Carbonyl Cyanide M-chlorophenylhydrazone Research Articles

Dependence of PINK1 accumulation on mitochondrial redox system.

Accumulation of PINK1 on the outer mitochondrial membrane (OMM) is necessary for PINK‐mediated mitophagy. The proton ionophores, like carbonyl cyanide m‐chlorophenylhydrazone (CCCP) and carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP), inhibit PINK1 import into mitochondrial matrix and induce PINK1 OMM accumulation. Here, we show that the CHCHD4/GFER disulfide relay system in the mitochondrial intermembrane space (IMS) is required for PINK1 stabilization when mitochondrial membrane potential is lost. Activation of CHCHD4/GFER system by mitochondrial oxidative stress or inhibition of CHCHD4/GFER system with antioxidants can promote or suppress PINK1 accumulation, respectively. Thus data suggest a pivotal role of CHCHD4/GFER system in PINK1 accumulation. The amyotrophic lateral sclerosis‐related superoxide dismutase 1 mutants dysregulated redox state and CHCHD4/GFER system in the IMS, leading to inhibitions of PINK1 accumulation and mitophagy. Thus, the redox system in the IMS is involved in PINK1 accumulation and damaged mitochondrial clearance, which may play roles in mitochondrial dysfunction‐related neurodegenerative diseases.

Supraphysiological Levels of Testosterone Induce Vascular Dysfunction via Activation of the NLRP3 Inflammasome.

Background: Both supraphysiological and subphysiological testosterone levels are associated with increased cardiovascular risk. Testosterone consumption at supraphysiological doses has been linked to increased blood pressure, left ventricular hypertrophy, vascular dysfunction, and increased levels of inflammatory markers. Activation of the NLRP3 inflammasome contributes to the production of proinflammatory cytokines, leading to cardiovascular dysfunction. We hypothesized that supraphysiological levels of testosterone, via generation of mitochondrial reactive oxygen species (mROS), activates the NLRP3 inflammasome and promotes vascular dysfunction.Methods: Male, 12 week-old C57Bl/6J (WT) and NLRP3 knockout (NLRP3−/−) mice were used. Mice were treated with testosterone propionate [TP (10 mg/kg) in vivo] or vehicle for 30 days. In addition, vessels were incubated with testosterone [Testo (10−6 M, 2 h) in vitro]. Testosterone levels, blood pressure, vascular function (thoracic aortic rings), pro-caspase-1/caspase-1 and interleukin-1β (IL-1β) expression, and generation of reactive oxygen species were determined.Results: Testosterone increased contractile responses and reduced endothelium-dependent vasodilation, both in vivo and in vitro. These effects were not observed in arteries from NLRP3−/− mice. Aortas of TP-treated WT mice (in vivo), as well as aortas from WT mice incubated with testo (in vitro), exhibited increased mROS levels and increased caspase-1 and IL-1β expression. These effects were not observed in arteries from NLRP3−/− mice. Flutamide [Flu, 10−5 M, androgen receptor (AR) antagonist], carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 10−6 M, mitochondrial uncoupler) and MCC950 (MCC950, 10−6 M, a NLRP3 receptor inhibitor) prevented testosterone-induced mROS generation.Conclusion: Supraphysiological levels of testosterone induce vascular dysfunction via mROS generation and NLRP3 inflammasome activation. These events may contribute to increased cardiovascular risk.

Inhibition of Efflux Pumps by Monoterpene (α-pinene) and Impact on Staphylococcus aureus Resistance to Tetracycline and Erythromycin.

Infectious diseases have been responsible for an increasing number of deaths worldwide. Staphylococcus aureus has been recognized as one of the most notable causative agents of severe infections, while efflux pump (EP) expression is one of the main mechanisms associated with S. aureus resistance to antibiotics. This study aimed to investigate the potential of α-pinene as an efflux pump inhibitor in species of S. aureus carrying the TetK and MrsA proteins. The minimum inhibitory concentrations (MIC) of α-pinene and other efflux pump inhibitors were assessed using serial dilutions of each compound at an initial concentration above 1024 μg/mL. Solutions containing culture medium and bacterial inoculums were prepared in test tubes and subsequently transferred to 96-well microdilution plates. The modulation of ethidium bromide (EtBr) and antibiotics (tetracycline and erythromycin) was investigated through analysis of the modification in their MICs in the presence of a subinhibitory concentration of α-pinene (MIC/8). Wells containing only culture medium and bacterial inoculums were used as negative control. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control. The MIC of ethidium bromide against S. aureus strains RN-4220 and IS-58 was reduced by association with α-pinene. This monoterpene potentiated the effect of tetracycline against the IS-58 strain but failed in modulating the antibacterial effect of erythromycin against RN-4220, suggesting a selective inhibitory effect on the TetK EP by α- pinene. In conclusion, α-pinene has promising effects against S.aureus strains, which should be useful in the combat of antibacterial resistance associated with EP expression. Nevertheless, further research is required to fully characterize its molecular mechanism of action as an EP inhibitor.

