Modulation of mitochondrial ion transport by inorganic polyphosphate - essential role in mitochondrial permeability transition pore

Inorganic polyphosphate (polyP) is a biopolymer of phosphoanhydride-linked orthophosphate residues. PolyP is involved in multiple cellular processes including mitochondrial metabolism and cell death. We used artificial membranes and isolated mitochondria to investigate the role of the polyP in mitochondrial ion transport and in activation of PTP. Here, we found that polyP can modify ion permeability of de-energised mitochondrial membranes but not artificial membranes. This permeability was selective for Ba²⁺ and Ca²⁺ but not for other monovalent and bivalent cations and can be blocked by inhibitors of the permeability transition pore – cyclosporine A or ADP. Lower concentrations of polyP modulate calcium dependent permeability transition pore opening. Increase in polyP concentrations and elongation chain length of the polymer causes calcium independent swelling in energized conditions. Physiologically relevant concentrations of inorganic polyP can regulate calcium dependent as well calcium independent mitochondrial permeability transition pore opening. This raises the possibility that cytoplasmic polyP can be an important contributor towards regulation of the cell death.     

Virus-induced permeability transition in mitochondria

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus-induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A-protein, or helical polymer, as well as supernatant of the heat-disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus-induced apoptosis is discussed.     

Mitochondrial regulation of synaptic plasticity in the hippocampus

Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used an in vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity.     

Comparison of beta-tubulin mRNA and protein levels in 12 human cancer cell lines

Antimitotic drugs are chemotherapeutic agents that bind tubulin and microtubules. Resistance to these drugs is a major clinical problem. One hypothesis is that the cellular composition of tubulin isotypes may predict the sensitivity of a tumor to antimitotics. Reliable and sensitive methods for measuring tubulin isotype levels in cells and tissues are needed to address this hypothesis. Quantitative measurements of tubulin isotypes have frequently relied upon inferring protein amounts from mRNA levels. To determine whether this approach is justified, protein and mRNA levels of beta-tubulin isotypes from 12 human cancer cell lines were measured. This work focused on only beta-tubulin isotypes because we had readily available monoclonal antibodies for quantitative immunoblots. The percentage of beta-tubulin isotype classes I, II, III, and IVa + IVb mRNA and protein were compared. For beta-tubulin class I that comprises >50% of the beta-tubulin protein in 10 of the 12 cell lines, there was good agreement between mRNA and protein percentages. Agreement between mRNA and protein was also found for beta-tubulin class III. For beta-tubulin classes IVa + IVb, we observed higher protein levels compared to mRNA levels.Beta-tubulin class II protein was found in only four cell lines and in very low abundance. We conclude that quantitative Western blotting is a reliable method for measuring tubulin isotype levels in human cancer cell lines. Inferring protein amounts from mRNA levels should be done with caution, since the correspondence is not one-to-one for all tubulin isotypes.     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.     

Dimethylaminopyridine derivatives of lupane triterpenoids cause mitochondrial disruption and induce the permeability transition

Triterpenoids are a large class of naturally occurring compounds, and some potentially interesting as anticancer agents have been found to target mitochondria. The objective of the present work was to investigate the mechanisms of mitochondrial toxicity induced by novel dimethylaminopyridine (DMAP) derivatives of pentacyclic triterpenes, which were previously shown to inhibit the growth of melanoma cells in vitro. MCF-7, Hs 578T and BJ cell lines, as well as isolated hepatic mitochondria, were used to investigate direct mitochondrial effects. On isolated mitochondrial hepatic fractions, respiratory parameters, mitochondrial transmembrane electric potential, induction of the mitochondrial permeability transition (MPT) pore and ion transport-dependent osmotic swelling were measured. Our results indicate that the DMAP triterpenoid derivatives lead to fragmentation and depolarization of the mitochondrial network in situ, and to inhibition of uncoupled respiration, induction of the permeability transition pore and depolarization of isolated hepatic mitochondria. The results show that mitochondrial toxicity is an important component of the biological interaction of DMAP derivatives, which can explain the effects observed in cancer cells.     

BMAP-28, an antibiotic peptide of innate immunity, induces cell death through opening of the mitochondrial permeability transition pore

BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca(2+) and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.     

