interrupting the vicious circle and the harmful effects of excessive NO formation. More clinical trials are needed to detect safety and effectiveness of iminobiotin and to establish its dose and optimal time of action.
... VDAC1 possesses several potential phosphorylatable residues, several of which were shown to undergo phosphorylation (Baines et al., 2003;Bera et al., 1995;Liberatori et al., 2004). VDAC is phosphorylated by protein kinase A (PKA), leading to reduced singlechannel current and open probability (Bera et al., 1995), and protein kinase Cε (PKCε), which also directly interacts with VDAC1 (Baines et al., 2003). ...
... VDAC is phosphorylated by protein kinase A (PKA), leading to reduced singlechannel current and open probability (Bera et al., 1995), and protein kinase Cε (PKCε), which also directly interacts with VDAC1 (Baines et al., 2003). VDAC1 and VDAC2 in guinea pig brain synaptosomes were phosphorylated at a Tyr residue under hypoxic conditions (Liberatori et al., 2004). Recently, phosphorylation sites in VDAC were identified by MALDI-TOF mass spectrometry, showing that under physiological conditions, rat liver VDAC1 is phosphorylated at Ser-12 and Ser-136, VDAC2 at Tyr-237 and VDAC3 at Thr-33 and Ser-241 (Distler et al., 2007). ...
Regulation of mitochondrial physiology requires an efficient exchange of molecules between mitochondria and the cytoplasm via the outer mitochondrial membrane (OMM). The voltage-dependent anion channel (VDAC) lies in the OMM and forms a common pathway for the exchange of metabolites between the mitochondria and the cytosol, thus playing a crucial role in the regulation of metabolic and energetic functions of mitochondria. VDAC is also recognized to function in mitochondria-mediated apoptosis and in apoptosis regulation via interaction with anti-apoptotic proteins, namely members of Bcl-2 family, and the pro-survival protein, hexokinase, overexpressed in many cancer types. Thus, VDAC appears to be a convergence point for a variety of cell survival and cell death signals, mediated by its association with various ligands and proteins. In this article, we review mammalian VDAC, specifically focusing on VDAC1, addressing its functions in cell life and the regulation of apoptosis and its involvement in several diseases. Additionally, we provide insight into the potential of VDAC1 as a rational target for novel therapeutics.
... Interestingly, Tyr62 is conserved in human, mouse, rat, bull and pig VDAC-1 (Fig. 1d), and is located at the caveolar scaffolding domain of this porin (Marin et al. 2008). The phosphorylation of pl-VDAC in these neurons is in agreement with previous observations in guinea pig brain synaptosomes, where VDAC-1 was also reported to undergo tyrosine phosphorylation which was increased under hypoxic situations (Liberatori et al. 2004). ...
... As previously pointed out, post-transductional modifications of mitochondrial VDAC may be a crucial aspect of its actions related to cell survival and cell death, mediated by its association with various ligands and proteins (Shoshan- Barmatz et al. 2010), whereas this aspect has been poorly explored for pl-VDAC. Even though the effects of mitochondrial VDAC phosphorylation in its activities have been mostly verified in vitro, evidences concluded that VDAC phosphorylation may contribute to the modulation of VDAC function under stressing conditions such as hypoxia (Liberatori et al. 2004), observing a reduction of its opening probability because of phosphorylation (Bera and Ghosh 2001). Therefore, it is conceivable that, related to AD pathology, modifications in VDAC associations at the neuronal membrane may alter its normal post-transductional pattern, contributing to channel impairment. ...
J. Neurochem. (2011) 116, 820–827.
The voltage-dependent anion channel, VDAC, is present at the neuronal membrane, where it appears to participate, among others, in the extrinsic apoptotic pathway and in the modulation of amyloid-beta induced injury, suggesting the involvement of this channel in Alzheimer's disease (AD) neurotoxicity. VDAC is also highly concentrated in neuronal lipid raft microdomains of different mouse and human cognitive areas, where it has been shown associated with estrogen receptor alpha (ERα), as a part of a `signalosome' that may activate some intracellular signal transduction. At the plasma membrane level, estrogens and antiestrogens (tamoxifen) have been demonstrated to exert rapid antagonist effects on the activation of VDAC, through their distinct effects on the channel post-transductional modulation. Therefore, part of the alternative mechanisms of estrogen related to neuroprotection against amyloid-beta may involve VDAC phosphorylation, in order to maintain the channel in an unactivated (closing) state. Interestingly, VDAC-ERα association has been shown to be disrupted in neuronal lipid rafts of AD brains, in correlation with the aberrant lipid composition observed in these microstructures, suggesting that disturbance of protein interactions may be related to variation in the physico-chemical properties of these microdomains.
... VDAC2 was separated into three, five, and two spots in rat, bovine, and chicken brain, respectively. Previous reports described VDAC isoforms with different pIs (Reymann et al. 1999;Yoo et al. 2001;Liberatori et al. 2004;Yamamoto et al. 2006;Distler et al. 2007;Alfonso et al. 2008). Yoo et al. (2001) reported the presence of three VDAC1 forms in post-mortem brain of Alzheimer's and Down syndrome patients, which differed in their isoelectric points. ...
... Yoo et al. (2001) reported the presence of three VDAC1 forms in post-mortem brain of Alzheimer's and Down syndrome patients, which differed in their isoelectric points. In the same way, Liberatori et al. (2004) identified charge heterogeneity of VDAC1 (two spots) and VDAC2 (three spots) in guinea pig brain synaptosomes. Both isoforms were found to contain phosphorylated tyrosines under hypoxic conditions. ...
Voltage-dependent anion channels (VDAC) are pore-forming proteins found in the outer mitochondrial membrane of eukaryotes. VDACs are known to play an essential role in cellular metabolism and in early stages of apoptosis. In mammals, three VDAC isoforms have been identified. A proteomic approach was exploited to study the expression of VDAC isoforms in rat, bovine, and chicken brain mitochondria. Given the importance of mitochondrially bound hexokinase in regulation of aerobic glycolysis in brain, we studied the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A/type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was significantly higher in bovine than in rat brain. Chicken brain mitochondria showed the highest VDAC1 expression and the lowest of VDAC2. Bovine brain mitochondria had the highest VDAC2 levels. We concluded that the nature of hexokinase binding site is not determined by the expression of a single VDAC isoform.
... 21,22 Recently, the activation of nPKCe has been reported to participate in the activation of other intracellular kinase or membrane receptor cascades by a series of functional proteomic studies, such as nPKCe-Src tyrosine kinase, nPKCe-MAPKs, and nPKCe-voltage-dependent anion channel interactions. [24][25][26] The activation of nPKCe protected the ischemic heart through activation of extracellular signal-regulated kinases (ERKs). 27,28 nPKCe and ERK1/2 are all activated in hippocampal slices after neuronal injury. ...
... nPKCe-triggered signaling pathways have been characterized by functional proteomics studies, such as nPKCe-Src tyrosine kinase, nPKCe-MAPKs, and nPKCe-voltage-dependent anion channel interactions. [24][25][26]43,44 The translocation of nPKCe to special sites, such as mitochondria, and the phosphorylation of ERK1/2 play a key role in ischemic tolerance of cardiomyocytes, the heart, primary neuronal cultures, and brain slices. 28,29,[43][44][45] Phosphorylated ERK1/2 is the last cytosolic step in a phosphorylation cascade, which translocates to the nucleus in response to ischemia and reperfusion in rat heart, 46 and exerts a neuroprotective effect in the brain. ...
Accumulated reports have suggested that activation of protein kinase C (PKC) isoforms may involve the activation of extracellular signal-regulated kinases (ERKs) in the neuronal response to ischemic/hypoxic stimuli. We have previously demonstrated that the membrane translocation of novel PKC (nPKC) epsilon increased in the early phase of cerebral ischemic/hypoxic preconditioning of mice. In this study, we used Western blot analysis and propidium iodide stain to determine whether the activations of nPKCepsilon and ERKs were involved in oxygen-glucose deprivation (OGD)-induced neuroprotection via N-methyl-D-aspartate (NMDA) receptors. The hippocampal slices of mice were exposed to OGD for 10 (OGD10) or 45 minutes (OGD45) to mimic mild (causing ischemic/hypoxic preconditioning) and severe (causing severe OGD) ischemia/hypoxia, respectively. We found that OGD10-induced nPKCepslilon membrane translocation was mediated by NMDA receptors, and both OGD10 and NMDA (1 microM, 30 min) pretreatment could protect Cornu Ammonis region 1 neurons against the subsequent severe OGD45. In addition, nPKCepsilon translocation inhibitor, epsilonV1-2 (1 microM, 30 min), and ERKs upstream mitogen-activated protein/extracellular signal regulated kinase kinase inhibitor, PD-98059 (20 microM, 30 min), could significantly inhibit OGD10 and NMDA-induced neuroprotection. These results suggest that OGD10-induced neuroprotection against severe OGD45 in the Cornu Ammonis region 1 region of the hippocampal slices was mediated by the activations of NMDA receptors, nPKCepsilon, and the downstream ERKs.
