Mitochondrial alternative oxidase contributes to successful tardigrade anhydrobiosis.

Anhydrobiosis can be described as an adaptation to lack of water that enables some organisms, including tardigrades, to survive extreme conditions, even some that do not exist on Earth. The cellular mechanisms underlying anhydrobiosis are still not completely explained including the putative contribution of mitochondrial proteins. Since mitochondrial alternative oxidase (AOX), described as a drought response element in plants, was recently proposed for various invertebrates including tardigrades, we investigated whether AOX is involved in successful anhydrobiosis of tardigrades. Milnesium inceptum was used as a model for the study. We confirmed functionality of M. inceptum AOX and estimated its contribution to the tardigrade revival after anhydrobiosis of different durations. We observed that AOX activity was particularly important for M. inceptum revival after the long-term tun stage but did not affect the rehydration stage specifically. The results may contribute to our understanding and then application of anhydrobiosis underlying mechanisms.

yVDAC2, the second mitochondrial porin isoform of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae genome is endowed with two distinct isoforms of Voltage-Dependent Anion Channel (VDAC). The isoform yVDAC2 is currently understudied with respect to the best known yVDAC1. Yet, since the discovery, the function of yVDAC2 was unclear, leading to the hypothesis that it might be devoid of a channel function. In this work we have elucidated, by bioinformatics modeling and electrophysiological analysis, the functional activity of yVDAC2. The conformation of yVDAC2 and, for comparison, of yVDAC1 were modeled using a multiple template approach involving mouse, human and zebrafish structures and both showed to arrange the sequences as the typical 19-stranded VDAC ß-barrel. Molecular dynamics simulations showed that yVDAC2, in comparison with yVDAC1, has a different number of permeation paths of potassium and chloride ions. yVDAC2 protein was over-expressed in the S. cerevisiae cells depleted of functional yVDAC1 (Δpor1 mutant) and, after purification, it was reconstituted in artificial membranes (planar lipid bilayer (PLB) system). The protein displayed channel-forming activity and the calculated conductance, voltage-dependence and ion selectivity values were similar to those of yVDAC1 and other members of VDAC family. This is the first time that yVDAC2 channel features are detected and characterized.

Cytoprotective activity of minocycline includes improvement of mitochondrial coupling: the importance of minocycline concentration and the presence of VDAC.

Available data indicate that minocycline, an antibiotic of the tetracycline family, has cytoprotective properties due to a direct interaction with mitochondria. Yet, the data in the case of isolated mitochondria suggest discrepant or even detrimental effect(s) of the interaction. We have studied the cytoprotective activity displayed by minocycline in the case of the yeast Saccharomyces cerevisiae cells pretreated with H2O2. We demonstrated that the activity of minocycline required the presence of VDAC (voltage-dependent anion-selective channel) and provided distinct improvement of mitochondrial coupling. In the case of isolated mitochondria, we verified that minocycline exhibited uncoupler activity when applied in micromolar concentrations. However, when added in nanomolar concentrations, minocycline was able to improve the level of coupling for isolated mitochondria. The coupling improvement effect was observed in mitochondria containing VDAC but not in Δpor1 mitochondria (depleted of VDAC1, termed here VDAC) and in both types of mitoplasts. Thus, properly low concentrations of minocycline within the cell in the vicinity of VDAC-containing mitochondria enable the improvement of energy coupling of mitochondria that contributes to cytoprotective activity of minocycline.

Minocycline exerts uncoupling and inhibiting effects on mitochondrial respiration through adenine nucleotide translocase inhibition.

The present study was aimed to provide a better understanding of the mitochondria-targeted actions of minocycline (MC), a second-generation tetracycline which has cytoprotective effects. Although the specific mechanisms underlying its activity remained elusive, considerable amounts of data indicated mitochondria as the primary pharmacological target of MC. Previous reports have shown that MC affects the oxygen-uptake rate by isolated mitochondria in different respiratory states. Here, we report on the effect of MC, in the range 50-200µM, on mitochondrial respiration. State 3 respiration titration with carboxyatractyloside revealed that MC inhibits the adenine nucleotide translocase. Furthermore, we analyze MC channel-forming capacity in the lipid membrane bilayer. Our results confirmed the crucial role of Δψ and showed a dependence on Ca(2+) for MC to have an effect on mitochondria. Our data also indicated that outer and inner mitochondrial membranes contribute differently to this effect, involving the presence of Δψ (the inner membrane) and VDAC (the outer membrane). Data from three isosmotic media indicate that MC does not increase the permeability of the inner membrane to protons or potassium. In addition, by using mitoplasts and ruthenium red, we showed that Ca(2+) uptake is not involved in the MC effect, suggesting involvement of VDAC in the MC interaction with the outer membrane. Our data contribute to unravel the mechanisms behind the mitochondria-targeted activity of the cytoprotective drug MC.

