Search Results (35)

Tris(2-chloroethyl) phosphate (TCEP), as an organophosphate contaminant, poses a significant threat to the growth and development of plants, especially roots. This study aimed to clarify the mechanisms of TCEP’s toxicity and damage to root systems, as well as the mechanisms of its damage to the respiration and energy metabolism of alfalfa root cells. The results showed that TCEP obviously affected the root length, root surface area, root volume, and root diameter of alfalfa. With increasing stress intensity, the total mitochondrial respiration rate and Cytochrome C Oxidase (COX) pathway respiration rate progressively declined, while the Alternative Oxidase (AOX) pathway respiration rate and its proportion of total respiration gradually rose. In addition, adenosine triphosphate (ATP) content and root vigor were significantly reduced. Moreover, with an increase in TCEP concentration, root superoxide anion radical content in alfalfa root cells was significantly elevated, while superoxide dismutase (SOD) and catalase (CAT) activities were significantly lowered, and ascorbate peroxidase (APX) and peroxidase (POD) activities were significantly enhanced. The present study indicated that respiration was disrupted, causing a lack of ATP in root cells under TCEP. Both the overproduction of reactive oxygen species (ROS) from the mitochondrial respiratory electron transport chain (mECT) and the deficiency of ROS-scavenging enzymes caused ROS accumulation, which led to the destruction of the cell membrane structure and exacerbated the disruption of the respiratory metabolism. The disruption of the conversion and reuse of energy by TCEP affected root growth and development. Full article

Brain ischemia is one of the major causes of chronic disability and death worldwide. It is related to insufficient blood supply to cerebral tissue, which induces irreversible or reversible intracellular effects depending on the time and intensity of the ischemic event. Indeed, neuronal function may be restored in some conditions, such as transient ischemic attack (TIA), which may be responsible for protecting against a subsequent lethal ischemic insult. It is well known that the brain requires high levels of oxygen and glucose to ensure cellular metabolism and energy production and that damage caused by oxygen impairment is tightly related to the brain’s low antioxidant capacity. Oxygen is a key player in mitochondrial oxidative phosphorylation (OXPHOS), during which reactive oxygen species (ROS) synthesis can occur as a physiological side-product of the process. Indeed, besides producing adenosine triphosphate (ATP) under normal physiological conditions, mitochondria are the primary source of ROS within the cell. This is because, in 0.2–2% of cases, the escape of electrons from complex I (NADPH-dehydrogenase) and III of the electron transport chain occurring in mitochondria during ATP synthesis leads to the production of the superoxide radical anion (O₂^•−), which exerts detrimental intracellular effects owing to its high molecular instability. Along with ROS, reactive nitrosative species (RNS) also contribute to the production of free radicals. When the accumulation of ROS and RNS occurs, it can cause membrane lipid peroxidation and DNA damage. Here, we describe the intracellular pathways activated in brain tissue after a lethal/sub lethal ischemic event like stroke or ischemic tolerance, respectively, highlighting the important role played by oxidative stress and mitochondrial dysfunction in the onset of the two different ischemic conditions. Full article

Mitochondria are most likely descendants of strictly aerobic prokaryotes from the class Alphaproteobacteria. The mitochondrial matrix is surrounded by two membranes according to its relationship with Gram-negative bacteria. Similar to the bacterial outer membrane, the mitochondrial outer membrane acts as a molecular sieve because it also contains diffusion pores. However, it is more actively involved in mitochondrial metabolism because it plays a functional role, whereas the bacterial outer membrane has only passive sieving properties. Mitochondrial porins, also known as eukaryotic porins or voltage-dependent anion-selective channels (VDACs) control the permeability properties of the mitochondrial outer membrane. They contrast with most bacterial porins because they are voltage-dependent. They switch at relatively small transmembrane potentials of 20 to 30 mV in closed states that exhibit different permeability properties than the open state. Whereas the open state is preferentially permeable to anionic metabolites of mitochondrial metabolism, the closed states prefer cationic solutes, in particular, calcium ions. Mitochondrial porins are encoded in the nucleus, synthesized at cytoplasmatic ribosomes, and post-translationally imported through special transport systems into mitochondria. Nineteen beta strands form the beta-barrel cylinders of mitochondrial and related porins. The pores contain in addition an α-helical structure at the N-terminal end of the protein that serves as a gate for the voltage-dependence. Similarly, they bind peripheral proteins that are involved in mitochondrial function and compartment formation. This means that mitochondrial porins are localized in a strategic position to control mitochondrial metabolism. The special features of the role of mitochondrial porins in apoptosis and cancer will also be discussed in this article. Full article

