Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target

Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O₂ with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.     

Synthesis and characterization of latex-protein complexes from different antigens of Toxoplasma gondii for immunoagglutination assays

The acute phase recombinant protein of Toxoplasma gondii P22Ag was expressed and purified and the homogenate of the parasite was obtained from an infected mouse. These antigens were used to produce latex-protein complexes (LPC) through physical adsorption and chemical coupling onto different latexes, with the aim of producing immunoagglutination (IA) reagents able to detect recently acquired toxoplasmosis. Polystyrene and “core-shell” latexes where employed, exhibiting varied particle size, functionality (carboxyl or epoxy), and charge density. In sensitization experiments for producing LPC, the recombinant protein showed better coupling efficiency onto the particles surface than the homogenate and this could be explained by the complex mixture of the homogenate, which includes a large number of proteins of different molecular mass, isoelectric points, and hydrophobicity. The synthesized LPC were employed in IA assays. To this effect, the agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. Experiments against control sera were performed to evaluate the performance of various LPC and it was observed that the IA test based on P22Ag and the carboxylated latex of 350 nm of particle diameter allowed a good discrimination between acute sera and chronic/negative ones. The proposed test is cheap, rapid, and easy to implement.     

Impact of Hydrogen Sulfide on Mitochondrial and Bacterial Bioenergetics

This review focuses on the effects of hydrogen sulfide (H₂S) on the unique bioenergetic molecular machines in mitochondria and bacteria—the protein complexes of electron transport chains and associated enzymes. H₂S, along with nitric oxide and carbon monoxide, belongs to the class of endogenous gaseous signaling molecules. This compound plays critical roles in physiology and pathophysiology. Enzymes implicated in H₂S metabolism and physiological actions are promising targets for novel pharmaceutical agents. The biological effects of H₂S are biphasic, changing from cytoprotection to cytotoxicity through increasing the compound concentration. In mammals, H₂S enhances the activity of F_oF₁-ATP (adenosine triphosphate) synthase and lactate dehydrogenase via their S-sulfhydration, thereby stimulating mitochondrial electron transport. H₂S serves as an electron donor for the mitochondrial respiratory chain via sulfide quinone oxidoreductase and cytochrome c oxidase at low H₂S levels. The latter enzyme is inhibited by high H₂S concentrations, resulting in the reversible inhibition of electron transport and ATP production in mitochondria. In the branched respiratory chain of Escherichia coli, H₂S inhibits the bo₃ terminal oxidase but does not affect the alternative bd-type oxidases. Thus, in E. coli and presumably other bacteria, cytochrome bd permits respiration and cell growth in H₂S-rich environments. A complete picture of the impact of H₂S on bioenergetics is lacking, but this field is fast-moving, and active ongoing research on this topic will likely shed light on additional, yet unknown biological effects.     

Mitochondrial Carriers and Substrates Transport Network: A Lesson from Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is one of the most widely used model organisms for investigating various aspects of basic cellular functions that are conserved in human cells. This organism, as well as human cells, can modulate its metabolism in response to specific growth conditions, different environmental changes, and nutrient depletion. This adaptation results in a metabolic reprogramming of specific metabolic pathways. Mitochondrial carriers play a fundamental role in cellular metabolism, connecting mitochondrial with cytosolic reactions. By transporting substrates across the inner membrane of mitochondria, they contribute to many processes that are central to cellular function. The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, most of which have been functionally characterized. The aim of this review is to describe the role of the so far identified yeast mitochondrial carriers in cell metabolism, attempting to show the functional connections between substrates transport and specific metabolic pathways, such as oxidative phosphorylation, lipid metabolism, gluconeogenesis, and amino acids synthesis. Analysis of the literature reveals that these proteins transport substrates involved in the same metabolic pathway with a high degree of flexibility and coordination. The understanding of the role of mitochondrial carriers in yeast biology and metabolism could be useful for clarifying unexplored aspects related to the mitochondrial carrier network. Such knowledge will hopefully help in obtaining more insight into the molecular basis of human diseases.     