Bicarbonate suppresses mitochondrial membrane depolarization induced by conventional uncouplers

Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues. However, bicarbonate could not restore the ATP synthesis suppressed by DNP or CCCP in mitochondria. Bicarbonate neither altered the depolarizing action of DNP and FCCP on proteoliposomes with reconstituted cytochrome c oxidase, nor affected the protonophoric activity of DNP and FCCP in artificial lipid membranes as measured with pyranine-loaded liposomes, thereby showing that the bicarbonate-induced reversal of the depolarizing action of DNP and FCCP on mitochondria did not result from direct interaction of bicarbonate with the uncouplers.

Efflux MexAB-Mediated Resistance in P. aeruginosa Isolated from Patients with Healthcare Associated Infections.

Today, one of the most important challenges for physicians is the adequate treatment of infections due to multidrug resistant organism (MDR). Pseudomonas aeruginosa is considered an opportunistic organism causing different types of healthcare associated infections (HAIs). We aimed to investigate the MDR and pandrug resistance (PDR) rate in P. aeruginosa in our region and detect efflux-pump mexAB genes and the proposed binding interactions of five different categories of antimicrobial agents with the mexB pump. A total of 180 non-duplicated P. aeruginosa strains were isolated from patients with HAIs in the Suez Canal University Hospital. Phenotypically, minimum inhibitory concentration (MIC) was done for all MDR and PDR strains before and after addition of efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Molecular detection of mexA and mexB genes was done by using polymerase chain reaction (PCR). Most of the isolated strains (126 strains) were MDR (70%); only 10 samples (5.5%) were PDR. MexA and mexB genes were detected in 88.2% (120 strains) and 70.5% (96 strains) of stains, respectively. All PDR strains (10 stains) carried both mexA and mexB genes. Efflux mexAB genes were detected in all MDR and PDR strains (136 strains). Molecular modeling studies were performed to investigate the modes of intermolecular binding interactions between the antimicrobial agents and mexB key amino acids that resulted in MDR and PDR. The current study reported high prevalence of MDR and PDR P. aeruginosa in patients with HAIs in the Suez Canal University Hospitals.

Cyclo(Phe-Pro) produced by Vibrio species passes through biological membranes by simple diffusion.

Cyclo(Phe-Pro) (cFP), produced by the Vibrio species, plays the dual roles of being a signaling molecule and a virulence factor. Acting modes of this compound have recently been characterized at the molecular level. Nevertheless, the method by which this compound passes across biological membranes remains obscure. Using radiolabeled cFP, we examined the kinetics of transport for this compound across membranes using V. vulnificus, Escherichia coli, and sheep red blood cells. We observed that cFP was taken up by these cells in a concentration-dependent manner and was not affected by the addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that cFP is taken up by passive transport. The kinetics of uptake of cFP by the above three types of cells revealed no significant differences, indicating that no specific protein is involved in this process. When the intracellular accumulation of cFP in the tested cells was measured, the concentrations did not exhibit significant differences between the 1-min and 10-min time points after cFP was added to the culture. In contrast, the intracellular concentration of fumarate, which is well known to be taken up by cells via active transport, was significantly higher at the 10-min than at the 1-min time point after addition. Taken together, this study shows that cFP is a diffusible molecule that does not require energy for transportation across biological membranes, and that cFP does not need membrane machinery in order to cross membranes and consequently act as a virulence factor or signal. KEY POINTS: • Kinetics of cFP uptake into cells of V. vulnificus, E. coli, or RBS was studied. • The uptake was not saturated and required no energy, indicating passive transport. • The lack of cell specificity in cFP uptake means no specific protein is needed. • Therefore, the cFP moves across the biological membrane by simple diffusion.