The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H₂O₂)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of Megalobrama amblycephala. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 μM of H₂O₂ for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 μg/mL) for 3 h, and then stimulated with 100 μM of H₂O₂ for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs’ antioxidant capacities. The 200 μg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H₂O₂-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (p < 0.05). The over-production of reactive oxygen species, depletion of nicotinamide adenine dinucleotide phosphate, and collapse of the mitochondrial membrane potential were significantly inhibited in the 200-MLP pre-treatment group (p < 0.05). The expressions of endoplasmic reticulum stress-related genes (forkhead box O1α (foxO1α), binding immunoglobulin protein (bip), activating transcription factor 6 (atf6), and C/EBP-homologous protein (chop)), Ca²⁺ transport-related genes (voltage-dependent anion-selective channel 1 (vdac1), mitofusin 2 (mfn2), and mitochondrial Ca²⁺ uniporter (mcu)), and interleukin 6 (il-6) and bcl2-associated x (bax) were significantly lower in the 200-MLP pre-treatment group than in the HP group (p < 0.05), which rebounded to normal levels in the NC group (p > 0.05). These results indicated that MLP pre-treatment attenuated H₂O₂-induced PBL oxidative damage in the M. amblycephala by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for M. amblycephala, as they protect against oxidative damage. Full article

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein–lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane. Full article

Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative Acucp gene of the free-living protozoan amoeba, A. castellanii, encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete Acucp coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the S. cerevisiae strain InvSc1. Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the ΔSOD1 strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the A. castellanii UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins. Full article

In respiring mitochondria, the proton gradient across the inner mitochondrial membrane is used to drive ATP production. Mitochondrial uncouplers, which are typically weak acid protonophores, can disrupt this process to induce mitochondrial dysfunction and apoptosis in cancer cells. We have shown that bisaryl urea-based anion transporters can also mediate mitochondrial uncoupling through a novel fatty acid-activated proton transport mechanism, where the bisaryl urea promotes the transbilayer movement of deprotonated fatty acids and proton transport. In this paper, we investigated the impact of replacing the urea group with squaramide, amide and diurea anion binding motifs. Bisaryl squaramides were found to depolarise mitochondria and reduce MDA-MB-231 breast cancer cell viability to similar extents as their urea counterpart. Bisaryl amides and diureas were less active and required higher concentrations to produce these effects. For all scaffolds, the substitution of the bisaryl rings with lipophilic electron-withdrawing groups was required for activity. An investigation of the proton transport mechanism in vesicles showed that active compounds participate in fatty acid-activated proton transport, except for a squaramide analogue, which was sufficiently acidic to act as a classical protonophore and transport protons in the absence of free fatty acids. Full article

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism. Full article

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-^{Tspotm1GuWu(GuwiyangWurra)}-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo. Full article

Mitochondrial oxidative stress and dysfunction are strongly implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Glutathione (GSH) is an endogenous antioxidant that exists as distinct cytosolic and mitochondrial pools. The status of the mitochondrial GSH pool is reliant on transport from the cytosol through the 2-oxoglutarate carrier (OGC), an inner membrane anion carrier. We have previously reported that the outer mitochondrial membrane protein, Bcl-2, directly binds GSH and is a key regulator of OGC-dependent mitochondrial GSH transport. Here, we show that G93A mutant SOD1 (Cu, Zn-superoxide dismutase) reduces the binding of GSH to Bcl-2 and disrupts mitochondrial GSH uptake in vitro. In the G93A mutant hSOD1 mouse model of ALS, mitochondrial GSH is significantly depleted in spinal cord of end-stage mice. Finally, we show that OGC is heavily S-nitrosylated in the spinal cord of end-stage mice and consequently, the GSH uptake capacity of spinal cord mitochondria isolated from these mutant mice is significantly diminished. Collectively, these findings suggest that spinal cord GSH depletion, particularly at the level of the mitochondria, plays a significant role in ALS pathogenesis induced by mutant SOD1. Furthermore, the depletion of mitochondrial GSH in the G93A mutant hSOD1 mouse model may be caused by the S-nitrosylation of OGC and the capacity of mutant SOD1 to disrupt the Bcl-2/GSH interaction, resulting in a disruption of mitochondrial GSH transport. Full article