Direct Analysis of Mitochondrial Damage Caused by Misfolded/Destabilized Proteins

Protein quality control is essential for cellular homeostasis. In this study, we examined the effect of improperly folded proteins that do not form amyloid fibrils on mitochondria, which play important roles in ATP production and cell death. First, we prepared domain 3 of the dengue envelope protein in wild type and four mutants with widely different biophysical properties in misfolded/aggregated or destabilized states. The effects of the different proteins were detected using fluorescence microscopy and Western blotting, which revealed that three of the five proteins disrupted both inner and outer membrane integrity, while the other two proteins, including the wild type, did not. Next, we examined the common characteristics of the proteins that displayed toxicity against mitochondria by measuring oligomer size, molten globule-like properties, and thermal stability. The common feature of all three toxic proteins was thermal instability. Therefore, our data strongly suggest that thermally unstable proteins generated in the cytosol can cause cellular damage by coming into direct contact with mitochondria. More importantly, we revealed that this damage is not amyloid-specific.     

The Biochemical Assessment of Mitochondrial Respiratory Chain Disorders

Mitochondrial respiratory chain (MRC) disorders are a complex group of diseases whose diagnosis requires a multidisciplinary approach in which the biochemical investigations play an important role. Initial investigations include metabolite analysis in both blood and urine and the measurement of lactate, pyruvate and amino acid levels, as well as urine organic acids. Recently, hormone-like cytokines, such as fibroblast growth factor-21 (FGF-21), have also been used as a means of assessing evidence of MRC dysfunction, although work is still required to confirm their diagnostic utility and reliability. The assessment of evidence of oxidative stress may also be an important parameter to consider in the diagnosis of MRC function in view of its association with mitochondrial dysfunction. At present, due to the lack of reliable biomarkers available for assessing evidence of MRC dysfunction, the spectrophotometric determination of MRC enzyme activities in skeletal muscle or tissue from the disease-presenting organ is considered the ‘Gold Standard’ biochemical method to provide evidence of MRC dysfunction. The purpose of this review is to outline a number of biochemical methods that may provide diagnostic evidence of MRC dysfunction in patients.     

Insights into Domain Organization and Regulatory Mechanism of Cystathionine Beta-Synthase from Toxoplasma gondii

Cystathionine beta-synthase (CBS) is a key regulator of homocysteine metabolism. Although eukaryotic CBS have a similar domain architecture with a catalytic core and a C-terminal Bateman module, their regulation varies widely across phyla. In human CBS (HsCBS), the C-terminus has an autoinhibitory effect by acting as a cap that avoids the entry of substrates into the catalytic site. The binding of the allosteric modulator AdoMet to this region alleviates this cap, allowing the protein to progress from a basal toward an activated state. The same activation is obtained by artificial removal or heat-denaturation of the Bateman module. Recently, we reported the crystal structure of CBS from Toxoplasma gondii (TgCBS) showing that the enzyme assembles into basket-like dimers similar to the basal conformers of HsCBS. These findings would suggest a similar lid function for the Bateman module which, as in HsCBS, should relax in the absence of the C-terminal module. However, herein we demonstrate that, in contrast with HsCBS, removal of the Bateman module in TgCBS through deletion mutagenesis, limited proteolysis, or thermal denaturation has no effects on its activity, oligomerization, and thermal stability. This opposite behavior we have now found in TgCBS provides evidence of a novel type of CBS regulation.     

Progesterone Can Directly Inhibit the Life Activities of Toxoplasma gondii In Vitro through the Progesterone Receptor Membrane Component (PGRMC)

Toxoplasma gondii (T. gondii), as an opportunistic pathogen, has special pathogenic effects on pregnant animals and humans. Progesterone (P4) is a critical hormone that supports pregnancy, and its levels fluctuate naturally during early pregnancy. However, little is known about the association of host P4 levels with the infectivity and pathogenicity of T. gondii. Our study showed that P4 significantly inhibited the invasion and proliferation of tachyzoites, resulting in abnormal cytoskeletal daughter budding and subsequent autophagy in vitro. To investigate the underlying mechanism, we identified a Toxoplasma gondii progesterone membrane receptor protein (TgPGRMC) that was localized to the mitochondrion and closely related to the effect of P4 on tachyzoites. The knockout of the pgrmc gene conferred resistance to P4 inhibitory effects. Our results prove the direct relationship between P4 single factors and T. gondii in vitro and demonstrate that TgPGRMC is an important link between T. gondii and P4, providing a new direction for research on T. gondii infection during pregnancy.     

Ubiquitin C-Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine-Based Inhibitor

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.     

Molecular Hydrogen Enhances Proliferation of Cancer Cells That Exhibit Potent Mitochondrial Unfolded Protein Response

Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.     