Exploring the Impact of PARK2 Mutations on the Total and Mitochondrial Proteome of Human Skin Fibroblasts.

Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson’s disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.

Human telomerase reverse transcriptase positively regulates mitophagy by inhibiting the processing and cytoplasmic release of mitochondrial PINK1

Mutations in the phosphatase and tensin homologue-induced putative kinase 1 (PINK1) gene have been linked to an early-onset autosomal recessive form of familial Parkinson′s disease (PD). PINK1, a mitochondrial serine/threonine-protein kinase, plays an important role in clearing defective mitochondria by mitophagy – the selective removal of mitochondria through autophagy. Evidence suggests that alteration of the PINK1 pathway contributes to the pathogenesis of PD, but the mechanisms by which the PINK1 pathway regulates mitochondrial quality control through mitophagy remain unclear. Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase that functions in telomere maintenance as well as several non-telomeric activities. For example, hTERT has been associated with cellular immortalization, cell growth control, and mitochondrial regulation. We determined that hTERT negatively regulates the cleavage and cytosolic processing of PINK1 and enhances its mitochondrial localization by inhibiting mitochondrial processing peptidase β (MPPβ). Consequently, hTERT promotes mitophagy following carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondrial dysfunction and improves the function of damaged mitochondria by modulating PINK1. These findings suggest that hTERT positively regulates PINK1 function, leading to increased mitophagy following mitochondrial damage.

Cloning and Functional Characterization of Putative Escherichia coli ABC Multidrug Efflux Transporter YddA.

A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drugsensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.

Phenotypic Activity of Efflux Pumps by Carbonyl Cyanide M-Chlorophenyl Hydrazone (CCCP) and Mutations in GyrA and ParC Genes Among Ciprofloxacin-Resistant Acinetobacter baumannii Isolates

Background: Acinetobacter baumannii is one of the major bacteria causing nosocomial infections. The emergence of multidrug-resistant (MDR) isolates causes morbidity and mortality and lead to financial burdens for patients and public health systems. Objectives: Regarding the use of ciprofloxacin and increase of ciprofloxacin resistance, this study was aimed to investigate the role of the efflux pump and mutations in gyrA and parC genes as the mechanisms of ciprofloxacin-resistance. Methods: This study was conducted on 55 strains of A. baumannii isolated from the patients hospitalized in Milad Hospital, Tehran. The isolates were identified by biochemical tests, and their antibiotic susceptibility was assessed by the disc diffusion method. To investigate the role of the efflux pump, minimum inhibitory concentration (MIC) of ciprofloxacin was determined in the presence and absence of the carbonyl cyanide m-chlorophenyl hydrazone (CCCP) inhibitor. The PCR was used for amplification of gyrA, and parC genes and sequencing of its products was carried out to track the mutations. Results: The highest and lowest antibiotic resistance were observed in ciprofloxacin (100%) and tobramycin (52.7%), respectively. Moreover, 96% of the A. baumannii isolates exhibited MDR. Five percent of strains showed 4-fold MIC reduction after the use of the inhibitor and were reported as the strains with high activity efflux pump. In 100% of the isolates, gyrA and parC genes were detected, and only one mutation was observed in parC and gyrA genes in the studied isolates that altered amino acid type. Conclusions: Reduction in MIC in the presence of an efflux pump inhibitor confirmed its role in enhancing the resistance to ciprofloxacin in some isolates. The results of this study also revealed no significant relationship between the ciprofloxacin resistance of the studied isolates and mutation in the encoding region of parC and gyrA genes. Thus, the significance of other mechanisms, such as the expression of genes encoding efflux pump proteins, should not be neglected.

Programmed switch in the mitochondrial degradation pathways during human retinal ganglion cell differentiation from stem cells is critical for RGC survival