... The second group of studies provided an improvement from the first group by indicating the exact site of phosphorylation in VDAC (and also the particular isoform) under a certain stimulus (or a disease). Tyr phosphorylation of pig VDAC1 and VDAC2 were reported using immunoblotting followed by mass spectrometry (MS) under hypoxia, with no specific site identified (Liberatori et al., 2004). Using anti-Phospho-Tyr antibodies, Schwertz et al. (2007) showed that in rabbits, VDAC1 underwent Tyr phosphorylation during myocardial ischemia by p38 MAPK. ...
The voltage-dependent anion channels (VDACs) are the most abundant proteins present on the outer mitochondrial membrane. They serve a myriad of functions ranging from energy and metabolite exchange to highly debatable roles in apoptosis. Their role in molecular transport puts them on the center stage as communicators between cytoplasmic and mitochondrial signaling events. Beyond their general role as interchangeable pores, members of this family may exhibit specific functions. Even after nearly five decades of their discovery, their role in plant systems is still a new and rapidly emerging field. The information on biochemical regulation of VDACs is limited. Various interacting proteins and post-translational modifications (PTMs) modulate VDAC functions, amongst these, phosphorylation is quite noticeable. In this review, we have tried to give a glimpse of the recent advancements in the biochemical/interactional regulation of plant VDACs. We also cover a critical analysis on the importance of PTMs in the functional regulation of VDACs. Besides, the review also encompasses numerous studies which can identify VDACs as a connecting link between Ca²⁺ and reactive oxygen species signaling in special reference to the plant systems.
... Closing is a slow process and is thus well resolved in a time scale of 1 s ( Fig. 3.5). The opening process occurs in a millisecond time scale and thus seems to occur instantly in This variation may be related to the existence of different forms of VDAC [isoforms (Song et al., 1998b) or splice variants (Liberatori et al., 2004), or other modified versions]. ...
... Regulation of Gnas gave an indication for a higher sympathetic nervous system activity in C3H.Pde6b+ mice. Voltage-dependent anion channel (VDAC) proteins are small, abundant, pore-forming proteins and play an essential role in cellular metabolism and in early stages of apoptosis (39). VDAC1 is involved in permeability transition pore (PTP) activity and/or regulation and thus an important player in retinal degeneration associated with PTP-mediated mitochondrial dysfunction (40). ...
... Liberatori et al has shown that VDAC proteins have an isoform-specific pattern of phosphorylation. Using 2D immunoblotting with anti-VDAC and anti-phosphotyrosine antibodies, they found that one of two spots identified as VDAC1, and two out of three spots identified as VDAC2 were endogenously phosphorylated (VDAC3 was not detected) (Liberatori et al. 2004). The sites of phosphorylation have yet to be determined, but the isoform-specific phosphorylation suggests that the isoforms may have different functional roles and may be involved in different processes. ...
... In line with this, we have recently observed this channel in murine neuronal caveolae in a complex with estrogen receptor alpha (mERα), where it participates in the toxic effect provoked by Aβ exposure (Marin et al., 2007). The modulation of pl-VDAC is largely unknown, and only a few data in cellular models have demonstrated that, in general, Maxi Cl − channels can be modulated by phosphorylation (Liberatori et al., 2004), G-proteins (Schwiebert et al., 1990;McGill et al., 1993;Mitchell et al., 1997), fatty acids (Riquelme and Parra, 1999), amyloid beta (Jalonen et al., 1997), and estrogens and anti-estrogens (Li et al., 2000;Diaz et al., 2001;Valverde et al., 2002), the latter suggesting that estrogen receptors (ERs) may be involved in VDAC functionality at the plasma membrane. ...
Membrane estrogen receptor Caveolae Caveolin-1 Human brain Alzheimer's disease Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERα), which participates in neuroprotection against amyloid beta (Aβ), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERα, in a complex with scaffolding caveolin-1 which likely provides mERα stability at the plasma membrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERα was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Aβ-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.
... In line with this, we have recently observed this channel in murine neuronal caveolae in a complex with estrogen receptor alpha (mERα), where it participates in the toxic effect provoked by Aβ exposure (Marin et al., 2007). The modulation of pl-VDAC is largely unknown, and only a few data in cellular models have demonstrated that, in general, Maxi Cl − channels can be modulated by phosphorylation (Liberatori et al., 2004), G-proteins (Schwiebert et al., 1990;McGill et al., 1993;Mitchell et al., 1997), fatty acids (Riquelme and Parra, 1999), amyloid beta (Jalonen et al., 1997), and estrogens and anti-estrogens (Li et al., 2000;Diaz et al., 2001;Valverde et al., 2002), the latter suggesting that estrogen receptors (ERs) may be involved in VDAC functionality at the plasma membrane. ...
Membrane estrogen receptor Caveolae Caveolin-1 Human brain Alzheimer's disease Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERα), which participates in neuroprotection against amyloid beta (Aβ), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERα, in a complex with scaffolding caveolin-1 which likely provides mERα stability at the plasma membrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERα was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Aβ-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.
... For example proteomic approach has been used by our group to identify mechanisms involved in hypoxic brain injury in guinea pig brain synaptosomes. Voltage-dependent anion channel (VDAC) protein isoforms, selectively undergo hypoxiainduced tyrosine phosphorylation, suggesting that tyrosine phosphorylation may contribute to the modulation of VDAC protein function/conformation or interaction with other proteins in hypoxic conditions [5,6]. ...
... In recent years VDAC was identified as a target of different kinases, such as GSK3β [46], PKA [53], PKCε [49], Nek1 [55], and p38 Map kinase [56]. A few VDAC phosphorylation sites have been identified using a proteomic approach5758. Here we are making an attempt to relate the suggested phosphorylation sites to our functional data on phosphorylated VDAC by mapping them on the available VDAC folding pattern of mouse VDAC1 determined by x-ray crystallography [18] and presented inFig. 5. Pastorino and coauthors [46] suggested that GSK3β phosphorylates VDAC at amino acids 51 to 55 because the mutation of Thr-51 to alanine abolished the ability of GSK3β to phosphorylate VDAC. ...
Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm, and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. For example, one of the long-standing puzzles was that in permeabilized cells, adenine nucleotide translocase is less accessible to cytosolic ADP than in isolated mitochondria. Still another puzzle was that, according to channel-reconstitution experiments, voltage regulation of VDAC is limited to potentials exceeding 30mV, which are believed to be much too high for MOM. We have solved these puzzles and uncovered multiple new functional links by identifying a missing player in the regulation of VDAC and, hence, MOM permeability - the cytoskeletal protein tubulin. We have shown that, depending on VDAC phosphorylation state and applied voltage, nanomolar to micromolar concentrations of dimeric tubulin induce functionally important reversible blockage of VDAC reconstituted into planar phospholipid membranes. The voltage sensitivity of the blockage equilibrium is truly remarkable. It is described by an effective "gating charge" of more than ten elementary charges, thus making the blockage reaction as responsive to the applied voltage as the most voltage-sensitive channels of electrophysiology are. Analysis of the tubulin-blocked state demonstrated that although this state is still able to conduct small ions, it is impermeable to ATP and other multi-charged anions because of the reduced aperture and inversed selectivity. The findings, obtained in a channel reconstitution assay, were supported by experiments with isolated mitochondria and human hepatoma cells. Taken together, these results suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC interaction with tubulin at the mitochondria-cytosol interface. Immediate physiological implications include new insights into serine/threonine kinase signaling pathways, Ca(2+) homeostasis, and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation. In the present review, we speculate how these findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect). This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
... There are emerging data that VDAC is a target of different kinases, in addition to GSK3band PKA, such as Nek1 [24], PKCe [25], p38 MAP kinase [26], and endostatin reduced hexokinase 2 [27]. A few VDAC post-translational phosphorylation sites have been identified using proteomic approach [28,29] . This phosphorylation contributes to the VDAC total phosphorylation level and could be involved in different regulatory processes. ...
Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.
... The Edman degradation of the corresponding peptide should have had revealed the presence of VDAC2 contamination but it did not, indicating that the protein purified in that experiment was only VDAC1. Also the few reports showing 2-D analysis of purified VDAC1 did not show the presence of alternative spots later found to be VDAC2 or VDAC3 [35,36]. ...