Verification of Hypsibius exemplaris Gasiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research.

Anhydrobiosis is considered to be an adaptation of important applicative implications because it enables resistance to the lack of water. The phenomenon is still not well understood at molecular level. Thus, a good model invertebrate species for the research is required. The best known anhydrobiotic invertebrates are tardigrades (Tardigrada), considered to be toughest animals in the world. Hypsibius. exemplaris is one of the best studied tardigrade species, with its name "exemplaris" referring to the widespread use of the species as a laboratory model for various types of research. However, available data suggest that anhydrobiotic capability of the species may be overestimated. Therefore, we determined anhydrobiosis survival by Hys. exemplaris specimens using three different anhydrobiosis protocols. We also checked ultrastructure of storage cells within formed dormant structures (tuns) that has not been studied yet for Hys. exemplaris. These cells are known to support energetic requirements of anhydrobiosis. The obtained results indicate that Hys. exemplaris appears not to be a good model species for anhydrobiosis research.

Communication between mitochondria and nucleus: putative role for VDAC in reduction/oxidation mechanism.

Voltage dependent anion channel (VDAC) was identified in 1976 and since that time has been extensively studied. It is well known that VDAC transports metabolites across the outer mitochondrial membrane. The simple transport function is indispensable for proper mitochondria functions and, consequently for cell activity, and makes VDAC crucial for a range of cellular processes including ATP rationing, Ca2+ homeostasis and apoptosis execution. Here, we review recent data obtained for Saccharomyces cerevisiae cells used as a model system concerning the putative role of VDAC in communication between mitochondria and the nucleus. The S. cerevisiae VDAC isoform known as VDAC1 (termed here YVDAC) mediates the cytosol reduction/oxidation (redox) state that contributes to regulation of expression and activity of cellular proteins including proteins that participate in protein import into mitochondria and antioxidant enzymes. Simultaneously, copper-and-zinc-containing superoxide dismutase (CuZnSOD) plays an important role in controlling YVDAC activity and expression levels. Thus, it is proposed that VDAC constitutes an important component of a regulatory mechanism based on the cytosol redox state.

Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis.

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-X(L) and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a "bioenergetic crisis." A decreased level of cellular energy may predispose cells to necrotic-like cell death.

Redox-Sensitive VDAC: A Possible Function as an Environmental Stress Sensor Revealed by Bioinformatic Analysis.

Voltage-dependent anion-selective channel (VDAC) allows the exchange of small metabolites and inorganic ions across the mitochondrial outer membrane. It is involved in complex interactions that regulate mitochondrial and cellular functioning. Many organisms have several VDAC paralogs that play distinct but poorly understood roles in the life and death of cells. It is assumed that such a large diversity of VDAC-encoding genes might cause physiological plasticity to cope with abiotic and biotic stresses known to impact mitochondrial function. Moreover, cysteine residues in mammalian VDAC paralogs may contribute to the reduction-oxidation (redox) sensor function based on disulfide bond formation and elimination, resulting in redox-sensitive VDAC (rsVDAC). Therefore, we analyzed whether rsVDAC is possible when only one VDAC variant is present in mitochondria and whether all VDAC paralogs present in mitochondria could be rsVDAC, using representatives of currently available VDAC amino acid sequences. The obtained results indicate that rsVDAC can occur when only one VDAC variant is present in mitochondria; however, the possibility of all VDAC paralogs in mitochondria being rsVDAC is very low. Moreover, the presence of rsVDAC may correlate with habitat conditions as rsVDAC appears to be prevalent in parasites. Thus, the channel may mediate detection and adaptation to environmental conditions.

The tardigrade Hypsibius exemplaris has the active mitochondrial alternative oxidase that could be studied at animal organismal level.

Mitochondrial alternative oxidase (AOX) is predicted to be present in mitochondria of several invertebrate taxa including tardigrades. Independently of the reason concerning the enzyme occurrence in animal mitochondria, expression of AOX in human mitochondria is regarded as a potential therapeutic strategy. Till now, relevant data were obtained due to heterologous AOX expression in cells and animals without natively expressed AOX. Application of animals natively expressing AOX could importantly contribute to the research. Thus, we decided to investigate AOX activity in intact specimens of the tardigrade Hypsibius exemplaris. We observed that H. exemplaris specimens' tolerance to the blockage of the mitochondrial respiratory chain (MRC) cytochrome pathway was diminished in the presence of AOX inhibitor and the inhibitor-sensitive respiration enabled the tardigrade respiration under condition of the blockage. Importantly, these observations correlated with relevant changes of the mitochondrial inner membrane potential (Δψ) detected in intact animals. Moreover, detection of AOX at protein level required the MRC cytochrome pathway blockage. Overall, we demonstrated that AOX activity in tardigrades can be monitored by the animals' behavior observation as well as by measurement of intact specimens' whole-body respiration and Δψ. Furthermore, it is also possible to check the impact of the MRC cytochrome pathway blockage on AOX level as well as AOX inhibition in the absence of the blockage on animal functioning. Thus, H. exemplaris could be consider as a whole-animal model suitable to study AOX.

