Mitocentricity.

Worldwide, interest in mitochondria is constantly growing, as evidenced by scientific statistics, and studies of the functioning of these organelles are becoming more prevalent than studies of other cellular structures. In this analytical review, mitochondria are conditionally placed in a certain cellular center, which is responsible for both energy production and other non-energetic functions, without which the existence of not only the eukaryotic cell itself, but also the entire organism is impossible. Taking into account the high multifunctionality of mitochondria, such a fundamentally new scheme of cell functioning organization, including mitochondrial management of processes that determine cell survival and death, may be justified. Considering that this issue is dedicated to the memory of V. P. Skulachev, who can be called mitocentric, due to the history of his scientific activity almost entirely aimed at studying mitochondria, this work examines those aspects of mitochondrial functioning that were directly or indirectly the focus of attention of this outstanding scientist. We list all possible known mitochondrial functions, including membrane potential generation, synthesis of Fe-S clusters, steroid hormones, heme, fatty acids, and CO2. Special attention is paid to the participation of mitochondria in the formation and transport of water, as a powerful biochemical cellular and mitochondrial regulator. The history of research on reactive oxygen species that generate mitochondria is subject to significant analysis. In the section "Mitochondria in the center of death", special emphasis is placed on the analysis of what role and how mitochondria can play and determine the program of death of an organism (phenoptosis) and the contribution made to these studies by V. P. Skulachev.

Mitochondrial Network: Electric Cable and More.

Mitochondria in a cell can unite and organize complex, extended structures that occupy the entire cellular volume, providing an equal supply with energy in the form of ATP synthesized in mitochondria. In accordance with the chemiosmotic concept, the oxidation energy of respiratory substrates is largely stored in the form of an electrical potential difference on the inner membrane of mitochondria. The theory of the functioning of extended mitochondrial structures as intracellular electrical wires suggests that mitochondria provide the fastest delivery of electrical energy through the cellular volume, followed by the use of this energy for the synthesis of ATP, thereby accelerating the process of ATP delivery compared to the rather slow diffusion of ATP in the cell. This analytical review gives the history of the cable theory, lists unsolved critical problems, describes the restructuring of the mitochondrial network and the role of oxidative stress in this process. In addition to the already proven functioning of extended mitochondrial structures as electrical cables, a number of additional functions are proposed, in particular, the hypothesis is put forth that mitochondrial networks maintain the redox potential in the cellular volume, which may vary depending on the physiological state, as a result of changes in the three-dimensional organization of the mitochondrial network (fragmentation/fission-fusion). A number of pathologies accompanied by a violation of the redox status and the participation of mitochondria in them are considered.

Isn't It Time for Establishing Mitochondrial Nomenclature Breaking Mitochondrial Paradigm?

In this work, we decided to initiate a discussion concerning heterogeneity of mitochondria, suggesting that it is time to build classification of mitochondria, like the one that exists for their progenitors, α-proteobacteria, proposing possible separation of mitochondrial strains and maybe species. We continue to adhere to the general line that mitochondria are friends and foes: on the one hand, they provide the cell and organism with the necessary energy and signaling molecules, and, on the other hand, participate in destruction of the cell and the organism. Current understanding that the activity of mitochondria is not only limited to energy production, but also that these alternative non-energetic functions are unique and irreplaceable in the cell, allowed us to speak about the strong subordination of the entire cellular metabolism to characteristic functional manifestations of mitochondria. Mitochondria are capable of producing not only ATP, but also iron-sulfur clusters, steroid hormones, heme, reactive oxygen and nitrogen species, participate in thermogenesis, regulate cell death, proliferation and differentiation, participate in detoxification, etc. They are a mandatory attribute of eukaryotic cells, and, so far, no eukaryotic cells performing a non-parasitic or non-symbiotic life style have been found that lack mitochondria. We believe that the structural-functional intracellular, intercellular, inter-organ, and interspecific diversity of mitochondria is large enough to provide grounds for creating a mitochondrial nomenclature. The arguments for this are given in this analytical work.