VDACs (Voltage Dependent Anion selective Channels) are a family of pore-forming proteins discovered in the mitochondrial outer membrane. In the animal kingdom, mammals show a conserved genetic organization of the VDAC genes, corresponding to a group of three active genes. Three VDAC protein isoforms thus exist. From a historically point of view most of the data collected about this protein refer to the VDAC1 isoform, the first to be identified and also the most abundant in the organisms. In this work we compare the information available about the three VDAC isoforms, with a special emphasis upon the human proteins, here considered prototypical of the group, and we try to shed some light on specific functional roles of this apparently redundant group of proteins. A new hypothesis about the VDAC(s) involvement in ROS control is proposed. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
... In this regard, it is worth noting that excepting DD16, a protein that has been scarcely studied, all differential proteins identified in the present report are proteins that had been previously reported to change in different cell death paradigms or to be related to cell death mechanisms. Four of these proteins (HSC70, malate dehydrogenase, NDRG1 and VDAC1) showed altered levels in hypoxia [33][34][35][36] and five of them, HSC70 [37,38], calretinin [39], coronin 1a [40], PHB2 [41] and TCTP [42][43], have been reported to be able to rescue cells from cell death. Two of the identified proteins (malate dehydrogenase and VDCA1) have been reported to induce cell death [44,45], and the effect of ENO1 and NDGR1 on cell fate might be dependent on concentration or PTMs of the protein [46][47][48][49]. ...
Cell death induced by over-activation of glutamate receptors occurs in different neuropathologies. Cholesterol depletors protect from neurotoxic over-activation of glutamate receptors, and we have recently reported that this neuroprotection is associated with a reduction of the N-methyl-D-aspartate subtype of glutamate receptors in detergent-resistant membrane domains (DRM). In the present study we used comparative proteomics to further identify which proteins, besides the N-methyl-D-aspartate receptor, change its percentage of association to DRM after treatment of neurons with simvastatin. We detected 338 spots in neuronal DRM subjected to 2-DE; eleven of these spots changed its intensity after treatment with simvastatin. All 11 differential spots showed reduced intensity in simvastatin-treated samples and were identified as adipocyte plasma membrane associated protein, enolase, calretinin, coronin 1a, f-actin capping protein alpha1, f-actin capping protein alpha2, heat shock cognate protein 71, malate dehydrogenase, n-myc downregulated gene 1, prohibitin 2, Rab GDP dissociation inhibitor, translationally controlled tumor protein and voltage dependent anion selective channel protein 1. The proteins tested colocalized with the lipid raft marker caveolin-1. Interestingly, the proteins we have identified in the present study had been previously reported to play a role in cell fate and, thus, they might represent novel targets for neuroprotection.
... Once properly denatured, these proteins are fairly soluble in the conditions prevailing in IEF, and can thus be analyzed with high resolution. this is true for bacterial porins [41] but also for eukaryotic porins [42]. ...
The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field.
... In addition, protein kinase C epsilon binds and phosphorylates VDAC, an effect causing inhibition of the mitochondrial permeability transition [10]. VDAC isoforms in guinea pig brain synaptosomes also undergo tyrosine phosphorylation in response to hypoxia [52]. Thus, phosphorylation may be an important regulator of VDAC function. ...
Despite a detailed understanding of their metabolism, mitochondria often behave anomalously. In particular, global suppression of mitochondrial metabolism and metabolite exchange occurs in apoptosis, ischemia and anoxia, cytopathic hypoxia of sepsis and multiple organ failure, alcoholic liver disease, aerobic glycolysis in cancer cells (Warburg effect) and unstimulated pancreatic beta cells. Here, we propose that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane accounts for global mitochondrial suppression. In anoxia, cytopathic hypoxia and ethanol treatment, reactive oxygen and nitrogen species, cytokines, kinase cascades and increased NADH act to inhibit VDAC conductance and promote selective oxidation of membrane-permeable respiratory substrates like short chain fatty acids and acetaldehyde. In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin-secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. We propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release. In support of the overall proposal, ethanol treatment of isolated rat hepatocytes inhibited mitochondrial respiration and accessibility to adenylate kinase in the intermembrane space, effects that were overcome by digitonin permeabilization of the outer membrane. Overall, these considerations suggest that VDAC is a dynamic regulator, or governator, of global mitochondrial function both in health and disease.
... These proteins have been shown to be involved in the loss of mitochondrial potential and neuronal degeneration during ischemia reperfusion. Liberatori et al. [179] examined the expression pattern of VDAC isoforms in guinea pig brain synaptosomes, under both normoxic and hypoxic conditions. Hypoxia ischemia was also studied on rats in correlation with proteomics [68,127,170]. ...
Proteomics technologies have been widely used in the investigation of neurodegenerative and psychiatric disorders, and in particular in the detection of differences between healthy individuals and patients suffering from such diseases. Thus, brain and cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease, Down syndrome, Pick's disease, Parkinson's disease, schizophrenia, and other disorders as well as brain and CSF from animals serving as models of neurological disorders have been analyzed by proteomics. 2-DE followed by MALDI-TOF-MS has been mainly applied as this proteomics approach provides the possibility of convenient quantification of protein levels and detection of post-translational modifications. About 330 unique proteins with deranged levels and modifications have been detected by proteomics approaches to be related to neurodegeneration and psychiatric disorders. They are mainly involved in metabolism pathways, cytoskeleton formation, signal transduction, guidance, detoxification, transport, and conformational changes. In this article, we provide a summary of the major contributions of proteomics technologies in the study of neurodegenerative and psychiatric diseases, in particular, in the detection of changes in protein levels and modifications related to these disorders.
... VDAC is phosphorylated by protein kinase A (PKA) (22), protein kinase Cε (PKCε) (23), and tyrosine kinases (24). The phosphorylation of mitochondrial proteins by PKCε was correlated with the inhibition of PTP opening and may be related to protection against ischemic injury (23). ...
In work spanning more than a century, mitochondria have been recognized for their multifunctional roles in metabolism, energy transduction, ion transport, inheritance, signaling, and cell death. Foremost among these tasks is the continuous production of ATP through oxidative phosphorylation, which requires a large electrochemical driving force for protons across the mitochondrial inner membrane. This process requires a membrane with relatively low permeability to ions to minimize energy dissipation. However, a wealth of evidence now indicates that both selective and nonselective ion channels are present in the mitochondrial inner membrane, along with several known channels on the outer membrane. Some of these channels are active under physiological conditions, and others may be activated under pathophysiological conditions to act as the major determinants of cell life and death. This review summarizes research on mitochondrial ion channels and efforts to identify their molecular correlates. Except in a few cases, our understanding of the structure of mitochondrial ion channels is limited, indicating the need for focused discovery in this area.
... 14 It has been suggested that the observed hypoxia-induced tyrosine phosphorylation of VDAC1 and VDAC2 might be crucial for the modulation of proteineprotein interactions both in metabolic and proapoptotic/anti-apoptotic equilibrium, leading to activation/ inactivation of cellular processes related to cellular apoptosis, neuronal degeneration or survival. 14 Under conditions of stress, the mitochondrial permeability transition pore in the mitochondrial inner membrane, which allows the free passage of any molecule of <1.5 kDa, can open. 15,16 This loss of the inner membrane permeability barrier allows all small-molecular-weight solutes to move freely across the membrane, which uncouples oxidative phosphorylation. ...
Oxidative stress plays an important role in causing organ injury in the compromised fetus and neonate. Recent experimental research and clinical studies have clarified important pathways in the production of reactive oxygen and nitrogen species. Free radicals are involved in causing cerebral damage after perinatal hypoxia-ischemia affecting membrane lipids, proteins, and DNA. Anti-oxidant strategies can be used as add-on neuroprotective therapy after perinatal oxidative stress. Selective inhibitors of neuronal and inducible nitric oxide synthase, allopurinol, melatonin, and erythropoietin are among the first compounds that are ready for clinical trials.
... Even though the role of this channel at the plasma membrane has not been still elucidated, some recent data have suggested a dual action, on one hand, to participate in the maintenance of normal redox homeostasis and, on the other hand, to modulate apoptotic events (Elinder et al. 2005). The modulation of VDAC at the plasma membrane level is still largely unknown, and only a few data in a variety of cell types have suggested that overall Maxi Cl ( channels may be modulated by different factors such as unsaturated fatty acids (Riquelme & Parra 1999), GTP-binding proteins (Schwiebert et al. 1990, McGill et al. 1993, nucleotides (Schwiebert et al. 1992, Mitchell et al. 1997, phosphorylation (Pahapill & Schlichter 1992, Liberatori et al. 2004, and direct interaction with hormones and anti-hormones (Kajita et al. 1995, Li et al. 2000, Díaz et al. 2001, Valverde et al. 2002. ...
Voltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria and participation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane, some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normal redox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDAC antibodies prior to amyloid beta (Abeta) peptide was observed to prevent neurotoxicity. In these cell lines, we identified a VDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor alpha (mERalpha) (80 and 67 kDa), known in SN56 cells to participate in estrogen-induced neuroprotection against Abeta injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the first time that both VDAC and mERalpha interact at the plasma membrane of these neurons as well as in microsomal fractions of the corresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, thereby corroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERalpha association at the plasma membrane level may participate in the modulation of Abeta-induced cell death.
CAMP is an important messenger in neurohormonal regulation of the heart. By activating its effectors, cAMP regulates many cellular functions such as gene expression, excitation-contraction coupling and cellular metabolism. In mammals, cAMP is produced by a family of adenylyl cyclase with various subcellular locations and membrane anchorage. The existence and role of cyclic nucleotide signaling in mitochondria has been postulated, but has not yet been demonstrated. Moreover, its implication in the regulation of cell death is still unknown. In this thesis, we demonstrated the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1) and showed a protective role for sACt against cell death, apoptosis as well as necrosis, in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3-) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increased the mitochondrial membrane potential (∆Ψm) and ATP production. cAMP is rate-limiting for matrix Ca2+ entry via the mitochondrial calcium uniporter (MCU) and, as a consequence, prevented mitochondrial permeability transition (MPT). In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3- rescued the sensitization of mitochondria to Ca2+-induced MPT. We also found that PDE2, 3 and 4 families are located in cardiac mitochondria. They form a local signaling pathway with soluble AC in the matrix, which regulates cardiac mitochondrial functions. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism, some PDEs and cell death in the heart, which is independent of cytosolic cAMP signaling. This might constitute a novel cardioprotective mechanism through mitochondrial function preservation in pathophysiological conditions.
Voltage dependent anion channels (VDAC) are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through mitochondrial membranes. The functional role of VDAC has been investigated in many studies, and different functions of VDAC have been shown.
Posttranslational modifications of VDAC are significant for its regulation. The aim of our research was to develop approach for characterization of primary structure and posttranslational modifications of VDACs and other membrane proteins.
Mouse brain membranes were isolated from mouse brains by differential centrifugation. Primary structure of mitochondrial isoform VDAC1 from mouse brain membranes has been identified almost completely (95%, 258 of 283 amino acids) by combination of SDS-PAGE and LTQ-FTMS mapping of peptide mixtures after proteolytic degradation with trypsin. Sequence of each found peptide of VDAC has been analysed and confirmed according to accurate mass, isotopic distribution and MS/MS tandem analysis. Posttranslational modifications of VDAC’s peptides have been shown. High sequence coverage of VDAC has been
obtained, including 11 transmembranes domains. Extensive sequence coverage has been also detected for some other proteins at 30-34 kDa. A repressor of estrogen receptor activity has been identified with 76% coverage, malate dehydrogenase with 55 % sequence coverage, syntaxin 1A and syntaxin 1B2 have been sequenced
with 60% and 65% coverage, respectively. These results demonstrated that mass spectrometric mapping is reliable and sensitive approach for characterization of primary structure membrane proteins and identification of their posttranslational modification.
This review presents current knowledge related to VDAC1 as a multi-functional mitochondrial protein acting on both sides of the coin, regulating cell life and death, and highlighting these functions in relation to disease. It is now recognized that VDAC1 plays a crucial role in regulating the metabolic and energetic functions of mitochondria. The location of VDAC1 at the outer mitochondrial membrane (OMM) allows the control of metabolic cross-talk between mitochondria and the rest of the cell and also enables interaction of VDAC1 with proteins involved in metabolic and survival pathways. Along with regulating cellular energy production and metabolism, VDAC1 is also involved in the process of mitochondria-mediated apoptosis by mediating the release of apoptotic proteins and interacting with anti-apoptotic proteins. VDAC1 functions in the release of apoptotic proteins located in the mitochondrial intermembrane space via oligomerization to form a large channel that allows passage of cytochrome c and AIF and their release to the cytosol, subsequently resulting in apoptotic cell death. VDAC1 also regulates apoptosis via interactions with apoptosis regulatory proteins, such as hexokinase, Bcl2 and Bcl-xL, some of which are also highly expressed in many cancers. This review also provides insight into VDAC1 function in Ca2+ homeostasis, oxidative stress, and presents VDAC1 as a hub protein interacting with over 100 proteins. Such interactions enable VDAC1 to mediate and regulate the integration of mitochondrial functions with cellular activities. VDAC1 can thus be considered as standing at the crossroads between mitochondrial metabolite transport and apoptosis and hence represents an emerging cancer drug target.
Laryngeal carcinoma related gene LCRG1, cloned by the laboratory using mRNA differential display, has the suppressive function to none expression LCRG1 Hep-2 cell line. Bioinformatics analysis using software showed LCRG1 may play function in cellular signal transduction. In order to further elucidate the function of LCRG1, RT-PCR and colony efficiency were used to identify whether LCRG1 expressed and had the tumor suppressive function in incubated Hep-2/LCRG1 cell lines. The results suggested LCRG1 was expressed in Hep-2/LCRG1 cell lines and had the significant suppressive proliferation ability. Hence, the total proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1 (+) cell lines were separated by immobilized pH gradient (IPG)-based two-dimensional gel electrophoresis(2DGE), coupled with anti-tyrosine phosphorylated antibody immunoblotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) identifying tyrosine-phosphorylated proteins. The well-resolved, reproducible 2DGE patterns of Hep-2/LCRG1 and Hep-2/pcDNA3.1 (+) cell lines were established and 13 differential tyrosine-phosphorylated proteins were identified using immunoblotting, analysis software and MALDI-TOF-MS methods. These proteins were involved in the signal transduction and cell cycle. So it was speculated that LCRG1 may be involved in the processes of cellular proliferation, metabolic pathways and apoptosis etc. and play tumor suppressive functions through regulating the phosphorylation/ dephosphorylation status of these proteins. These data will be helpful to elucidate the molecular mechanism of LCRG1 tumor suppressive function.
Lifelong disabilities such as cerebral palsy, epilepsy, and behavioral and learning disorders are some of the consequences of brain injury acquired in fetal life or the perinatal and neonatal periods. Although there are multiple etiologies for neonatal encephalopathy in the newborn period, hypoxia-ischemia is largely recognized as the major cause of brain damage and subsequent neurologic disability. Perinatal deficiency in oxygen supply can be due to two major mechanisms-namely, hypoxemia and ischemia. It is becoming more evident that hypoxia-ischemia is the final common endpoint for a complex convergence of events, some genetically determined and some triggered by an in utero (but not necessarily intrapartum) stressor. At the cell level, cerebral hypoxia-ischemia sets in motion a cascade of biochemical events commencing with a shift from oxidative to anaerobic metabolism, which leads to an accumulation of NADH, FADH, lactic acid, and H+ ions. The chapter explains various mechanisms involved in hypoxic brain injury, including those involving free iron, oxidative stress (OS), inflammatory cytokines, excitotoxicity, or infection. Neonatologists have several methods of determining whether brain damage occurred in the prenatal, perinatal, or neonatal period.
Immunoblotting technique, also dubbed “Western blotting,” exploits antibody specificity to identify target proteins among a number of unrelated protein species (1,2). Traditionally used for mapping known proteins on electrophoretically-resolved mixtures, in the proteome era immunoblotting has been combined with 2-D gel electrophoresis for rapid visualization and identification of target proteins (3), protein adducts with drugs (4) and antigenic proteins associated to pathogens, allergens as well as tumours, with patient serum used as primary antibody (5–9). In the postgenomic era, gene expression is becoming a focus of attention and it is widely accepted that one gene does not necessarily encode a single protein product, as molecular mechanisms generating different isoforms from the same gene have been described in many different organisms. Although the relation between number of genes and number of potentially encoded proteins has yet to be clarified, it is usually recognized that the number of polypeptides expressed by a genome is greater than expected on the basis of calculated gene numbers. In fact, it has been estimated that the average number of alternates spliced from the transcript of a single mammalian gene may be two to three or more (10) and that many proteins may be subject to co- and posttranslational modifications and proteolytic processing. For example, up to 20% of protein is acetylated in yeast (11). Several preliminary proteome projects have also shown that prokaryotic organisms can express more than one protein isoform from a single gene and posttranslationally modified gene products have been reported (12).
