Biomedical subjects

Marco Colombini

Publications and source records attributed to Marco Colombini.

18 recordsLinked to original sources

PubMed2006

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP.

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K(i) between 0.2 and 0.5 microM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 microM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 microM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.

Adenosine Diphosphate↗

PubMed2006

Sphingosine, a product of ceramide hydrolysis, influences the formation of ceramide channels.

Ceramides are known to have a regulatory function in apoptosis, including the release of cytochrome c and other proapoptotic factors from the mitochondrial intermembrane space. Ceramides can form large, stable channels in the outer mitochondrial membrane, leading to the proposal that ceramide channels are the pathway through which these proteins are released. Here, we report that sphingosine, a product of ceramide hydrolysis by ceramidase, is capable of destabilizing ceramide channels, leading to their disassembly. Sphingosine is directly responsible for the disassembly of ceramide channels in planar membrane experiments and markedly reduces the ability of ceramide to induce the release of intermembrane space proteins from mitochondria in vitro. Low concentrations of both L and D sphingosine potentiate the release of intermembrane space proteins by long-chain ceramide and channel formation in liposomes. These results provide evidence for a mechanism by which the disassembly of ceramide channels, as initiated by ceramidase, could be accelerated by the direct interaction of the hydrolysis product with the ceramide channels themselves. This mechanism therefore could form a positive feedback loop for rapid shut-down of ceramide channels. However, potentiation of ceramide channel formation is also possible and thus both effects could influence the propensity for mitochondria-mediated apoptosis.

Ceramides↗

PubMed2006

A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC.

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.

Base Sequence↗

PubMed2006

Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations.

Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.

Animals↗

PubMed2005

The use of yeast mitochondria to study the properties of wild-type and mutant human mitochondrial ornithine transporter.

Yeast deletion models have general utility for the study of a variety of inherited metabolic disorders. Mutations in the mitochondrial ornithine transporter result in hyperammonemia, hyperornithinemia, homocitrullinuria syndrome, a disorder of the urea cycle. To study the effects of mutations in a model system that more closely resembles the in vivo environment, we have developed an expression system based on a yeast strain lacking its endogenous ornithine transporter homologue. Wild-type human ornithine transporter and a recurrent mutation, DeltaF 188, were expressed and characterized with this system. The wild-type transporter appeared to insert into yeast mitochondria in the same orientation as in mammalian mitochondria. It showed stereospecificity, strong antiport activity and ornithine transport was competed by citrulline and arginine. The DeltaF 188 mutant was not incorporated into the membrane to the same extent as wild type, but retained significant residual activity and lost stereospecificity. In these isolated mitochondria, samarium chloride was found to be a potent blocker of transport compared to previously reported sulfhydryl-based inhibitors. A low-affinity background transport activity that promoted the exchange of ornithine for either acidic or basic amino acids was observed. This yeast model can readily be extended to the study of protein:protein interactions. In this manner, the use of yeast deletion strains can serve as a general framework to perform metabolic pathway analysis.

Amino Acid Transport Systems, Basic↗

PubMed2005

New insights into the mechanism of permeation through large channels.

The mitochondrial channel, VDAC, regulates metabolite flux across the outer membrane. The open conformation has a higher conductance and anionic selectivity, whereas closed states prefer cations and exclude metabolites. In this study five mutations were introduced into mouse VDAC2 to neutralize the voltage sensor. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity, and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for wild-type VDAC2. The outer membranes of mitochondria containing the mutant are permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from an anionic to a cationic preference does not substantially influence the flux of negatively charged metabolites. This finding supports our previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall.

Adenosine Diphosphate↗

PubMed2005

Proapoptotic triterpene electrophiles (avicins) form channels in membranes: cholesterol dependence.