A Walk in the Memory,from the First Functional Approach up to Its Regulatory Role of Mitochondrial Bioenergetic Flow in Health and Disease: Focus on the Adenine Nucleotide Translocator

The mitochondrial adenine nucleotide translocator (ANT) plays the fundamental role of gatekeeper of cellular energy flow, carrying out the reversible exchange of ADP for ATP across the inner mitochondrial membrane. ADP enters the mitochondria where, through the oxidative phosphorylation process, it is the substrate of Fo-F1 ATP synthase, producing ATP that is dispatched from the mitochondrion to the cytoplasm of the host cell, where it can be used as energy currency for the metabolic needs of the cell that require energy. Long ago, we performed a method that allowed us to monitor the activity of ANT by continuously detecting the ATP gradually produced inside the mitochondria and exported in the extramitochondrial phase in exchange with externally added ADP, under conditions quite close to a physiological state, i.e., when oxidative phosphorylation takes place. More than 30 years after the development of the method, here we aim to put the spotlight on it and to emphasize its versatile applicability in the most varied pathophysiological conditions, reviewing all the studies, in which we were able to observe what really happened in the cell thanks to the use of the “ATP detecting system” allowing the functional activity of the ANT-mediated ADP/ATP exchange to be measured.     

Diverse Roles of Mitochondria in Renal Injury from Environmental Toxicants and Therapeutic Drugs

Mitochondria are well-known to function as the primary sites of ATP synthesis in most mammalian cells, including the renal proximal tubule. Other functions have also been associated with different mitochondrial activities, including the regulation of redox status and the initiation of mitophagy and apoptosis. Mechanisms for the membrane transport of glutathione (GSH) and various GSH-derived metabolites across the mitochondrial inner membrane of renal proximal tubular cells are critical determinants of these functions and may serve as pharmacological targets for potential therapeutic approaches. Specific interactions of reactive intermediates, derived from drug metabolism, with molecular components in mitochondria have been identified as early steps in diverse forms of chemically-induced nephrotoxicity. Applying this key observation, we developed a novel hypothesis regarding the identification of early, sensitive, and specific biomarkers of exposure to nephrotoxicants. The underlying concept is that upon exposure to a diverse array of environmental contaminants, as well as therapeutic drugs whose efficacy is limited by nephrotoxicity, renal mitochondria will release both high- and low-molecular-weight components into the urine or the extracellular medium in an in vitro model. The detection of these components may then serve as indicators of exposure before irreversible renal injury has occurred.     

Chemistry of Lipoquinones: Properties,Synthesis, and Membrane Location of Ubiquinones,Plastoquinones, and Menaquinones

Lipoquinones are the topic of this review and are a class of hydrophobic lipid molecules with key biological functions that are linked to their structure, properties, and location within a biological membrane. Ubiquinones, plastoquinones, and menaquinones vary regarding their quinone headgroup, isoprenoid sidechain, properties, and biological functions, including the shuttling of electrons between membrane-bound protein complexes within the electron transport chain. Lipoquinones are highly hydrophobic molecules that are soluble in organic solvents and insoluble in aqueous solution, causing obstacles in water-based assays that measure their chemical properties, enzyme activities and effects on cell growth. Little is known about the location and ultimately movement of lipoquinones in the membrane, and these properties are topics described in this review. Computational studies are particularly abundant in the recent years in this area, and there is far less experimental evidence to verify the often conflicting interpretations and conclusions that result from computational studies of very different membrane model systems. Some recent experimental studies have described using truncated lipoquinone derivatives, such as ubiquinone-2 (UQ-2) and menaquinone-2 (MK-2), to investigate their conformation, their location in the membrane, and their biological function. Truncated lipoquinone derivatives are soluble in water-based assays, and hence can serve as excellent analogs for study even though they are more mobile in the membrane than the longer chain counterparts. In this review, we will discuss the properties, location in the membrane, and syntheses of three main classes of lipoquinones including truncated derivatives. Our goal is to highlight the importance of bridging the gap between experimental and computational methods and to incorporate properties-focused considerations when proposing future studies relating to the function of lipoquinones in membranes.     

Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation,Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells

Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC.     

Bioenergetics and Reactive Nitrogen Species in Bacteria

The production of reactive nitrogen species (RNS) by the innate immune system is part of the host’s defense against invading pathogenic bacteria. In this review, we summarize recent studies on the molecular basis of the effects of nitric oxide and peroxynitrite on microbial respiration and energy conservation. We discuss possible molecular mechanisms underlying RNS resistance in bacteria mediated by unique respiratory oxygen reductases, the mycobacterial bcc-aa₃ supercomplex, and bd-type cytochromes. A complete picture of the impact of RNS on microbial bioenergetics is not yet available. However, this research area is developing very rapidly, and the knowledge gained should help us develop new methods of treating infectious diseases.     