The voltage-dependent anion channel (VDAC) or porin is a major membrane protein integrated into the mitochondrial outer membrane in eukaryotes. It is encoded as three isoforms (VDAC1 to 3), which play differential roles in metabolism and cell death. As a channel, VDAC mediates metabolites, ions and water movements trough the outer membrane in physiological conditions, but it can also participate to mitochondrial membrane permeabilization, an apoptotic checkpoint in stress and pathological conditions. Indeed, due to its subcellular location, VDAC interacts with many molecules as diverse as NAD+, lipids and cytosolic proteins such as hexokinase, tubulin, GSK3, Bax and Bcl-2 family members and mitochondrial proteins, such as the adenine nucleotide translocase (ANT). All these interactions can influence VDAC role in cell fate determination. In the recent past, major efforts focused on VDAC1 channel function and regulation by calcium and reactive oxygen species, and comparatively, fewer studies have been undertaken on VDAC2 and 3 and their pathophysiological involvement. Here, we review recent insights into the role of VDAC isoforms in cell death, its regulation by phosphorylation or protein-lipid interactions and discuss the putative consequences of this post-translational modification on cell fate, notably in the context of lipid accumulation. This might have important implications for the understanding of basic mechanisms of mitochondrial lipid sensing and might contribute to define novel therapeutic target for future investigation.
G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite to understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.
Introduction Oxidant-Sensitive Cys Challenges in Redox Proteomics Iodoacetamide-Based Redox Proteomics ICAT Approach to Redox Proteomics Validation of the ICAT Approach Using the Recombinant Protein Creatine Kinase ICAT Approach to the Complex Protein Mixtures Perspectives
Depression is a severe and life-threatening psychiatric illness whose pathogenesis is still essentially unknown. Proteomic
analysis of synaptic terminals (synaptoproteomics) in animal models of depression is a powerful approach to gain insight into
the molecular mechanisms underlying vulnerability to mood disorders and the long-term action of drug treatments. Here, we
employed two different animal models of depression, the Learned Helplessness rats (a classical behavioral model of depression)
and a new model of depression with gene—environment interaction (Flinders Sensitive Line rats subjected to early life stress).
Both animal models were treated with the antidepressant escitalopram. Analysis of their synaptoproteomic profile revealed
a number of protein spots differently regulated by basic vulnerability and/or early life stress. Using this approach, we obtained
information regarding biomarkers that may represent predictors of pathology or response/resistance to drug treatment, as well
as potential targets for novel pharmacological and therapeutic strategies.
There is a wealth of information indicating that estradiol exerts rapid actions involved in neuroprotection and cognitive-enhancing effects. Some of these effects appear to delay onset, or even ameliorate, the neuropathology of Alzheimer's disease (AD), although some controversy exists about the beneficial brain effects of estrogen therapies. Therefore, it is crucial to better understand the mechanisms developed by 17β-estradiol to signal in the brain. At the neuronal membrane, the hormone can rapidly interact with estrogen receptors (mERs) or activate other receptors, such as G protein-coupled and ionotropic receptors. And the list of membrane signalling molecules modulated by estradiol in neurons is increasing. VDAC is a voltage-dependent anion channel, known as a mitochondrial porin which is also found at the neuronal membrane, where it appears to be involved in redox regulation, extrinsic apoptosis and amyloid beta neurotoxicity. Moreover, VDAC is present in neuronal lipid rafts, where it is associated with estrogen receptor α-like (mER), forming part of a macromolecular complex together with caveolin-1 and other signalling proteins related to neuronal preservation. Interestingly, we have recently found that 17β-estradiol rapidly promotes VDAC phosphorylation through the activation of protein kinase A (PKA) and Src-kinase, which may be relevant to maintain this channel inactivated. On the contrary, tamoxifen, a selective estrogen receptor modulator (SERM), provokes the dephosphorylation of VDAC, and eventually its opening, by activating a cascade of phosphatases, including protein phosphatase 2 (PP2A). This review will focus on the relevance of these novel findings in the alternative estrogen mechanisms to achieve neuroprotection related to AD.
Although depression is a severe and life-threatening psychiatric illness, its pathogenesis still is essentially unknown. Recent studies highlighted the influence of environmental stress factors on an individual’s genetic predisposition to develop mood disorders. In the present study, we employed a well-validated stress-induced animal model of depression, Learned Helplessness paradigm, in rats. Learned helpless (LH) and non-learned helpless (NLH) rats were treated with nortriptyline, a tricyclic antidepressant. The resulting 4 groups (LH vs. NLH, treated vs. non-treated), were subjected to global analysis of protein expression, a powerful approach to gain insight into the molecular mechanisms underlying vulnerability to psychiatric disorders and the long-term action of drug treatments. Many of the biological targets of antidepressant drugs are localized at synapses. Thus, to reduce the complexity of the proteome analyzed and to enrich for less abundant synaptic proteins, purified nerve terminals (synaptosomes) from prefrontal/frontal cortex (P/FC) and hippocampus (HPC) of LH-NLH rats were used. Synaptosomes were purified by differential centrifugation on Percoll gradients and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-DE). Protein spots differently regulated in the various comparisons were excised from gels and identified by mass spectrometry. Proteins involved in energy metabolism and cellular remodeling were primarily dysregulated, when LH and NLH rats were compared. Moreover, several proteins (aconitate hydratase, pyruvate dehydrogenase E1, dihydropyrimidinase-related protein-2 and stathmin) were found to be regulated in opposite directions by stress and drug treatment. These proteins could represent new molecular correlates of both vulnerability to stress and response to drugs, and putative targets for the development of novel drugs with antidepressant action.
VDAC (voltage-dependent anion channel) ist ein kleines, Poren-formendes Protein, welches synonym als Porin bezeichnet wird. Es kommt sowohl in Bakterien, als auch in der äußeren mitochondrialen Membran sowie in der Plasmamembran somatischer Zellen vor.
Die Ziele dieser Arbeit waren 1.) Reinigung spezifischer anti-VDAC Antikörper und deren Charakterisierung bezüglich ihrer Affinität und ihrer Isoform-Spezifität, 2.) Lokalisation von VDAC1, 2, und 3 in bovinen ejakulierten Spermatozoen und 3.) Untersuchungen zur funktionellen Bedeutung von VDAC für die Spermatozoenmotilität und bezüglich des akrosomalen Status mit Hilfe der anti-VDAC Antikörper.
Die Spezifität der Antikörper wurde durch Immunoblots mit rekombinantem VDAC1, 2 und 3 überprüft. Soweit bekannt, sind mit dieser Arbeit die ersten mono-spezifischen AK gegen alle drei VDAC Isoformen charakterisiert worden.
Die immunzytochemischen Untersuchungen zeigten, dass VDAC1 im akrosomalen Bereich und in der fibrösen Hülle des Flagellum-Hauptstücks lokalisiert werden konnte. VDAC2 wurde im Flagellum und VDAC3 in den Mantelfasern (ODF = outer dense fibers) detektiert. Die Präsenz von VDAC2 und 3 in den ODF konnte mit Hilfe des Immunoblots bestätigt werden. Alle drei VDAC Isoformen konnten somit in bovinen Spermatozoen lokalisiert werden und zwar wie beschrieben in unterschiedlichen Kompartimenten. Der Nachweis von VDAC2 und 3 in den ODF ist zugleich der erstmalige Nachweis von VDAC in einer nicht membranären Struktur. Dies lässt die Hypothese zu, dass es sich bei VDAC nicht nur um ein Kanal bildendes Protein handeln könnte und es in ODF eine andere Funktion als in Mitochondrien oder Plasmamembranen haben könnte.
Die Analyse der Spermatozoenmotilität zeigte nach Inkubation mit anti-VDAC2 Antikörper eine statistisch signifikante Abnahme der Gesamtmotilität. Für diesen Effekt konnte eine Dosis-Wirkungs-Abhängigkeit gezeigt werden.
Weiterhin riefen alle drei anti-VDAC Antikörper unterschiedlich stark ausgeprägte morphologische Veränderungen im akrosomalen Bereich des Kopfes hervor: Nach 4h Inkubation zeigte sich ein höherer Verlust an intakten Akrosomen im Vergleich zur Kontrolle. VDAC scheint somit wichtig für die Spermatozoenmotilität und die morphologische Integrität des Akrosoms zu sein.
In der Rasterelektronenmikroskopie konnten die durch anti-VDAC Antikörper hervorgerufene Veränderungen der Spermatozoenköpfe in Form von Membranablösungen und einer Bläschenbildung im Bereich des Akrosoms bestätigt werden. Die distalen Anteile des Flagellums waren häufig eingerollt. Dies sind Hinweise darauf, dass VDAC in die Volumenregulation von Spermatozoen involviert sein könnte.
Weitere Untersuchungen sind notwendig, um die die physiologische Bedeutung von VDAC in Spermatozoen näher zu definieren. VDACs (voltage-dependent anion channels) or porins are small pore-forming proteins first identified in outer mitochondrial membranes. VDAC was also found in bacterial membranes as well as in the plasma membrane of somatic cells.
The aims of this study were 1) to purify and subsequently characterise specific anti-VDAC antibodies, 2) to localise VDAC1, 2 and 3 in ejaculated bovine spermatozoa and 3) to determine the effect of anti-VDAC antibodies upon bovine sperm function, e.g. motility and acrosomal status.