Avicins, a family of triterpenoid saponins from Acacia victoriae, can regulate the innate stress response in human cells. Their ability to induce apoptosis in transformed cells makes them potential anticancer agents. We report that avicins can form channels in membranes. The conductance reached a steady state after each addition, indicating a dynamic equilibrium between avicin in solution and in the membrane. The high power dependence (up to 10) of the membrane conductance on the avicin concentration indicates the formation of multimeric channels, consistent with the estimated pore radius of 1.1 nm. This radius is too small to allow protein flux across the mitochondrial outer membrane, a process known to initiate apoptosis. Channel formation is lost when avicin's amphipathic side chain is removed, implicating this as the channel-forming region. A small difference in this side chain results in strong cholesterol dependence of channel formation in avicin G that is not found in avicin D. In neutral membranes, avicin channels are nonselective, but negatively-charged lipids confer cation selectivity (5:1, K(+):Cl(-)), indicating that phospholipids form part of the permeation pathway. Avicin channels in the mitochondrial outer membrane may favor apoptosis by altering the potential across this membrane and the intermembrane space pH.

Acacia↗

PubMed2005

On the role of VDAC in apoptosis: fact and fiction.

Research on VDAC has accelerated as evidence grows of its importance in mitochondrial function and in apoptosis. New investigators entering the field are often confounded by the VDAC literature and its many apparent conflicts and contradictions. This review is an effort to shed light on the situation and identify reliable information from more questionable claims. Our views on the most important controversial issues are as follows: VDAC is only present in the mitochondrial outer membrane. VDAC functions as a monomer. VDAC functions normally with or without Ca(2+). It does not form channels that mediate the flux of proteins through membranes (peptides and unfolded proteins are excluded from this statement). Closure of VDAC, not VDAC opening, leads to mitochondria outer membrane permeabilization and apoptosis.

Animals↗

PubMed2005

Sphingosine forms channels in membranes that differ greatly from those formed by ceramide.

Ceramide channels formed in the outer membrane of mitochondria have been proposed to be the pathways by which proapoptotic proteins are released from mitochondria during the early stages of apoptosis. We report that sphingosine also forms channels in membranes, but these differ greatly from the large oligomeric barrel-stave channels formed by ceramide. Sphingosine channels have short open lifetimes and have diameters less than 2 nm, whereas ceramide channels have long open lifetimes, enlarge in size reaching diameters in excess of 10 nm. Unlike ceramide, sphingosine forms channels in erythrocyte plasma membranes that vary in size with concentration, but with a maximum possible channel diameter of 2 nm. In isolated mitochondria, a large proportion of the added sphingosine was rapidly metabolized to ceramide in the absence of externally added fatty acids or fatty-acyl-CoAs. The ceramide synthase inhibitor, fumonisin B1 failed to prevent sphingosine metabolism to ceramide and actually increased it. However, partial inhibition of conversion to ceramide was achieved in the presence of ceramidase inhibitors, indicating that reverse ceramidase activity is at least partially responsible for sphingosine metabolism to ceramide. A small amount of cytochrome c release was detected. It correlated with the level of ceramide converted from sphingosine. Thus, sphingosine channels, unlike ceramide channels, are not large enough to allow the passage of proapoptotic proteins from the intermembrane space of mitochondria to the cytoplasm.

Animals↗

PubMed2004

Potassium channel openers are uncoupling protonophores: implication in cardioprotection.

Excessive build-up of mitochondrial protonic potential is harmful to cellular homeostasis, and modulation of inner membrane permeability a proposed countermeasure. Here, we demonstrate that structurally distinct potassium channel openers, diazoxide and pinacidil, facilitated transmembrane proton translocation generating H(+)-selective current through planar phospholipid membrane. Both openers depolarized mitochondria, activated state 4 respiration and reduced oxidative phosphorylation, recapitulating the signature of mitochondrial uncoupling. This effect was maintained in K(+)-free conditions and shared with the prototypic protonophore 2,4-dinitrophenol. Diazoxide, pinacidil and 2,4-dinitrophenol, but not 2,4-dinitrotoluene lacking protonophoric properties, preserved functional recovery of ischemic heart. The identified protonophoric property of potassium channel openers, thus, implicates a previously unrecognized component in their mechanism of cardioprotection.

Animals↗

PubMed2004

Bid, but not Bax, regulates VDAC channels.