Is the Mitochondrial Membrane Potential (∆Ψ) Correctly Assessed? Intracellular and Intramitochondrial Modifications of the ∆Ψ Probe, Rhodamine 123.

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.

Do Extracellular Vesicles Derived from Mesenchymal Stem Cells Contain Functional Mitochondria?

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV.

Mitochondrial membrane potential.

The mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III and IV) is an essential component in the process of energy storage during oxidative phosphorylation. Together with the proton gradient (ΔpH), ΔΨm forms the transmembrane potential of hydrogen ions which is harnessed to make ATP. The levels of ΔΨm and ATP in the cell are kept relatively stable although there are limited fluctuations of both these factors that can occur reflecting normal physiological activity. However, sustained changes in both factors may be deleterious. A long-lasting drop or rise of ΔΨm vs normal levels may induce unwanted loss of cell viability and be a cause of various pathologies. Among other factors, ΔΨm plays a key role in mitochondrial homeostasis through selective elimination of dysfunctional mitochondria. It is also a driving force for transport of ions (other than H+) and proteins which are necessary for healthy mitochondrial functioning. We propose additional potential mechanisms for which ΔΨm is essential for maintenance of cellular health and viability and provide recommendations how to accurately measure ΔΨm in a cell and discuss potential sources of artifacts.

Intercellular Signalling Cross-Talk: To Kill, To Heal and To Rejuvenate.

Intercellular cross-talk is a fundamental process for spreading cellular signals between neighbouring and distant cells to properly regulate their metabolism, to coordinate homeostasis, adaptation and survival as a functional tissue and organ. In this review, we take a close molecular view of the underpinning molecular mechanisms of such complex intercellular communications. There are several studied forms of cell-to-cell communications considered crucial for the maintenance of multicellular organisms. The most explored is paracrine signalling which is realised through the release of diffusible signalling factors (e.g., hormones or growth factors) from a donor cell and taken up by a recipient cell. More challenging is communication which also does not require the direct contact of cells but is organised through the release of named signalling factors embedded in membranous structures. This mode of cell-to-cell communication is executed through the transfer of extracellular vesicles. Two other types of cellular cross-communication require direct contact of communicating cells. In one type, cells are connected by gap junctions which regulate permeation of chemical signals addressed to a neighbouring cell. Another type of cell communication is organised to provide a cytosolic continuum of adjacent cells joined by different tiny cell membrane extensions coined tunnelling nanotubes. In this review, we consider the various cell communication modes in the heart, and examples of processes in non-cardiac cells which may have mechanistic parallels with cardiovascular cells.

Antibiotic streptolydigin requires noncatalytic Mg2+ for binding to RNA polymerase.

Multisubunit RNA polymerase, an enzyme that accomplishes transcription in all living organisms, is a potent target for antibiotics. The antibiotic streptolydigin inhibits RNA polymerase by sequestering the active center in a catalytically inactive conformation. Here, we show that binding of streptolydigin to RNA polymerase strictly depends on a noncatalytic magnesium ion which is likely chelated by the aspartate of the bridge helix of the active center. Substitutions of this aspartate may explain different sensitivities of bacterial RNA polymerases to streptolydigin. These results provide the first evidence for the role of noncatalytic magnesium ions in the functioning of RNA polymerase and suggest new routes for the modification of existing and the design of new inhibitors of transcription.

The mitochondrion as a key regulator of ischaemic tolerance and injury.