Retinal ganglion cell (RGC) degeneration is the root cause for vision loss in glaucoma as well as in other forms of optic neuropathy. A variety of studies have implicated abnormal mitochondrial quality control (MQC) as contributing to RGC damage and degeneration in optic neuropathies. The ability to differentiate human pluripotent stem cells (hPSCs) into RGCs provides an opportunity to study RGC MQC in great detail. Degradation of damaged mitochondria is a critical step of MQC, and here we have used hPSC-derived RGCs (hRGCs) to analyze how altered mitochondrial degradation pathways in hRGCs affect their survival. Using pharmacological methods, we have investigated the role of the proteasomal and endo-lysosomal pathways in degrading damaged mitochondria in hRGCs and their precursor stem cells. We found that upon mitochondrial damage induced by the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), hRGCs more efficiently degraded mitochondria than did their precursor stem cells. We further identified that for degrading damaged mitochondria, stem cells predominantly use the ubiquitine-proteasome system (UPS) while hRGCs use the endo-lysosomal pathway. UPS inhibition causes apoptosis and cell death in stem cells, while hRGC viability is dependent on the endo-lysosomal pathway but not on the UPS pathway. These findings suggest that manipulation of the endo-lysosomal pathway could be therapeutically relevant for RGC protection in treating optic neuropathies associated with mitophagy defects. Endo-lysosome dependent cell survival is also conserved in other human neurons as we found that differentiated human cerebral cortical neurons also degenerated upon endo-lysosomal inhibition but not with proteasome inhibition.

Comparative studies on measurement of membrane potential of bacterial cells treated with ZnO nanoparticles by Spectrofluorometry, fluorescence microscopy and flowcytometry

Many methods are developed to assess antimicrobial action of ZnO nanoparticles (NPs). A large number of methods associated with the use of fluorescent probes are developed, including Spectrofluorometry, fluorescence microscopy, and cytometry. In this study, flowcytometry, Spectrofluorometry and fluorescent microscopy was used to measure membrane potential variation of E. coli and S. aureus cells treated with two different sizes of zinc oxide (ZnO) NPs and were compared with conventional methods. In order to estimate change in membrane potential, E. coli and S. aureus cells were treated with iopnophore agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) and membrane potential was evaluated using fluorescent probe 3,3′-Diethyloxacarbocyanine, iodide (DIOC2(3)). All the three methods showed similar results and among these Spectrofluorometry was easy to use and inexpensive to assess the viability of bacterial cells via their membrane potential.

Utilizing Synergistic Potential of Mitochondria-Targeting Drugs for Leukemia Therapy.

Acute myeloid leukemia (AML) is an aggressive group of cancers with high mortality rates and significant relapse risks. Current treatments are insufficient, and new therapies are needed. Recent discoveries suggest that AML may be particularly sensitive to chemotherapeutics that target mitochondria. To further investigate this sensitivity, six compounds that target mitochondria [IACS-010759, rotenone, cytarabine, etoposide, ABT-199 (venetoclax), and carbonyl cyanide m-chlorophenylhydrazone] were each paired with six compounds with other activities, including tyrosine kinase inhibitors (midostaurin and dasatinib), glycolytic inhibitors (2-deoxy-D-glucose, 3-bromopyruvate, and lonidamine), and the microtubule destabilizer vinorelbine. The 36 resulting drug combinations were tested for synergistic cytotoxicity against MOLM-13 and OCI-AML2 AML cell lines. Four combinations (IACS-010759 with vinorelbine, rotenone with 2-deoxy-D-glucose, carbonyl cyanide m-chlorophenylhydrazone with dasatinib, and venetoclax with lonidamine) showed synergistic cytotoxicity in both AML cell lines and were selective for tumor cells, as survival of healthy PBMCs was dramatically higher. Among these drug pairs, IACS-010759/vinorelbine decreased ATP level and impaired mitochondrial respiration and coupling efficiency most profoundly. Some of these four treatments were also effective in K-562, KU812 (chronic myelogenous leukemia) and CCRF-CEM, MOLT-4 (acute lymphoblastic leukemia) cells, suggesting that these treatments may have value in treating other forms of leukemia. Finally, two of the four combinations retained high synergy and strong selectivity in primary AML cells from patient samples, supporting the potential of these treatments for patients.