The specificity of the antibodies was demonstrated by immunoblotting using recombinant VDAC1, 2 and 3. The results obtained in this thesis revealed that monospecific antibodies for all three VDAC isoforms were generated. Immunofluorescence microscopy suggests that VDAC1 protein is present in the sperm acrosome and in the fibrous sheath of the flagellum. The data showed that anti-VDAC2 and anti-VDAC3 antibodies bound to the sperm flagellum, in particular to the ODF (= outer dense fibers). The presence of VDAC2 and 3 in ODF was strengthened by immunoblotting results. All three VDAC isoforms could be identified in bovine spermatozoa and were located in different sperm compartments. Extra membranous localisation of VDAC2 and 3 in the ODF of the sperm flagellum could be demonstrated for the first time. We propose that VDAC might have different physiological functions in ODF than in mitochondria or cell plasmamembranes.
Sperm motion analysis after incubation with anti-VDAC2 antibodies yielded a decrease of sperm motility. A dose-dependency could be demonstrated for this effect. After incubation of spermatozoa with anti-VDAC antibodies it could be observed that antibodies against all three VDAC isoformes caused morphological alterations at the acrosome that finally led to an acrosomal loss.
These morphological changes of the acrosome observed in light microscopy studies could be confirmed by scanning electron microscopical studies. Furthermore, morphological alterations of the sperm tail were apparent. Pig tail-like structures at the tip of the flagellum, commonly seen when spermatozoa are subjected to hypoosmotic conditions, suggest that VADAC might be involved in sperm volume regulation.
It can be concludeed that VDAC Isoformes in bovine spermatozoa are involved in regulation of different sperm functions, e.g. the maintenance of acrosomal integrity and possibly in sperm volume regulation. However, the potential functional roles of VDACs in spermatozoa need to be evaluated further and will be subject of future scientific investigations.
Despite decades of extensive research, the large-scale analysis of membrane proteins remains a difficult task. This is due to the fact that membrane proteins require a carefully balanced hydrophilic and lipophilic environment, which optimum varies with different proteins, while most protein chemistry methods work mainly, if not only, in water-based media. Taking this review [Santoni, Molloy and Rabilloud, Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21, 1054-1070] as a pivotal paper, the current paper analyzes how the field of membrane proteomics exacerbated the trend in proteomics, i.e. developing alternate methods to the historical two-dimensional electrophoresis, and thus putting more and more pressure on the mass spectrometry side. However, in the case of membrane proteins, the incentive in doing so is due to the poor solubility of membrane proteins. This review also shows that in some situations, where this solubility problem is less acute, two-dimensional electrophoresis remains a method of choice. Last but not least, this review also critically examines the alternate approaches that have been used for the proteomic analysis of membrane proteins.
An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.
Immunoblotting techniques use antibodies (or other specific ligands in related techniques) to identify target proteins among a number of unrelated protein species. They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions. Proteins are typically separated by electrophoresis and transferred onto membranes (usually nitrocellulose). The membrane is overlaid with a primary antibody for a specific target and then with a secondary antibody labeled, for example, with enzymes or with radioisotopes. When the ligand is not an antibody, the reaction can be visualized using a ligand that is directly labeled. Dot blot is a simplified procedure in which protein samples are not separated by electrophoresis but are spotted directly onto membrane. Immunoblotting is now widely used in conjunction with two-dimensional polyacrylamide gel electrophoresis, not only for traditional goals, such as the immunoaffinity identification of proteins and analysis of immune responses but also as a genome-proteome interface technique.
Proteomics methodologies hold great promise in basic renal research and clinical nephrology. The classical approach for proteomic analysis couples two-dimensional gel electrophoresis (2-DE) with protein identification by mass spectrometry, to produce more global information regarding normal protein expression and alterations in different physiological and pathological states. In this report we have expanded the identification of proteins in the renal cortex, improving the previously published map to facilitate the study of different diseases affecting the human kidney. About 250 spots were analyzed by peptide mass fingerprinting, 89 proteins and 74 isoforms for some of them were identified and implemented in the normal human renal cortex 2-DE reference map. This more comprehensive view of the proteome of the human renal cortex could be of invaluable help to the differential proteomic display of urological diseases.
The synucleins are a family of presynaptic proteins that are abundant in neurons and include alpha-, beta, and gamma-synuclein. Alpha-synuclein (ASN) is involved in several neurodegenerative age-related disorders but its relevance in physiological aging is unknown. In the present study we investigated the expression of ASN mRNA and protein in the different brain parts of the adult (4-month-old) and aged (24-month-old) rats by using RT-PCR technique and Western blot, respectively. Our results indicated that mRNA expression and immunoreactivity of ASN is similar in brain cortex, hippocampus and striatum but markedly lower in cerebellum comparing to the other brain parts. Aging lowers ASN mRNA expression in striatum and cerebellum by about 40%. The immunoreactivity of ASN in synaptic plasma membranes (SPM) from aged brain cortex, hippocampus and cerebellum is significantly lower comparing to adult by 39%, 24% and 65%, respectively. Beta-synuclein (BSN) was not changed in aged brain comparing to adult. Age-related alteration of ASN may affect the nerve terminals structure and function.
Mitochondria are multifunctional organelles that participate in a range of cellular processes such as energy production, proliferation, death, and senescence. The involvement of mitochondria in such distinct aspects of cell life requires the existence of an integrated system of signals that enter and exit the organelle according to the diverse needs of the cell. The recent discovery of several protein kinases and phosphatases that localize partially or predominantly inside mitochondria opens new perspectives into the regulation of these signals. This review focuses on tyrosine phosphorylation in mitochondria. A description of the protein tyrosine kinases and phosphatases which regulate this process along with the mitochondrial tyrosine-phosphorylated proteins identified to date is followed by a discussion of the possible involvement of tyrosine phosphorylation in mitochondrial signaling and future perspectives for developments in this emerging field.
In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [alpha-(32)P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [alpha-(32)P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of approximately 17- and approximately 14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotide-binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.
Conventional two-dimensional electrophoresis (2DE) is the main technique used for protein profiling of tissues and cells, however separation of strongly acidic, basic or highly insoluble proteins is still limited. A series of methods have been proposed to cope with this problem and the use of discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) with subsequent SDS-PAGE followed by mass spectrometry showed that results from 2DE can be complemented by this approach. It was the aim of this study to separate and identify proteins from whole mouse brain that were not demonstrated by 2DE. For this purpose samples were homogenised, soluble proteins were removed by ultracentrifugation and the water-insoluble pellet was resuspended in a mixture containing urea, 16-BAC, glycerol, pyronine Y and dithiothreitol. Electrophoresis was run in the presence of 16-BAC, the strip from the gel containing separated proteins was cut out and was re-run on SDS-PAGE. Protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. One hundred and six individual proteins represented by 187 spots were unambiguously identified consisting of 42 proteins with predicted pI values of pI>8.0, 25 with a 6.0<pI<8.0 and 39 with a pI<6.0. Twelve proteins with transmembrane domains (ranging from 1 to 8) including channels and carriers were identified. The generated map revealed a series of important brain proteins that were not separated and identified previously. Therefore, this system may be relevant for protein chemical determination of channels and carriers independent of antibody availability and specificity. The fact that transmembrane, basic, acidic as well as hydrophobic proteins with a positive Gravy Index can be resolved warrants work on further improvement of this analytical tool.
G3139, an 18-mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA, can induce caspase-dependent apoptosis via the intrinsic mitochondrial pathway in 518A2 and other melanoma cells. G3139-mediated apoptosis appears to be independent of its ability to down-regulate the expression of Bcl-2 protein, because the release of mitochondrial cytochrome c precedes in time the down-regulation of Bcl-2 protein expression. In this study, we demonstrate the ability of G3139 and other phosphorothioate oligonucleotides to bind directly to mitochondria isolated from 518A2 cells. Furthermore, we show that this interaction leads to the release of cytochrome c in the absence of a mitochondrial membrane permeability transition. Our data further demonstrate that there is an interaction between G3139 and VDAC, a protein that can facilitate the physiologic exchange of ATP and ADP across the outer mitochondrial membrane. Evidence from the electrophysiologic evaluation of VDAC channels reconstituted into phospholipid membranes demonstrates that G3139 is capable of producing greatly diminished channel conductance, indicating a closed state of the VDAC. This effect is oligomer length-dependent, and the ability of phosphorothioate homopolymers of thymidine of variable lengths to cause the release of cytochrome c from isolated mitochondria of 518A2 melanoma cells can be correlated with their ability to interact with VDAC. Because it has been suggested that the closure of VDAC leads to the opening of another outer mitochondrial membrane channel through which cytochrome c can transit, thus initiating apoptosis, it appears that VDAC may be an important pharmacologic target of G3139.