Heterologous expression and folding analysis of a beta-tubulin isotype from the Antarctic ciliate Euplotes focardii.

Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1). To study the beta-tubulin folding in lower eukaryotes, an isotype of beta-tubulin (beta-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35S-labeled, denatured beta-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric beta-tubulin. We show for the first time in protozoa that beta-tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardiibeta-T1 competes with human beta5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardiibeta-T1 and beta5 tubulin are compared. Finally, the mitochondrial chaperonin mt-cpn60 binds to beta-T1 but is unable to release it in a native or quasi-native state.     

Changes in specific lipids regulate BAX-induced mitochondrial permeability transition

Recent evidence suggests the existence of lipid microdomains in mitochondria, apparently coexisting as structural elements with some of the mitochondrial permeability transition pore-forming proteins and members of the Bcl-2 family. The aim of this study was to investigate the relevance of the main components of membrane microdomains (e.g. cholesterol and sphingolipids) in activation of the mitochondrial permeability transition pore (mPTP) by recombinant BAX (rBAX). For this purpose, we used chemically modified renal cortex mitochondria and renal cortex mitochondria from hypothyroid rats that show a modified mitochondrial lipid composition in vivo. Oligomeric rBAX induced an enhanced permeability conformation in the mPTP of control mitochondria. rBAX failed to induce mPTP opening when the cholesterol and ganglioside content of mitochondria were modified with the chelator methyl-beta-cyclodextrin. Accordingly, hypothyroid mitochondria, with endogenously lower cholesterol and ganglioside content, showed resistance to mPTP opening induced by rBAX. These observations suggest that enriched cholesterol and ganglioside domains in the mitochondrial membranes may determine BAX interaction with the mPTP. An intriguing observation was that chemical extraction of cholesterol and ganglioside in control mitochondria did not have an effect on rBAX insertion. Conversely, in hypothyroid mitochondria, rBAX insertion was diminished dramatically compared with control mitochondria. The membrane and protein changes associated with thyroid status and their possible role in rBAX docking into the membranes are discussed.     

Rationalization of paclitaxel insensitivity of yeast ß-tubulin and human ßIII-tubulin isotype using principal component analysis

ABSTRACT: BACKGROUND: The chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among beta-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin, within a common theoretical framework, we have performed structural principal component analyses of beta-tubulin sequences across eukaryotes. RESULTS: The paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of beta-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in betaIII isotype was shown to be crucial for paclitaxel-resistance. CONCLUSIONS: The present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human betaIII tubulin isotype, through two common collective sequence vectors.     

Cyclosporin A-insensitive permeability transition in brain mitochondria: inhibition by 2-aminoethoxydiphenyl borate

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.     

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

Bax does not directly participate in the Ca(2+)-induced permeability transition of isolated mitochondria

The mitochondrial permeability transition pore and Bax have both been proposed to be involved in the release of pro-apoptotic factors from mitochondria in the "intrinsic" pathway of apoptosis. The permeability transition pore is widely thought to be a supramolecular complex including or interacting with Bax. Given the relevance of the permeability transition in vivo, we have verified whether Bax influences the formation and/or the properties of the Ca(2+)/P(i)-induced permeability transition by using mitochondriaisolated from isogenic human colon cancer bax(+/-) and bax(-/-) HCT116 cell lines. We used mitochondria isolated from both types of cells and from Bax(+) cells exposed to apoptotic stimuli, as well as Bax-less mitochondria into which exogenous Bax had been incorporated. All exhibited the same behavior and pharmacological profile in swelling and Ca(2+)-retention experiments. Mitochondria from a bax(-)/bak(-) cell line also underwent an analogous Ca(2+)/P(i)-inducible swelling. This similarity indicates that Bax hasno major role in regulating the Ca(2+)-induced mitochondrial permeability transition.     

A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro.

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase-3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl-xL, cyclosporin A and z-VAD.fmk. Moreover, caspase-3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl-xL- and cyclosporin A-inhibitable manner. These results suggest that caspase-3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.     

