VDAC as a Cellular Hub: Docking Molecules and Interactions.

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane [...].

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.

How the Geomagnetic Field Influences Life on Earth - An Integrated Approach to Geomagnetobiology.

Earth is one of the inner planets of the Solar System, but - unlike the others - it has an oxidising atmosphere, relatively stable temperature, and a constant geomagnetic field (GMF). The GMF does not only protect life on Earth against the solar wind and cosmic rays, but it also shields the atmosphere itself, thus creating relatively stable environmental conditions. What is more, the GMF could have influenced the origins of life: organisms from archaea to plants and animals may have been using the GMF as a source of spatial information since the very beginning. Although the GMF is constant, it does undergo various changes, some of which, e.g. a reversal of the poles, weaken the field significantly or even lead to its short-term disappearance. This may result in considerable climatic changes and an increased frequency of mutations caused by the solar wind and cosmic radiation. This review analyses data on the influence of the GMF on different aspects of life and it also presents current knowledge in the area. In conclusion, the GMF has a positive impact on living organisms, whereas a diminishing or disappearing GMF negatively affects living organisms. The influence of the GMF may also be an important factor determining both survival of terrestrial organisms outside Earth and the emergence of life on other planets.

The Open State Selectivity of the Bean Seed VDAC Depends on Stigmasterol and Ion Concentration.

The voltage-dependent anion channel (VDAC) is the major pathway for metabolites and ions transport through the mitochondrial outer membrane. It can regulate the flow of solutes by switching to a low conductance state correlated with a selectivity reversal, or by a selectivity inversion of its open state. The later one was observed in non-plant VDACs and is poorly characterized. We aim at investigating the selectivity inversion of the open state using plant VDAC purified from Phaseolus coccineus (PcVDAC) to evaluate its physiological role. Our main findings are: (1) The VDAC selectivity inversion of the open state occurs in PcVDAC, (2) Ion concentration and stigmasterol affect the occurrence of the open state selectivity inversion and stigmasterol appears to interact directly with PcVDAC. Interestingly, electrophysiological data concerning the selectivity inversion of the PcVDAC open state suggests that the phenomenon probably does not have a significant physiological effect in vivo.

The Diversity of the Mitochondrial Outer Membrane Protein Import Channels: Emerging Targets for Modulation.

The functioning of mitochondria and their biogenesis are largely based on the proper function of the mitochondrial outer membrane channels, which selectively recognise and import proteins but also transport a wide range of other molecules, including metabolites, inorganic ions and nucleic acids. To date, nine channels have been identified in the mitochondrial outer membrane of which at least half represent the mitochondrial protein import apparatus. When compared to the mitochondrial inner membrane, the presented channels are mostly constitutively open and consequently may participate in transport of different molecules and contribute to relevant changes in the outer membrane permeability based on the channel conductance. In this review, we focus on the channel structure, properties and transported molecules as well as aspects important to their modulation. This information could be used for future studies of the cellular processes mediated by these channels, mitochondrial functioning and therapies for mitochondria-linked diseases.

Integrative description of bisexual Paramacrobiotus experimentalis sp. nov. (Macrobiotidae) from republic of Madagascar (Africa) with microbiome analysis.

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological - specimen size dimorphism; (ii) structural (primary sexual characteristics) - females have an unpaired ovary while males have an unpaired testis and (iii) molecular - heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89-92%) and Firmicutes (6-7%). In the case of samples from tardigrades' laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51-90%) and Bacteroides (9-48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified - Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.

Insight into the Phytoremediation Capability of Brassica juncea (v. Malopolska): Metal Accumulation and Antioxidant Enzyme Activity.

Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. Brassica species are known to display hyperaccumulation capability. Brassica juncea (Indiana mustard) v. Malopolska plants were exposed to trace elements, i.e., cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn), at a concentration of 50 µM and were then harvested after 96 h for analysis. We observed a high index of tolerance (IT), higher than 90%, for all B. juncea plants treated with the four metals, and we showed that Cd, Cu, Pb, and Zn accumulation was higher in the above-ground parts than in the roots. We estimated the metal effects on the generation of reactive oxygen species (ROS) and the levels of protein oxidation, as well as on the activity and gene expression of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The obtained results indicate that organo-specific ROS generation was higher in plants exposed to essential metal elements (i.e., Cu and Zn), compared with non-essential ones (i.e., Cd and Pb), in conjunction with SOD, CAT, and APX activity and expression at the level of encoding mRNAs and existing proteins. In addition to the potential usefulness of B. juncea in the phytoremediation process, the data provide important information concerning plant response to the presence of trace metals.