[The role of mitochondria in the pathogenesis of Huntington's disease]. / Rola mitochondriów w patogenezie choroby Huntingtona.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative hereditary disorder that gradually robs affected individuals of memory, cognitive skills and normal movements. It is originated by the mutation of the gene encoding the huntingtin-protein (Htt). Htt with an abnormal stretch of above 35 glutamines in the N terminus (mHtt) results in HD. The observed symptoms result from the selective loss of neurons within the central nervous system, mainly in the striatum but also in the cortex. At present increasing numbers of data indicate that mitochondrial functioning is affected by mHtt and the resulting mitochondrial impairments may occur early enough to contribute to mHtt-induced toxicity and the HD pathogenic mechanism. Here, we review how mHtt might cause mitochondrial dysfunction by either perturbing transcription of nuclear-encoded mitochondrial proteins or by direct interaction with mitochondrial proteins. In addition, we discuss therapeutic opportunities for HD based on protection against mitochondrial dysfunction.

The TOM complex is involved in the release of superoxide anion from mitochondria.

Available data indicate that superoxide anion (O(2)(*-) ) is released from mitochondria, but apart from VDAC (voltage dependent anion channel), the proteins involved in its transport across the mitochondrial outer membrane still remain elusive. Using mitochondria of the yeast Saccharomyces cerevisiae mutant depleted of VDAC (Deltapor1 mutant) and the isogenic wild type, we studied the role of the TOM complex (translocase of the outer membrane) in the efflux of O(2)(*-) from the mitochondria. We found that blocking the TOM complex with the fusion protein pb(2)-DHFR decreased O(2)(*-) release, particularly in the case of Deltapor1 mitochondria. We also observed that the effect of the TOM complex blockage on O(2)(*-) release from mitochondria coincided with the levels of O(2)(*-) release as well as with levels of Tom40 expression in the mitochondria. Thus, we conclude that the TOM complex participates in O(2)(*-) release from mitochondria.

Voltage-dependent anion channel isoform 3 as a potential male contraceptive drug target.

Voltage-dependent anion channel isoform 3 (VDAC3), a channel in the mitochondrial outer membrane, has been suggested to play a role in the regulation of ATP transport and Ca2+ homeostasis. These processes are regarded as important for spermatozoa motility. Accordingly, in previous years, mutations in the VDAC3-encoding gene were detected in spermatozoa with low motility from infertile patients. Therefore, it can be assumed that these mutations would cause alteration of the structure and/or charge of the VDAC3 channel. The review is focused on current knowledge about contribution of VDAC3 activity to human spermatozoa motility and morphology. We also discuss the possibility of designing new molecules that could specifically block the VDAC3 channel and consequently act as male contraceptives.

Recombinant yeast VDAC2: a comparison of electrophysiological features with the native form.

Voltage-dependent anion channel isoform 2 of the yeast Saccharomyces cerevisiae (yVDAC2) was believed for many years to be devoid of channel activity. Recently, we isolated yVDAC2 and showed that it exhibits channel-forming activity in the planar lipid bilayer system when in its so-called native form. Here, we describe an alternative strategy for yVDAC2 isolation, through heterologous expression in bacteria and refolding in vitro. Recombinant yVDAC2, like its native form, is able to form voltage-dependent channels. However, some differences between native and recombinant yVDAC2 emerged in terms of voltage dependence and ion selectivity, suggesting that, in this specific case, the recombinant protein might be depleted of post-translational modification(s) that occur in eukaryotic cells.

VDAC-Targeted Drugs Affecting Cytoprotection and Mitochondrial Physiology in Cerebrovascular and Cardiovascular Diseases.

BACKGROUND: Cerebrovascular and cardiovascular diseases are caused by impairment of the brain and/or heart circulation. Insufficient blood flow results in decreased oxygen delivery (ischemia), which affects mitochondrial functioning and consequently leads to insufficient ATP production. The predominant mitochondrial outer membrane protein, the voltage dependent anion selective channel (VDAC), is considered to be crucial for mitochondrial functioning. In human mitochondria, as in other vertebrates, three isoforms of VDAC (VDAC1-VDAC3) are present, and they likely play different roles. OBJECTIVE: In this review, we summarize the available data concerning VDAC involvement in cardiovascular and cerebrovascular diseases with regard to VDAC isoforms and discuss the use of possible VDAC-related intervention targets as well as known VDAC-interacting and cytoprotection- conferring molecules in the treatment of cerebrovascular and cardiovascular diseases. METHOD AND RESULTS: The suitable references on disorders defined as cerebrovascular and cardiovascular diseases as well as VDAC contribution to these conditions were searched using PubMed and ClinicalTrials.gov databases. The review is based on the 138 carefully selected articles. CONCLUSION: Mitochondrial dysfunction triggered by changes in VDAC properties undoubtedly contributes to cell death and related diseases, including cerebrovascular and cardiovascular diseases. Thus, beside diagnostic application, modulation of VDAC activity, including its isoforms, is thus of great importance for the development of efficient therapeutic interventions. Moreover, identification of VDAC-interacting molecules that protect against mitochondrial dysfunction and cell death seems to be of great importance.