The voltage-dependent anion channel (VDAC) is the main passageway for ions and metabolites over the outer mitochondrial membrane. It was associated with many physiological processes, including apoptosis and modulation of intracellular Ca²⁺ signaling. The protein is formed by a barrel of 19 beta-sheets with an N-terminal helix lining the inner pore. Despite its large diameter, the channel can change its selectivity for ions and metabolites based on its open state to regulate transport into and out of mitochondria. VDAC was shown to be regulated by a variety of cellular factors and molecular partners including proteins, lipids and ions. Although the physiological importance of many of these modulatory effects are well described, the binding sites for molecular partners are still largely unknown. The highly symmetrical and sleek structure of the channel makes predictions of functional moieties difficult. However, one residue repeatedly sticks out when reviewing VDAC literature. A glutamate at position 73 (E73) located on the outside of the channel facing the hydrophobic membrane environment was repeatedly proposed to be involved in channel regulation on multiple levels. Here, we review the distinct hypothesized roles of E73 and summarize the open questions around this mysterious residue. Full article

Cultivated soybean (Glycine max (L.)), the world’s most important legume crop, has high-to-moderate salt sensitivity. Being the frontier for sensing and controlling solute transport, membrane proteins could be involved in cell signaling, osmoregulation, and stress-sensing mechanisms, but their roles in abiotic stresses are still largely unknown. By analyzing salt-induced membrane proteomic changes in the roots and leaves of salt-sensitive soybean cultivar (C08) seedlings germinated under NaCl, we detected 972 membrane proteins, with those present in both leaves and roots annotated as receptor kinases, calcium-sensing proteins, abscisic acid receptors, cation and anion channel proteins, proton pumps, amide and peptide transporters, and vesicle transport-related proteins etc. Endocytosis, linoleic acid metabolism, and fatty acid biosynthesis pathway-related proteins were enriched in roots whereas phagosome, spliceosome and soluble NSF attachment protein receptor (SNARE) interaction-related proteins were enriched in leaves. Using label-free quantitation, 129 differentially expressed membrane proteins were found in both tissues upon NaCl treatment. Additionally, the 140 NaCl-induced proteins identified in roots and 57 in leaves are vesicle-, mitochondrial-, and chloroplast-associated membrane proteins and those with functions related to ion transport, protein transport, ATP hydrolysis, protein folding, and receptor kinases, etc. Our proteomic results were verified against corresponding gene expression patterns from published C08 RNA-seq data, demonstrating the importance of solute transport and sensing in salt stress responses. Full article

Experimental liver injury with hepatocelluar necrosis and abnormal liver tests is caused by exposure to heavy metals (HMs) like aluminum, arsenic, beryllium, cadmium, chromium, cobalt, copper, iron, lead, mercury, molybdenum, nickel, platinum, thallium, titanium, vanadium, and zinc. As pollutants, HMs disturb the ecosystem, and as these substances are toxic, they may affect the health of humans and animals. HMs are not biodegradable and may be deposited preferentially in the liver. The use of animal models can help identify molecular and mechanistic steps leading to the injury. HMs commonly initiate hepatocellular overproduction of ROS (reactive oxygen species) due to oxidative stress, resulting in covalent binding of radicals to macromolecular proteins or lipids existing in membranes of subcellular organelles. Liver injury is facilitated by iron via the Fenton reaction, providing ROS, and is triggered if protective antioxidant systems are exhausted. Ferroptosis syn pyroptosis was recently introduced as mechanistic concept in explanations of nickel (Ni) liver injury. NiCl₂ causes increased iron deposition in the liver, upregulation of cyclooxygenase 2 (COX-2) protein and mRNA expression levels, downregulation of glutathione eroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), nuclear receptor coactivator 4 (NCOA4) protein, and mRNA expression levels. Nickel may cause hepatic injury through mitochondrial damage and ferroptosis, defined as mechanism of iron-dependent cell death, similar to glutamate-induced excitotoxicity but likely distinct from apoptosis, necrosis, and autophagy. Under discussion were additional mechanistic concepts of hepatocellular uptake and biliary excretion of mercury in exposed animals. For instance, the organic anion transporter 3 (Oat3) and the multidrug resistance-associated protein 2 (Mrp2) were involved in the hepatic handling of mercury. Mercury treatment modified the expression of Mrp2 and Oat3 as assessed by immunoblotting, partially explaining its impaired biliary excretion. Concomitantly, a decrease in Oat3 abundance in the hepatocyte plasma membranes was observed that limits the hepatic uptake of mercury ions. Most importantly and shown for the first time in liver injury caused by HMs, titanium changed the diversity of gut microbiota and modified their metabolic functions, leading to increased generation of lipopolysaccharides (LPS). As endotoxins, LPS may trigger and perpetuate the liver injury at the level of gut-liver. In sum, mechanistic and molecular steps of experimental liver injury due to HM administration are complex, with ROS as the key promotional compound. However, additional concepts such as iron used in the Fenton reaction, ferroptosis, modification of transporter systems, and endotoxins derived from diversity of intestinal bacteria at the gut-liver level merit further consideration. Full article