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.

The emerging picture of the mitochondrial protein import complexes of Amoebozoa supergroup.

BACKGROUND: The existence of mitochondria-related organelles (MROs) is proposed for eukaryotic organisms. The Amoebozoa includes some organisms that are known to have mitosomes but also organisms that have aerobic mitochondria. However, the mitochondrial protein apparatus of this supergroup remains largely unsampled, except for the mitochondrial outer membrane import complexes studied recently. Therefore, in this study we investigated the mitochondrial inner membrane and intermembrane space complexes, using the available genome and transcriptome sequences. RESULTS: When compared with the canonical cognate complexes described for the yeast Saccharomyces cerevisiae, amoebozoans with aerobic mitochondria, display lower differences in the number of subunits predicted for these complexes than the mitochondrial outer membrane complexes, although the predicted subunits appear to display different levels of diversity in regard to phylogenetic position and isoform numbers. For the putative mitosome-bearing amoebozoans, the number of predicted subunits suggests the complex elimination distinctly more pronounced than in the case of the outer membrane ones. CONCLUSION: The results concern the problem of mitochondrial and mitosome protein import machinery structural variability and the reduction of their complexity within the currently defined supergroup of Amoebozoa. This results are crucial for better understanding of the Amoebozoa taxa of both biomedical and evolutionary importance.

Protein import complexes in the mitochondrial outer membrane of Amoebozoa representatives.

BACKGROUND: An ancestral trait of eukaryotic cells is the presence of mitochondria as an essential element for function and survival. Proper functioning of mitochondria depends on the import of nearly all proteins that is performed by complexes located in both mitochondrial membranes. The complexes have been proposed to contain subunits formed by proteins common to all eukaryotes and additional subunits regarded as lineage specific. Since Amoebozoa is poorly sampled for the complexes we investigated the outer membrane complexes, namely TOM, TOB/SAM and ERMES complexes, using available genome and transcriptome sequences, including transcriptomes assembled by us. RESULTS: The results indicate differences in the organization of the Amoebozoa TOM, TOB/SAM and ERMES complexes, with the TOM complex appearing to be the most diverse. This is reflected by differences in the number of involved subunits and in similarities to the cognate proteins of representatives from different supergroups of eukaryotes. CONCLUSIONS: The obtained results clearly demonstrate structural variability/diversity of these complexes in the Amoebozoa lineage and the reduction of their complexity as compared with the same complexes of model organisms.

[Mitochondrial protein import complexes - a phylogenetic perspective]. / Organizacja aparatu importu bialka do mitochondriów - perspektywa filogenetyczna.

The proper functioning of mitochondria and consequently of eukaryotic cells requires protein import into mitochondria. The import proceeds due to the presence of different pathways formed by sophisticated complexes known as the import complexes. The complexes are located in all mitochondrial compartments including the both mitochondrial membranes. Here we collect data concerning the organization of the import complexes and available for representatives of currently distinguished eukaryotic lineages. Despite the lack of many data, the emerging picture indicates at differentiation of the complex organization, particularly observed for the TOM complex. This, in turn, implicates interesting issues for further discussion concerning mitochondria evolution and the knowledge practical application.

Molecular identification of first putative aquaporins in snails.

Aquaporins (AQPs), also known as water channel proteins, are members of a large protein family termed Major Intrinsic Proteins (MIP). The mammalian AQPs have been most comprehensively described, while knowledge about AQPs in invertebrates is limited mainly to insects. Not a single AQP protein has been described in snails to date. Consequently, we decided to search for the proteins in gastropod representatives, namely Lymnaea stagnalis, Catascopia occulta, and Stagnicola palustris (Mollusca; Gastropoda; Pulmonata; Lymnaeidae). Using the molecular approach, we identified L. stagnalis, C. occulta, and S. palustris open reading frames (ORFs) showing homology to AQP genes available in GenBank database, and characterized the encoded proteins, referred to as LsAQP1, CoAQP1, and SpAQP1, respectively. The putative snail aquaporins contain 299 amino acids, have a molecular mass of about 32 kDa, display the general AQP topology and three-dimensional structure congruent with orthodox AQPs, i.e., water-specific ones. Due to high levels of similarity in their characteristics, LsAQP1 was chosen for further studies, as the obtained results were supposed to be applicable for CoAQP1 and SpAQP1. Expression analysis revealed the presence of LsAQP1 transcript in the digestive tract, the cerebral ganglia, the kidney, the reproductive system, and the foot, suggesting that LsAQP1 as well as CoAQP1 and SpAQP1 are ubiquitous proteins and may play important roles in many essential water transport processes. The role appears to be confirmed by results of the yeast growth complementation assay pointing at functionality of LsAQP1. Thus, the obtained results support the AQP expression in gastropod tissues for the first time.