• cytochrome c
• mitochondria
• phosphorothioate
Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.
The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3.
We have cloned a protein from bovine brain, brain-derived voltage-dependent anion channel 1 (BR1-VDAC), that is identical to a recently sequenced plasmalemmal-bound porin from human lymphocytes. mRNA hybridization indicates that BR1-VDAC is widely distributed throughout nervous and nonnervous tissues. In situ localization substantiated that the BR1-VDAC is associated with the plasmalemma of astrocytes. A monoclonal antibody that recognizes the N terminus of the BR1-VDAC protein completely blocks an astrocytic high-conductance anion channel that has electrophysiological similarities with the mitochondrial VDAC. Since the high-conductance anion channel in astrocytes has been shown to respond to hypoosmotic solutions, its molecular identification provides the basis for a better understanding of volume regulation in brain tissue.
The voltage-dependent anion channel of the outer mitochondrial membrane, VDAC (also known as mitochondrial porin), is a small abundant protein which forms a voltage-gated pore when incorporated into planar lipid bilayers. This protein forms the primary pathway for movement of major metabolites through the outer membrane. Recently, it has been demonstrated that two human VDAC genes, HVDAC1 and HVDAC2, produce three proteins that differ most significantly at their amino termini. These results suggest that the distinct amino termini lead to the targeting of individual VDAC isoforms to different cellular compartments. Consistent with this hypothesis, recent reports suggest that HIV-DAC1 is found in the plasma membrane of mammalian cells. To define the subcellular location of HVDAC isoforms, HVDAC genes were modified so that the encoded proteins contain COOH-terminal epitopes recognized by either of two monoclonal antibodies. Introduction of these epitope tags had no effect on the function of modified VDAC proteins. Epitope-tagged proteins were then individually expressed in COS7 cells or rat astrocytes and the intracellular location of each isoform subsequently identified by subcellular fractionation, light level immunofluorescence, and immunoelectron microscopy. Our results demonstrate that each HVDAC protein is exclusively located in fractions or subcellular regions containing mitochondrial marker proteins. In addition, immunofluorescence and immunoelectron microscopy show that an individual mitochondrion can contain both HVDAC1 and HVDAC2. Our results call into question previous reports demonstrating VDAC molecules in the plasma membrane and suggest that functional differences between individual VDAC isoforms may result in distinct regulatory processes within a single mitochondrion.
Pores formed in the outer membrane of mitochondria by mitochondrial porin make it permeable to water-soluble metabolites smaller than approximately 5 kDa. We have isolated a full-length cDNA encoding a human porin. This probe detects a single approximately 1.5-kilobase mRNA species on Northern blots, but multiple hybridizing bands on genomic Southern blots. The open reading frame predicts a 38.1-kDa protein with a pI of 6.59 that is homologous but not identical to a previously reported protein sequence of a 31-kDa porin isolated from human lymphocytes (porin 31HL). The most striking difference between the two porins is that the sequence predicted by the cDNA is longer than the 31HL porin by 27 amino acids at the amino terminus and 38 amino acids at the carboxyl terminus. The porin cDNA directs the in vitro translation of two protein species of approximately 32 and approximately 36 kDa, which appear to result from alternate usage of AUG initiation codons. The 32-kDa protein is the predominant species imported into both rat and yeast mitochondria in vitro. Taken together, these results suggest that multiple porin proteins can be expressed in humans. Additionally, a porin consensus protein sequence has been identified that is conserved in eukaryotic organisms as diverse as yeast and man.
Little is known about the molecular organization and functions of the postsynaptic density (PSD), a cytoskeletal specialization on the postsynaptic membrane. In an attempt to elucidate the protein composition of PSD, we have sequenced a 35 kDa protein of the rat forebrain PSD fraction. Amino acid sequence information of the tryptic peptides and immunoblot analyses revealed that the protein is a voltage-dependent anion channel 1 (VDAC1). VDAC1 was enriched in the PSD fraction and was partially soluble in 1% n-octyl glucoside (NOG) or Triton X-100. Our data indicate that VDAC1, which is originally found in the outer mitochondrial membrane, is also present in the central nervous system (CNS) synapses in association with the PSD 'core'.
Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.
Voltage-dependent anion channels (VDACs) are pore-forming proteins (porins) that form the major pathway for movement of adenine nucleotides through the outer mitochondrial membrane. Electrophysiological studies indicate that VDAC-like channel activity is also prevalent in the cell membranes of many mammalian cells. However, the multitopological localization of porins outside the mitochondrion has remained an extremely controversial issue. Herein, we show that usage of two alternative first exons of the murine VDAC-1 gene leads to expression of two porins differing within their N termini. One porin (plasmalemmal VDAC-1) harboring a hydrophobic leader peptide is primarily targeted through the Golgi apparatus to the cell membrane. In contrast, the second isoform lacking the N-terminal leader (mitochondrial VDAC-1) is translocated more efficiently into the outer mitochondrial membrane. Thus, our data provide unique genetic evidence in favor of a multitopological localization of a mitochondrial porin.
The diffusion of metabolites across the outer mitochondrial membrane is essential for coupled cellular respiration. The outer membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the outer membrane without inducing the loss of cytochrome c from the intermembrane space. Restoration of outer membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the outer mitochondrial membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain outer mitochondrial membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure.
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.
Mitochondrial creatine kinase (MtCK) co-localizes with mitochondrial porin (voltage-dependent anion channel) and adenine nucleotide translocator in mitochondrial contact sites. A specific, direct protein-protein interaction between MtCK and mitochondrial porin was demonstrated using surface plasmon resonance spectroscopy. This interaction was independent of the immobilized binding partner (porin reconstituted in liposomes or MtCK) or the analyzed isoform (chicken sarcomeric MtCK or human ubiquitous MtCK, human recombinant porin, or purified bovine porin). Increased ionic strength reduced the binding of MtCK to porin, suggesting predominantly ionic interactions. By contrast, micromolar concentrations of Ca(2+) increased the amount of bound MtCK, indicating a physiological regulation of complex formation. No interaction of MtCK with reconstituted adenine nucleotide translocator was detectable in our experimental setup. The relevance of these findings for structure and function of mitochondrial contact sites is discussed.
Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.
Immunoblotting was first devised by Towbin et al. (1979) to exploit the specificity of the reaction between antibodies and proteins transferred from SDS- polyacrylamide electrophoretic gels onto nitrocellulose. Immunoreactive bands were evidenced by labelled “second antibody” or protein A.
Oxygen plays such a critical role in the central nervous system that a specialized mechanism of oxygen delivery to neurons is required. Reduced oxygen tension, or hypoxia, may have severe detrimental effects on neuronal cells. Several studies suggest that hypoxia can induce cellular adaptive responses that overcome apoptotic signals in order to minimize hypoxic injury or damage. Adaptive responses of neuronal cells to hypoxia may involve activation of various ion channels, as well as induction of specific gene expression. For example, ATP sensitive K+ channels are activated by hypoxia in selective neuronal cells, and may play a role in cell survival during hypoxia/anoxia. Additionally, hypoxia-induced c-Jun, bFGF and NGF expression appear to be associated with prevention (or delay) of neuronal cell apoptosis. In this paper, these adaptive responses to hypoxia in neuronal cells are discussed to examine the possible role of hypoxia in pathophysiology of diseases.
Coupled cellular respiration requires that ATP and ADP be efficiently exchanged between the cytosol and the mitochondrial
matrix. When growth factors are withdrawn from dependent cells, metabolism is disrupted by a defect in ATP/ADP exchange across
the mitochondrial membranes. Unexpectedly, we find that this defect results from loss of outer mitochondrial membrane permeability
to metabolic anions. This decrease in anion permeability correlates with the changes in conductance properties that accompany
closure of the voltage-dependent anion channel (also known as mitochondrial porin). Loss of outer membrane permeability (i) results in the accumulation of stored metabolic energy within the intermembrane space in the form of creatine phosphate,
(ii) is prevented by the outer mitochondrial membrane proteins Bcl-xL and Bcl-2, and (iii) can be reversed by growth factor readdition. If outer membrane impermeability persists, the disruption of mitochondrial
homeostasis culminates in loss of outer mitochondrial membrane integrity, cytochrome c redistribution, and apoptosis. The recognition that outer membrane permeability is regulated under physiological conditions
has important implications for the understanding of bioenergetics and cell survival.