The multifunctional protein, voltage-dependent anion channel 1 (VDAC1), is located on the mitochondrial outer membrane. It is a pivotal protein that maintains mitochondrial function to power cellular bioactivities via energy generation. VDAC1 is involved in regulating energy production, mitochondrial oxidase stress, Ca²⁺ transportation, substance metabolism, apoptosis, mitochondrial autophagy (mitophagy), and many other functions. VDAC1 malfunction is associated with mitochondrial disorders that affect inflammatory responses, resulting in an up-regulation of the body’s defensive response to stress stimulation. Overresponses to inflammation may cause chronic diseases. Mitochondrial DNA (mtDNA) acts as a danger signal that can further trigger native immune system activities after its secretion. VDAC1 mediates the release of mtDNA into the cytoplasm to enhance cytokine levels by activating immune responses. VDAC1 regulates mitochondrial Ca²⁺ transportation, lipid metabolism and mitophagy, which are involved in inflammation-related disease pathogenesis. Many scientists have suggested approaches to deal with inflammation overresponse issues via specific targeting therapies. Due to the broad functionality of VDAC1, it may become a useful target for therapy in inflammation-related diseases. The mechanisms of VDAC1 and its role in inflammation require further exploration. We comprehensively and systematically summarized the role of VDAC1 in the inflammatory response, and hope that our research will lead to novel therapeutic strategies that target VDAC1 in order to treat inflammation-related disorders. Full article

Mitochondrial electron transport chain (ETC) inhibition is a phenomenon interesting in itself and serves as a tool for studying various cellular processes. Despite the fact that searching the term “rotenone” in PubMed returns more than 6900 results, there are many discrepancies regarding the directions of changes reported to be caused by this RTC inhibitor in the delicate redox balance of the cell. Here, we performed a multifaceted study of the popular ETC inhibitors rotenone and antimycin A, involving assessment of mitochondrial membrane potential and the production of hydrogen peroxide and superoxide anions at cellular and mitochondrial levels over a wide range of inhibitor concentrations (1 nmol/dm³–100 µmol/dm³). All measurements were performed with whole cells, with accompanying control of ATP levels. Antimycin A was more potent in hindering HepG2 cells’ abilities to produce ATP, decreasing ATP levels even at a 1 nmol/dm³ concentration, while in the case of rotenone, a 10,000-times greater concentration was needed to produce a statistically significant decrease. The amount of hydrogen peroxide produced in the course of antimycin A biological activity increased rapidly at low concentrations and decreased below control level at a high concentration of 100 µmol/dm³. While both inhibitors influenced cellular superoxide anion production in a comparable manner, rotenone caused a greater increase in mitochondrial superoxide anions compared to a modest impact for antimycin A. IC50 values for rotenone and antimycin A with respect to HepG2 cell survival were of the same order of magnitude, but the survival curve of cells treated with rotenone was clearly biphasic, suggesting a concentration-dependent mode of biological action. We propose a clear experimental setup allowing for complete and credible analysis of the redox state of cells under stress conditions which allows for better understanding of the effects of ETC inhibition. Full article