Vascular pathologies pose a significant health problem because of their wide prevalence and high impact on the rate of mortality. Blockade of blood flow in major blood vessels leads to ischaemia associated with oxidative stress, where mitochondria act as a major source of reactive oxygen species (ROS). While low levels of ROS perform a necessary function in normal cellular signalling and metabolism, elevated levels under pathological conditions are detrimental both at the cell and organ level. While cellular oxygenation is necessary to maintain tissue viability, a key pathological occurrence when restoring blood flow to ischaemic tissues is the subsequent burst of ROS generation following reoxygenation, resulting in a cascade of ROS-induced ROS release. This oxygen 'paradox' is a constraint in clinical practice, that is, the need for rapid and maximal restoration of blood flow while at the same time minimising the harmful impact of reperfusion injury on damaged tissues. Mitochondria play a central role in many signalling pathways, including cardioprotection against ischaemic injury and ROS signalling, thus the main target of any anti-ischaemic protective or post-injury therapeutic strategy should include mitochondria. At present, one of the most effective strategies that provide mitochondrial tolerance to ischaemia is ischaemic preconditioning. In addition, pharmacological preconditioning which mimics intrinsic natural protective mechanisms has proven effective at priming biological mechanisms to confront ischaemic damage. This review will discuss the role of mitochondria in contributing to acute ischaemia-reperfusion (IR) injury, and mechanisms of cardioprotection in respect to mitochondrial signalling pathways.

Targeting Mitochondria for Cancer Treatment.

There is an increasing accumulation of data on the exceptional importance of mitochondria in the occurrence and treatment of cancer, and in all lines of evidence for such participation, there are both energetic and non-bioenergetic functional features of mitochondria. This analytical review examines three specific features of adaptive mitochondrial changes in several malignant tumors. The first feature is characteristic of solid tumors, whose cells are forced to rebuild their energetics due to the absence of oxygen, namely, to activate the fumarate reductase pathway instead of the traditional succinate oxidase pathway that exists in aerobic conditions. For such a restructuring, the presence of a low-potential quinone is necessary, which cannot ensure the conventional conversion of succinate into fumarate but rather enables the reverse reaction, that is, the conversion of fumarate into succinate. In this scenario, complex I becomes the only generator of energy in mitochondria. The second feature is the increased proliferation in aggressive tumors of the so-called mitochondrial (peripheral) benzodiazepine receptor, also called translocator protein (TSPO) residing in the outer mitochondrial membrane, the function of which in oncogenic transformation stays mysterious. The third feature of tumor cells is the enhanced retention of certain molecules, in particular mitochondrially directed cations similar to rhodamine 123, which allows for the selective accumulation of anticancer drugs in mitochondria. These three features of mitochondria can be targets for the development of an anti-cancer strategy.

Mitochondrial ATP Synthase and Mild Uncoupling by Butyl Ester of Rhodamine 19, C4R1.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the ß-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and ß-subunits, close to the anionic amino acid residues of the ß-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

Stepwise mechanism for transcription fidelity.

BACKGROUND: Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive. RESULTS: Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH. CONCLUSIONS: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

Neuroprotective Potential of Mild Uncoupling in Mitochondria. Pros and Cons.

There has been an explosion of interest in the use of uncouplers of oxidative phosphorylation in mitochondria in the treatment of several pathologies, including neurological ones. In this review, we analyzed all the mechanisms associated with mitochondrial uncoupling and the metabolic and signaling cascades triggered by uncouplers. We provide a full set of positive and negative effects that should be taken into account when using uncouplers in experiments and clinical practice.

Lessons from the Discovery of Mitochondrial Fragmentation (Fission): A Review and Update.

Thirty-five years ago, we described fragmentation of the mitochondrial population in a living cell into small vesicles (mitochondrial fission). Subsequently, this phenomenon has become an object of general interest due to its involvement in the process of oxidative stress-related cell death and having high relevance to the incidence of a pathological phenotype. Tentatively, the key component of mitochondrial fission process is segregation and further asymmetric separation of a mitochondrial body yielding healthy (normally functioning) and impaired (incapable to function in a normal way) organelles with subsequent decomposition and removal of impaired elements through autophagy (mitophagy). We speculate that mitochondria contain cytoskeletal elements, which maintain the mitochondrial shape, and also are involved in the process of intramitochondrial segregation of waste products. We suggest that perturbation of the mitochondrial fission/fusion machinery and slowdown of the removal process of nonfunctional mitochondrial structures led to the increase of the proportion of impaired mitochondrial elements. When the concentration of malfunctioning mitochondria reaches a certain threshold, this can lead to various pathologies, including aging. Overall, we suggest a process of mitochondrial fission to be an essential component of a complex system controlling a healthy cell phenotype. The role of reactive oxygen species in mitochondrial fission is discussed.