During apoptosis, cytochrome c is released from mitochondria into the cytosol, where it participates in caspase activation. Various and often conflicting mechanisms have been proposed to account for the increased permeability of the mitochondrial outer membrane that is responsible for this process. The voltage-dependent anion channel (VDAC) is the major permeability pathway for metabolites in the mitochondrial outer membrane and therefore is a very attractive candidate for cytochrome c translocation. Here, we report that properties of VDAC channels reconstituted into planar phospholipid membranes are unaffected by addition of the pro-apoptotic protein Bax under a variety of conditions. Contrary to other reports (Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483-487; Shimizu, S., Ide, T., Yanagida, T., and Tsujimoto, Y. (2000) J. Biol. Chem. 275, 12321-12325; Shimizu, S., Konishi, A., Kodama, T., and Tsujimoto, Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3100-3105), we found no electrophysiologically detectable interaction between VDAC channels isolated from mammalian mitochondria and either monomeric or oligomeric forms of Bax. We conclude that Bax does not induce cytochrome c release by acting on VDAC. In contrast to Bax, another pro-apoptotic protein (Bid) proteolytically cleaved with caspase-8 affected the voltage gating of VDAC by inducing channel closure. We speculate that by decreasing the probability of VDAC opening, Bid reduces metabolite exchange between mitochondria and the cytosol, leading to mitochondrial dysfunction.

Animals↗

PubMed2004

The physiological properties of a novel family of VDAC-like proteins from Drosophila melanogaster.

VDAC, a major protein of the mitochondrial outer membrane, forms voltage-dependent, anion-selective channels permeable to most metabolites. Although multiple isoforms of VDAC have been found in different organisms, only one isoform (porin/DVDAC) has been previously reported for Drosophila melanogaster. We have examined the physiological properties of three other Drosophila proteins (CG17137, CG17139, and CG17140) whose primary sequences have significant homology to DVDAC. A comparison of their hydropathy profiles (beta-pattern) with known VDAC sequences indicates the same fundamental folding pattern but with major insertions and deletions. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137 and CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 was not able to form channels. DVDAC and CG17137 were able to rescue the temperature-sensitive conditional-lethal phenotype of VDAC-deficient yeast, whereas CG17139 and CG17140 demonstrated no complementation. Similar structure and channel formation indicate that VDAC-like proteins are part of the larger VDAC family but the modifications are indicative of specialized functions.

Amino Acid Sequence↗

PubMed2003

Enlargement and contracture of C2-ceramide channels.

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that ceramide, but not dihydroceramide, forms large and stable channels in phospholipid membranes and outer membranes of isolated mitochondria. C(2)-ceramide channel formation is characterized by conductance increments ranging from <1 to >200 nS. These conductance increments often represent the enlargement and contracture of channels rather than the opening and closure of independent channels. Enlargement is supported by the observation that many small conductance increments can lead to a large decrement. Also the initial conductances favor cations, but this selectivity drops dramatically with increasing total conductance. La(+3) causes rapid ceramide channel disassembly in a manner indicative of large conducting structures. These channels have a propensity to contract by a defined size (often multiples of 4 nS) indicating the formation of cylindrical channels with preferred diameters rather than a continuum of sizes. The results are consistent with ceramides forming barrel-stave channels whose size can change by loss or insertion of multiple ceramide columns.

Biophysical Phenomena↗

PubMed2002

Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins.

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C(2)- and C(16)-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001) J. Biol. Chem. 275, 38640-38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochrome c was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C(2)-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C(2)-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochrome c.

Adenylate Kinase↗

PubMed2002

Catalyzed insertion of proteins into phospholipid membranes: specificity of the process.