Recovery from anhydrobiosis in the tardigrade Paramacrobiotus experimentalis: Better to be young than old and in a group than alone.

Desiccation-tolerant organisms can survive dehydration in a state of anhydrobiosis. Tardigrades can recover from anhydrobiosis at any life stage and are considered among the toughest animals on Earth. However, the factors that influence recovery from anhydrobiosis are not well understood. The study aimed to evaluate the effect of sex, age, the presence of other individuals and the combination of the number and duration of anhydrobiosis episodes on the recovery of Paramacrobiotus experimentalis. The activity of 1200 individuals for up to 48 h after rehydration was evaluated using analysis of variance (ANOVA). Age was the main factor influencing return to activity, followed by the combination of number and duration of anhydrobiosis episodes, influence of the presence of other individuals, and sex. More individuals returned to activity after repeated short than repeated long anhydrobiosis episodes and older individuals were less likely to recover than younger individuals. In addition, when compared to single animals, the presence of other individuals resulted in higher number of active animals after dehydration and rehydration. The effect of sex was significant, but there was no general tendency for one sex to recover from anhydrobiosis better than the other one. The results contribute to a better understanding of the anhydrobiosis ability of Paramacrobiotus experimentalis and provide background for full explanation of molecular, cellular and environmental mechanisms of anhydrobiosis.

Potential oxidative stress related targets of mitochondria-focused therapy of PTSD.

Post-traumatic stress disorder (PTSD) remains a highly prevalent, under-diagnosed, and under-treated psychiatric disorder that often deteriorates over time, and is highly comorbid with major depressive disorder, suicidality, and substance use disorder. Several biomarkers have been proposed but have yet to be implemented into clinical practice. Treatments, including selective serotonin reuptake inhibitors, are efficacious in only a small number of patients, which underscores the need to develop novel, efficient treatments. Mitochondrial dysfunction resulting from chronic oxidative stress has been linked with both altered neurotransmitter signaling and the inflammatory response. Hereinafter, we discuss mechanisms by which mitochondrial dysfunction may contribute to the development of PTSD symptoms, and how these may even increase PTSD susceptibility. We also highlight possible therapeutic targets to reduce oxidative stress to prevent or treat PTSD symptoms.

The effect of hypomagnetic field on survival and mitochondrial functionality of active Paramacrobiotus experimentalis females and males of different age.

Even for tardigrades, often called the toughest animals on Earth, a hypomagnetic field (HMF) is an extreme environment. However, studies on the effect of HMF on tardigrades and other invertebrates are scarce. Mitochondria play an important role in an organism's response to extreme conditions. The effect of HMF on the mitochondrial inner membrane potential (Δψ), a well-known marker of mitochondria functionality, shows that mitochondria are very sensitive to HMF. To measure the HMF effect on Paramacrobiotus experimentalis, we calculated the tardigrade survival rate and Δψ level after HMF treatments of different durations. We also estimated the relationship between the age and sex of the tardigrade and the HMF effect. We observed age- and sex-related differences in Δψ and found that Δψ changes after HMF treatment were dependent on its duration as well as the animal's age and sex. Furthermore, active P. experimentalis individuals displayed a high survival rate after HMF treatment. The data may contribute to the understanding of tardigrade aging and their resistance to extreme conditions including HMF, which in turn may be useful for future space explorations.

How long can tardigrades survive in the anhydrobiotic state? A search for tardigrade anhydrobiosis patterns.

Anhydrobiosis is a desiccation tolerance that denotes the ability to survive almost complete dehydration without sustaining damage. The knowledge on the survival capacity of various tardigrade species in anhydrobiosis is still very limited. Our research compares anhydrobiotic capacities of four tardigrade species from different genera, i.e. Echiniscus testudo, Paramacrobiotus experimentalis, Pseudohexapodibius degenerans and Macrobiotus pseudohufelandi, whose feeding behavior and occupied habitats are different. Additionally, in the case of Ech. testudo, we analyzed two populations: one urban and one from a natural habitat. The observed tardigrade species displayed clear differences in their anhydrobiotic capacity, which appear to be determined by the habitat rather than nutritional behavior of species sharing the same habitat type. The results also indicate that the longer the state of anhydrobiosis lasts, the more time the animals need to return to activity.

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.