The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP Synthase: Emerging New Roles in Mitochondrial Pathophysiology

The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial F_OF₁ ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F₁ sector, it makes stable contacts with both F₁ and the peripheral stalk, ensuring the structural and functional coupling between F_O and F₁, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca²⁺ required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca²⁺-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death.     

Improving Consistency of Photobiomodulation Therapy: A Novel Flat-Top Beam Hand-Piece versus Standard Gaussian Probes on Mitochondrial Activity

The tremendous therapeutic potential of photobiomodulation therapy in different branches of medicine has been described in the literature. One of the molecular mechanisms for this treatment implicates the mitochondrial enzyme, cytochrome C oxidase. However, the efficacy and consistency of clinical outcomes with photobiomodulation treatments has been fiercely debated. This work was motivated by this need to improve photobiomodulation devices and delivery approaches. We designed a novel hand-piece with a flat-top beam profile of irradiation. We compared the beam profile versus a standard hand-piece and a fibre probe. We utilized isolated mitochondria and performed treatments at various spots within the beam, namely, the centre, left and right edge. We examined mitochondrial activity by assessing ATP synthesis with the luciferin/luciferase chemiluminescent method as a primary endpoint, while mitochondrial damage was assessed as the secondary endpoint. We observed a uniform distribution of the power density with the flat-top prototype compared to a wide Gaussian beam profile with the standard fibre and standard hand-piece. We noted increased production of ATP in the centre of all three beams with respect to the non-treated controls (p < 0.05). Both the fibre and standard hand-piece demonstrated less increase in ATP synthesis at the edges than the centre (p < 0.05). In contrast, ATP synthesis was increased homogenously in the flat-top handpiece, both in the centre and the edges of the beam. Fibre, standard hand-piece and the flat-top hand-piece prototype have discrete beam distribution characteristics. This significantly affected the mitochondrial activity with respect to their position within the treated areas. Flat-top hand-piece enhances the uniformity of photobiomodulation treatments and can improve the rigour and reproducibility of PBM clinical outcomes.     

Dual Mode of Mitochondrial ROS Action during Reprogramming to Pluripotency

Essential changes in cell metabolism and redox signaling occur during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this paper, using genetic and pharmacological approaches, we have investigated the role of electron transport chain (ETC) complex-I (CI) of mitochondria in the process of cell reprogramming to pluripotency. Knockdown of NADH-ubiquinone oxidoreductase core subunits S1 (Ndufs1) or subunit B10 (Ndufb10) of the CI or inhibition of this complex with rotenone during mouse embryonic fibroblast (MEF) reprogramming resulted in a significantly decreased number of induced pluripotent stem cells (iPSCs). We have found that mitochondria and ROS levels due course of the reprogramming tightly correlate with each other, both reaching peak by day 3 and significantly declining by day 10 of the process. The transient augmentation of mitochondrial reactive oxygen species (ROS) could be attenuated by antioxidant treatment, which ameliorated overall reprogramming. However, ROS scavenging after day 3 or during the entire course of reprogramming was suppressive for iPSC formation. The ROS scavenging within the CI-deficient iPSC-precursors did not improve, but further suppressed the reprogramming. Our data therefore point to distinct modes of mitochondrial ROS action during the early versus mid and late stages of reprogramming. The data further substantiate the paradigm that balanced levels of oxidative phosphorylation have to be maintained on the route to pluripotency.     

Mitochondrial a Kinase Anchor Proteins in Cardiovascular Health and Disease: A Review Article on Behalf of the Working Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology

Second messenger cyclic adenosine monophosphate (cAMP) has been found to regulate multiple mitochondrial functions, including respiration, dynamics, reactive oxygen species production, cell survival and death through the activation of cAMP-dependent protein kinase A (PKA) and other effectors. Several members of the large family of A kinase anchor proteins (AKAPs) have been previously shown to locally amplify cAMP/PKA signaling to mitochondria, promoting the assembly of signalosomes, regulating multiple cardiac functions under both physiological and pathological conditions. In this review, we will discuss roles and regulation of major mitochondria-targeted AKAPs, along with opportunities and challenges to modulate their functions for translational purposes in the cardiovascular system.