The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase.

Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg2+ ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg2+ ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the beta' subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg2+ binding to free RNAP below detection limits, the apparent affinity to Mg2+ in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg2+.

Mitochondrial Aging: Is There a Mitochondrial Clock?

Fragmentation (fission) of mitochondria, occurring in response to oxidative challenge, leads to heterogeneity in the mitochondrial population. It is assumed that fission provides a way to segregate mitochondrial content between the "young" and "old" phenotype, with the formation of mitochondrial "garbage," which later will be disposed. Fidelity of this process is the basis of mitochondrial homeostasis, which is disrupted in pathological conditions and aging. The asymmetry of the mitochondrial fission is similar to that of their evolutionary ancestors, bacteria, which also undergo an aging process. It is assumed that mitochondrial markers of aging are recognized by the mitochondrial quality control system, preventing the accumulation of dysfunctional mitochondria, which normally are subjected to disposal. Possibly, oncocytoma, with its abnormal proliferation of mitochondria occupying the entire cytoplasm, represents the case when segregation of damaged mitochondria is impaired during mitochondrial division. It is plausible that mitochondria contain a "clock" which counts the degree of mitochondrial senescence as the extent of flagging (by ubiquitination) of damaged mitochondria. Mitochondrial aging captures the essence of the systemic aging which must be analyzed. We assume that the mitochondrial aging mechanism is similar to the mechanism of aging of the immune system which we discuss in detail.

The role of myoglobin degradation in nephrotoxicity after rhabdomyolysis.

The fate of myoglobin in renal cells was explored in an animal model of rhabdomyolysis known as the pathology highly related to oxidative stress resulting in impairment of renal functioning. The working hypothesis was that the proper degradation of myoglobin in rhabdomyolytic kidney can activate the reparative processes in the tissue. We found that incubation of myoglobin with kidney cells causes its accumulation in the cytoplasm. In rhabdomyolytic rats, the level of heme and free iron in cytoplasm and mitochondria of kidney cells is remarkably increased while inhibition of proteolysis results in further elevation of myoglobin content in the renal tissue. Heme oxygenase and ferritin levels were found to be increased in the kidney tissue at rhabdomyolysis and simulating conditions performed by i/v injection of myoglobin. In addition, the level of peroxidized lipids was high in rhabdomyolytic kidney and became even higher after inhibition of proteolysis by aprotinin. Elevated levels of carbonylated proteins were also observed after rhabdomyolysis, however, if prior to induction of rhabdomyolysis the injection of myoglobin was done, the level of carbonylated proteins dropped versus unprimed kidney tissue thus affording protection to the kidney against oxidative stress. Injection of myoglobin to the rat results in impairment of renal functioning and inhibition of myoglobin degradation in the rhabdomyolytic animal aggravates acute renal failure, demonstrating that degradation of myoglobin is somehow beneficial although it may result in undesired release of free iron which can participate in toxic redox cycling.

Stimulation of kainate toxicity by zinc in cultured cerebellar granule neurons and the role of mitochondria in this process.

Zinc chloride (0.01 mM kept for 3h) is not toxic to cultured cerebellar granule neurons (CGNs) while kainate (0.1mM kept for 3h) demonstrates some but very low toxicity towards these cells. Measurements of the relative intraneuronal zinc ion concentration showed that increase in [Zn(2+)](i) under the simultaneous action of ZnCl(2) and kainate was significantly stronger compared to their separate action. Simultaneous treatment of CGNs with kainate and zinc chloride caused the swelling of neuronal mitochondria and consequent intensive neuronal death, which was totally prevented by NBQX (an AMPA/kainate-receptors blocker) or ruthenium red (a mitochondrial Ca(2+) uniporter blocker). These data imply that Zn(2+) synergistically to kainate increase their separate toxic effects on mitochondria leading to rapid neuronal death.

Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin.

Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria.

Structural basis of transcription inhibition by antibiotic streptolydigin.

Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 A resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 A away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.