The process of insertion of intrinsic proteins into phospholipid membranes conjures up the thought of enormous energy barriers but is a routine occurrence in cells. Proteinaceous complexes responsible for protein targeting/translocation/insertion into membranes have been studied intensively. However, the mitochondrial voltage-dependent anion channel (VDAC), can insert into phospholipid membranes by an auto-catalytic process called "auto-directed insertion." This process results in an oriented insertion of VDAC channels and an increase in insertion rate per unit area of 10 orders of magnitude. Here we report that VDAC catalyzes the insertion of PorA/C1 and KcsA by increasing their calculated insertion rate per unit area by 9 orders of magnitude with no detectable effect on the insertion of alpha-hemolysin. This was measured as a reduction in the delay before the first insertion of these proteins. Gramicidin and PorA/C1 accelerate the calculated insertion rate per unit area of VDAC by 8 and 9 orders of magnitude, respectively. Only PorA/C1 increases the overall rate of VDAC insertion (50-fold) over the self-catalyzed rate. Our results indicate that catalyzed insertion of proteins into phospholipid membranes does not arise simply from disturbance of the phospholipid membrane because it shows strong specificity.

Bacterial Proteins↗

PubMed2002

Dynamics of nucleotides in VDAC channels: structure-specific noise generation.

Nucleotide penetration into the voltage-dependent mitochondrial ion channel (VDAC) reduces single-channel conductance and generates excess current noise through a fully open channel. VDAC channels were reconstituted into planar phospholipid membranes bathed in 1.0 M NaCl. At a given nucleotide concentration, the average decrease in small-ion channel conductance induced by mononucleotides ATP, ADP, AMP, and UTP and dinucleotides beta- and alpha-NADH, NAD, and NADPH are very close. However, the excess current noise is about seven times higher in the presence of NADPH than in the presence of ATP and is about 40 times higher than in the presence of UTP. The nucleotide-generated low-frequency noise obeys the following sequence: beta-NADPH > beta-NADH = alpha-NADH > ATP > ADP > beta-NAD > or = AMP > UTP. Measurements of bulk-phase diffusion coefficients and of the effective charge of the nucleotides in 1.0 M NaCl suggest that differences in size and charge cannot be the major factors responsible for the ability to generate current noise. Thus, although the ability of nucleotides to partition into the channel's pore, as assessed by the reduction in conductance, is very similar, the ability to generate current noise involves a detailed recognition of the three-dimensional structure of the nucleotide by the VDAC channel. A possible mechanism for this selectivity is two noise-generating processes operating in parallel.

Adenine Nucleotides↗

PubMed2002

Electrostatics explains the shift in VDAC gating with salt activity gradient.

We have analyzed voltage-dependent anion-selective channel (VDAC) gating on the assumption that the states occupied by the channel are determined mainly by their electrostatic energy. The voltage dependence of VDAC gating both in the presence and in the absence of a salt activity gradient was explained just by invoking electrostatic interactions. A model describing this energy in the main VDAC states has been developed. On the basis of the model, we have considered how external factors cause the redistribution of the channels among their conformational states. We propose that there is a difference in the electrostatic interaction between the voltage sensor and fixed charge within the channel when the former is located in the cis side of membrane as opposed to the trans. This could be the main cause of the shift in the probability curve. The theory describes satisfactorily the experimental data (Zizi et al., Biophys. J. 1998. 75:704-713) and explains some peculiarities of VDAC gating. The asymmetry of the probability curve was related to the apparent location of the VDAC voltage sensor in the open state. By analyzing published experimental data, we concluded that this apparent location is influenced by the diffusion potential. Also discussed is the possibility that VDAC gating at high voltage may be better described by assuming that the mobile charge consists of two parts that have to overcome different energetic barriers in the channel-closing process.

Biophysical Phenomena↗

PubMedDate not supplied

VDAC: the channel at the interface between mitochondria and the cytosol.

The mitochondrial outer membrane is not just a barrier but a site of regulation of mitochondrial function. The VDAC family of proteins are the major pathways for metabolite flux through the outer membrane. These can be regulated in a variety of ways and the integration of these regulatory inputs allows mitochondrial metabolism to be adjusted to changing cellular conditions. This includes total blockage of the flux of anionic metabolites leading to permeabilization of the outer membrane to small proteins followed by apoptotic cell death.

Amino Acid Sequence↗