Mitochondria from malignant tumor cell lines show a higher capability for hexokinase binding than those from normal liver . To explore possible differences in hexokinase binding sites of mitochondria between tumor cells and normal liver, we characterized porin isoforms expressed in tumor cells. Cloning experiments on the three porin isoforms, VDAC1, VDAC2 and VDAC3 from malignant tumor cell line AH130 clearly showed that their primary structures were completely identical to those of the corresponding VDACs of normal liver cells. Possible expression of the fourth porin isoform in AH130 cells was excluded by degenerate primer-based RT-PCR. However, the transcript levels of the three VDAC isoforms in AH130 cells were significantly higher than those in normal liver. These results suggest that the high hexokinase-binding capability of malignant tumor cell mitochondria was not due to any structural difference, but due to a quantitative difference in binding sites.
A rapid (less than 2h) method is described for the preparation of synaptosomes from rat brain by using a discontinuous Ficoll/sucrose gradient by a flotation technique. These synaptosomes are metabolically active and minimally (less than 5%) contaminated with 'free' mitochondria as judged by marker-enzyme assays and electron microscopy.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.
Background staining that is associated with silver detection of proteins and nucleic acids in polyacrylamide gels has been shown to be due mostly to the amide groups in methylenebisacrylamide, a commonly used gel crosslinker. In attempts to reduce this background staining, eight existing crosslinking agents were tested. All of these proved to be unsuitable. Six new crosslinking agents were synthesized and tested. Of these, diacrylylpiperazine provided increased physical strength, improved electrophoretic separation of proteins, and silver staining detection of proteins with reduced background stain.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
We have simplified the highly sensitive silver stain of R. C. Switzer III, C. R. Merril, and S. Shifrin (1979, Anal. Biochem.98, 231–237) for visualizing proteins in polyacrylamide gels. We have reduced the number of steps in the procedure from 10 to 6, simplified the reagents in each step, and reduced the amount of silver required by a factor of 10, thus greatly reducing the expense of the procedure. In common with the original silver stain, our procedure is 100 times more sensitive than Coomassie brilliant blue and is comparable in sensitivity to radioautography of radioactively labeled proteins.
The voltage dependent anion channel (VDAC) of the outer membrane of mitochondria is thought to play a role in transport of metabolites including ATP across mitochondrial membrane and modulate mitochondrial functions such as respiration. However, regulation of this anion channel is only poorly understood. In this paper we demonstrate that VDAC purified from rat liver mitochondria can be phosphorylated by the catalytic subunit of cAMP dependent protein kinase (PKA). PKA phosphorylates VDAC linearly up to fifteenfold in sixty minutes. The level of VDAC phosphorylation increases to twofold and sevenfold of control value after ten and thirty minutes of reaction, respectively. Data presented here suggest the possibility that voltage dependent anion channel of the outer membrane of mitochondria may be a target of PKA in vivo.
A new nonlinear immobilized pH gradient (IPG) is proposed as the first dimension for two-dimensional electrophoresis. In comparison to conventional carrier ampholyte techniques, it offers better resolution and greater reproducibility whilst allowing application of higher protein loads. Furthermore, we have checked and supplemented existing data on pK values for the immobilized groups in the presence of 8M urea. This is necessary for pH gradients to be defined in a pH scale relevant to the focusing conditions such that spot positions can be related to amino acid compositions. The data will allow definition of pH scales for the temperature range 10-25 degrees C and for a pH range covering the major part of the nonlinear pH gradient. With the latter, focusing positions are neither influenced by urea concentration nor by the choice or the concentration of detergent or carrier ampholyte. Temperature is the only parameter affecting focusing reproducibility and here any changes in focusing positions can be related to the amino acid compositions of peptides.
As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor (HIF)-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1alpha tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance.
Macrophage migration inhibitory factor (MIF) is an ubiquitous protein playing various immunological and hormonal roles. Theoretical electrophoretic coordinates calculated from protein sequence in the SWISS-PROT database (AC P14174) are 12 kDa and pI 8.24. Using two-dimensional (2-D) immunoblotting, we have detected isoelectric forms at ca. 11.9 kDa, with pI values of 7.8 and 6.98 in human liver tissue, breast tissue and a cell line and in preparations of human MIF expressed in E. coli. This evidence suggests that MIF charge heterogeneity originates from a post-translational modification not requiring eukaryote-specific enzymes. We have also detected in human liver a minor immunoreactive spot at pI 6.23, which coincides with the MIF spot in the liver map in SWISS-2DPAGE. The pI 6.23 isoform also conceivably derives from post-translational modification, as MIF is known to be encoded in the human genome by a single copy gene.
We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.
During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria, which causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm, which in turn activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Here we create liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast did not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which were inhibited by Bcl-x(L). Our results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis.
Early events in the hypoxia-induced response trigger tyrosine phosphorylation cascades involving a large number of enzymes and substrates. The resolving power of advanced two-dimensional gel electrophoresis, followed by immunoblotting with specific antibodies to phosphotyrosine, has been used to analyze hypoxia-induced modifications in guinea pig brain synaptosomes. These procedures, in conjunction with computer-aided image analysis, are useful in the differential display of gene products, providing comparison at the level of posttranslationally modified products. Studies were performed in cerebral cortical synaptosomes from three normoxic and three hypoxic newborn guinea pigs. To filter off background noise consisting of nonreproducible migrating protein spots, only reproducible features of electrophoretic patterns were considered. Immunoreactivity patterns obtained with anti-phosphotyrosine antibodies proved to be different in normoxic and hypoxic synaptosomes: of a total of 130 immunoreactive spots, 49 were tyrosine-phosphorylated in hypoxic synaptosomes only and 20 in the normoxic ones only. Our data suggest that hypoxia extensively remodels the signaling pathway by switching off tyrosine phosphorylation of some cellular components (i.e., alpha-internexin) and switching on tyrosine phosphorylation of some other proteins (i.e., heat shock cognate 70, aconitase, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and pyruvate kinase).
The control of complex cellular events such as DNA repair, actin rearrangement, programmed cell death and transcriptional regulation requires acute orchestration of many signalling molecules, including kinases, phosphatases, proteases, guanine-nucleotide-binding (G) proteins and transcription factors. These molecules must relocate to specific sites in the cell, and their activities need to be regulated in a timely manner, often involving interactions with other proteins. Several papers1, 2, 3, the latest by Durocher et al. in Molecular Cell1, have revealed a new twist on the mechanism by which protein phosphorylation regulates the assembly of such complexes.
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The precise molecular events of mitochondrial dysfunction, one of the last steps that irreversibly determines cellular degeneration and death, remain unknown. We introduce a novel strategy to isolate and assess the molecular mechanisms underlying mitochondrial dysfunction. Using an in vitro ischemia model, we obtained evidence for prolonged mitochondrial depolarization in rat organotypic hippocampal brain slices during reperfusion. Then, mitochondria were isolated from brain slices and mitochondrial proteins were purified on a cyclosporin-A affinity column. Cyclosporin-A is the most potent inhibitor of mitochondrial dysfunction, in particular the mitochondrial permeability transition, and therefore we hypothesized that it may interact with proteins involved in the permeability transition after mitochondria were subjected to manipulations that promote this event. Mitochondrial porin was reproducibly eluted from the affinity column using proteins from ischemic brain mitochondria, or from mitochondria exposed to oxidative stress that were used as a positive control. Anti-porin antibodies prevented mitochondrial depolarization and electrophysiological deterioration of hippocampal neurons during hypoxia-reperfusion, as measured by simultaneous fluorescence imaging and whole-cell recordings. These observations provide biochemical and functional evidence that porin is directly involved in mitochondrial dysfunction and neuronal impairment during ischemia-reperfusion, and indicate that porin could be a novel therapeutic target to prevent cellular degeneration.
Voltage-dependent anion-selective channel proteins (VDACs) are pore-forming proteins found in the other mitochondrial membrane of all eukaryotes and in brain postsynaptic membranes. VDACs regulate anion fluxes of a series of metabolites including ATP, thus regulating mitochondrial metabolic functions. We determined protein levels of VDACs in individual post-mortem brain regions of patients with Down Syndrome (DS) and Alzheimer's disease (AD) using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). VDAC1 (SWISS-PROT accession number P21796) and VDAC2 (P45880) were unambiguously identified and quantified, but VDAC3 was not found. The spots representing VDAC1 were separated with different p/s (p/7.5, 8.5, and 10.0) probably caused by post-translational modifications as, e.g., phosphorylation. In DS cerebellum, total VDAC1 protein was elevated significantly whereas VDAC2 did not show any significant alterations. In AD brains, VDAC1 p/10.0 was significantly reduced in temporal, frontal, and occipital cortex with the p/7.5 form elevated in occipital cortex. Total VDAC1 was significantly decreased in frontal cortex and thalamus. VDAC2 was significantly elevated in temporal cortex only. The biological meaning of our results may be derangement of voltage-dependent anion-selective channel function and reflecting impaired glucose, energy, and intermediary metabolism as well as apoptotic mechanisms.
- Buonocore G.