Granzyme B induces BID-mediated cytochrome c release and mitochondrial permeability transition

Many cell death pathways converge at the mitochondria to induce release of apoptogenic proteins and permeability transition, resulting in the activation of effector caspases responsible for the biochemical and morphological alterations of apoptosis. The death receptor pathway has been described as a triphasic process initiated by the activation of apical caspases, a mitochondrial phase, and then the final phase of effector caspase activation. Granzyme B (GrB) activates apical and effector caspases as well as promotes cytochrome c (cyt c) release and loss of mitochondrial membrane potential. We investigated how GrB affects mitochondria utilizing an in vitro cell-free system and determined that cyt c release and permeability transition are initiated by distinct mechanisms. The cleavage of cytosolic BID by GrB results in truncated BID, initiating mitochondrial cyt c release. BID is the sole cytosolic protein responsible for this phenomenon in vitro, yet caspases were found to participate in cyt c release in some cells. On the other hand, GrB acts directly on mitochondria in the absence of cytosolic S100 proteins to open the permeability transition pore and to disrupt the proton electrochemical gradient. We suggest that GrB acts by two distinct mechanisms on mitochondria that ultimately lead to mitochondrial dysfunction and cellular demise.     

Calcium release from the rat liver mitochondria during collapse of the membrane potential

Ca(2+)-release from rat liver mitochondria after protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP)-induced membrane depolarisation is studied. It is shown that the release of calcium is accompanied by an increase of the inner mitochondrial membrane permeability as the result of the opening of permeability transition pore (PTP). Calcium is released from mitochondria through the uniporter working in reverse mode and also by PTP mechanism which accounts for ruthenium red (RR)-insensitive component of total. Ca(2+)-release. Unlike Ca2+, the strontium release from the mitochondria is completely sensitive to RR, specific uniporter blocker, which shows the absence of rapid Sr(2+)-efflux mechanisms other than uniporter of bivalent cations. The data obtained also give an evidence that the lifetime of the open state of the pore is limited, and barrier properties of the mitochondrial membrane are restored after the closure of the pore.     

Coordinated behavior of mitochondria in both space and time: a reactive oxygen species-activated wave of mitochondrial depolarization

Reactive oxygen species (ROS) can trigger a transient burst of mitochondrial ROS production via ROS activation of the mitochondrial permeability transition pore (MPTP), a phenomenon termed ROS-induced ROS release (RIRR). The goal of this study was to investigate if the generation of ROS in a discrete region of a cardiomyocyte could serve to propagate RIRR-mediated mitochondrial depolarizations throughout a cell. Our experiments revealed that localized RIRR activated either RIRR-mediated fluctuations in mitochondrial membrane potential (time period: 3-10 min) or a traveling wave of depolarization of the cell's mitochondria (velocity: approximately 5 microm/min). Both phenomena appeared to be mediated by the mitochondrial permeability transition pore and eventually encompassed the majority of the mitochondrial population of both isolated rat and rabbit cardiomyocytes. Furthermore, depolarization was often reversible; the waves of depolarization were then followed by a rapid (approximately 40 microm/min) repolarization wave of the mitochondria. We show that the RIRR can function to communicate the mitochondrial permeability transition from one mitochondrion to another in the isolated adult cardiomyocyte.     

Mitochondrial permeability transition and cytochrome c release induced by selenite

Cytochrome c release and mitochondrial permeability transition (MPT) play important roles in apoptosis. In this study, we found that selenium, an essential trace element, induced mitochondrial membrane potential (Delta psi(m)) loss, swelling, and cytochrome c release in isolated mitochondria. All of the above observations were blocked by cyclosporin A (CsA), which is a specific inhibitor to permeability transition pore (PTP), indicating selenite-induced mitochondrial changes were mediated through the opening of PTP. In physiological concentration, selenite could induce mitochondria at low-conductance PTP 'open' probability, which is correlated to regulate the physiological function, whereas in toxic concentration, induce mitochondria at high-conductance PTP 'open' probability and rapidly undergo a process of osmotic swelling following diffusion toward matrix as for inducer (Ca(2+)/P(i)). Selenite also induced other mitochondrial marker enzymes including monoamine oxidase (MAO) and mitochondria aspartate aminotransferase (mAST). Oligomycin inhibited the selenite-induced cytochrome c release and Delta psi(m) loss, showing that F(0)F(1)-ATPase was important in selenite or Ca(2+)/P(i)-induced MPT.