Human VDAC isoforms differ in their capability to interact with minocycline and to contribute to its cytoprotective activity.

It has been previously demonstrated that cytoprotective activity displayed by minocycline in the case of the yeast Saccharomyces cerevisiae cells pretreated with H2O2 requires the presence of functional VDAC (YVDAC1). Thus, we decided to transform YVDAC1-depleted yeast cells (Δpor1 cells) with plasmids expressing human VDAC isoforms (HVDAC1, HVDAC2, HVDAC3) to estimate their involvement in the minocycline cytoprotective effect. We observed that only expression of HVDAC3 in Δpor1 cells provided minocycline-mediated cytoprotection against H2O2 although all human isoforms are functional in Δpor1 cells. The observation appears to be important for on-going discussion concerning VDAC isoform roles in mitochondria and cell functioning.

The Association of VDAC with Cell Viability of PC12 Model of Huntington's Disease.

It is becoming increasingly apparent that mitochondria dysfunction plays an important role in the pathogenesis of Huntington's disease (HD), but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning, it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48 h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability, we decided to estimate the effect of Htt and mHtt expression lasted for 48 h on VDAC functioning. Therefore, we isolated VDAC from the cells and tested the preparations by black lipid membrane system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations, we observed a dominant quantitative content of VDAC1, and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearances in cells, may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application.

The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.

Minocycline mediated mitochondrial cytoprotection: premises for therapy of cerebrovascular and neurodegenerative diseases.

In the last decades, emerging molecular targets for ischemic neuroprotection and regeneration have been postulated. This fact allowed that classical drugs with well established therapeutic applications might be used in cerebrovascular diseases as well as neurodegenerative diseases. Minocycline is a commonly used antibiotic of the tetracycline family (7-dimethylamino-6-dimethyl-6-deoxytetracycline) which reveals cytoprotective capability and potential use in treatment of different diseases. Here, we discuss the literature concerning minocycline. The available data indicate that the antibiotic has multi-faceted effects on cell functions and, consequently, a number of clinical properties that are useful and/or could be useful for treatment of different diseases including bacterial infections, cancer, autoimmune disorders, ischemia as well as neurodegenerative and psychiatric diseases. Thus, application of minocycline as a therapeutic agent is the subject of clinical trials for various diseases. It is also evident that minocycline-mediated cytoprotection, including neuroprotection, is an important aspect of its clinical application. Here, we have reviewed the basis of the minocycline activity as well as different studies indicating that minocycline can be used as potential therapeutic agent in both cerebrovascular and neurodegenerative diseases in human.

Phylogenetic analysis of mitochondrial outer membrane ß-barrel channels.

Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have ß-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial ß-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane ß-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta.

Viability of Saccharomyces cerevisiae cells following exposure to H2O2 and protective effect of minocycline depend on the presence of VDAC.

Proteins involved in apoptosis are still a matter of debate. Therefore, we decided to check the effect of the presence of VDAC (voltage dependent anion selective channel) on viability of Saccharomyces cerevisiae cells following their exposure to H(2)O(2) that is known to induce apoptosis both in S. cerevisiae and in mammalian cells. Mitochondria of S. cerevisiae contain only one channel-forming VDAC isoform (VDAC1), which simplifies studies on the channel. Using S. cerevisiae mutant depleted of VDAC1 (termed here VDAC) and the isogenic wild type, we have shown that VDAC is important for protection of S. cerevisiae cells against H(2)O(2) treatment, particularly in exponential growth phase that is known to be more affected by H(2)O(2). The increased viability of H(2)O(2) pretreated exponentially growing cells containing VDAC was accompanied by clear changes of the cytosol redox state and was potentiated by minocycline, an antibiotic of the tetracycline family that displays cytoprotective potency. The protective effect of minocycline also coincided with distinct changes of cytosol redox state. Thus, we conclude that the ability to change the cytosol redox state following exposure to H(2)O(2) or/and minocycline appears to be an intrinsic feature of exponentially growing cells (young cells) containing VDAC. Moreover, the ability seems to be crucial for both cell viability and protective effect of minocycline.