Endothelial Dysfunction Induced by Mitochondrial Uncoupling is prevented by Retinol Binding Protein 7, a PPARγ Target Gene

PPARγ is a nuclear receptor that prevents oxidative stress in the endothelium. Endothelium specific PPARγ target gene, retinol binding protein 7 (RBP7) is required for the protective effects of PPARγ. In response to a cardiovascular stressor (high fat diet or Ang II), transgenic mice carrying a dominant‐negative mutation in PPARγ in the endothelium (E‐V290M) and RBP7 knockout mice (RBP7KO) exhibited increased oxidative stress and endothelial dysfunction. We hypothesize that RBP7 has a protective role against endothelial dysfunction induced by the pro‐oxidant effects of mitochondrial uncoupling (MU). Acetylcholine (ACh) and sodium nitroprusside (SNP) induced vasodilation response were evaluated in carotid arteries from 4 and 8 month old RBP7KO mice, infused with either saline or a sub‐pressor dose of Ang II (120ng/kg/min) for 2 weeks. Ang II infusion did not alter systolic blood pressure either in wild type (WT) (4 months old: 116.2±4.2 vs 113.9±4.1 mmHg; 8 months old: 111.3±3.8 vs 109.5±2.9 mmHg) or RBP7KO (4 months old: 112.9±1.1 vs 111.8±5.2 mmHg; 8 months old: 112.2±4.2 vs 113.9±4.1 mmHg). Endothelium‐dependent relaxation to ACh was not affected by Ang II in either RBP7KO (4 month old: % relaxation ‐ saline, 89.7±1.3 vs Ang II, 93.8±2.0; 8 months old: % relaxation ‐ saline, 80.1±6.8 vs Ang II, 74.6±10.6) or WT littermates (4 months old: % relaxation ‐ saline, 98.1±0.3 vs Ang II, 94.5±1.7; 8 months old: % relaxation ‐ saline, 81.4±4.7 vs Ang II, 78.6±10.4). To test if RBP7 plays a role in MU, the vessels were pre‐incubated with the MU carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; 1uM for 30 min). Pre‐incubation with CCCP did not alter the relaxation responses to ACh in 4 month old mice in all groups. However, in 8 month old mice, there is a trend towards impaired endothelial function in the carotid artery from Ang II‐infused RBP7KO compared with genotype‐matched saline‐treated mice (% relaxation: 51.9±3.8 vs 81.8±5.3, p=0.06) as well as Ang II‐treated WT mice (% relaxation: 51.9±3.8 vs 77.1±2.0 p=0.08). Giving that, RBP7 may play a role in MU, we analyzed the protein expression (western blot) of fusion (OPA1) and fission (DRP1) markers in RBP7 KO and WT mouse lung endothelial cells (MLEC) incubated with CCCP (10 uM) for 15, 30 and 60 minutes. At baseline, both, long (L) (3.62±0.19 vs 3.41±0.13 AU) and short (S) (1.01±0.07 vs 0.97±0.04) isoform of OPA1 were equally expressed in WT and RBP7 KO MLEC respectively. The expression of L OPA1 was almost inhibited in WT (0.12 ± 0.01 AU) and RBP7 KO (0.24 ± 0.01 AU) MLEC after 60 min, while there was no effect in S OPA1. We speculate that differences in L OPA 1 expression may be observed with a lower dose of CCCP or in a recovery experiment where the CCCP is replaced by new media for couple of hours. No differences were observed in neither, the phosphorylation or expression of DRP1.Although the role of RBP7 in fusion/fission process still unclear, we conclude that Ang II alone was not able to impair vascular function, however, in 8 month old mice, Ang II increased the susceptibility to CCCP‐dependent endothelial impairment, and RBP7 was required to mediate vascular protection against mitochondrial uncoupling.

AMPK activation induces mitophagy and promotes mitochondrial fission while activating TBK1 in a PINK1-Parkin independent manner.

Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non‐native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry‐green fluorescence protein‐mtFIS1101-152). Here, we report that both carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read‐out of PTEN‐induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1‐Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK‐binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non‐muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK‐dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1‐Parkin independent manner.

Selenite reduction by the rhizobacterium Azospirillum brasilense, synthesis of extracellular selenium nanoparticles and their characterisation

Microbial reduction of selenium oxyanions has attracted attention in recent years. In this study, an original and simple method for the synthesis of extracellular selenium nanoparticles (Se NPs) of relatively uniform size has been developed using strains Sp7 and Sp245 of the ubiquitous plant-growth promoting rhizobacterium Azospirillum brasilense, both capable of selenite (SeO32−) reduction. In addition, a reliable purification protocol for the recovery of the Se NPs has been perfected, which could be applied with minor modifications to cultures of other microbial species. Importantly, it was found that, by changing the conditions of bacterial reduction of selenite, extracellularly localised Se NPs can be obtained using bacteria which would otherwise produce intracellular Se NPs. In particular, bacterial cultures grown up to the end of the logarithmic growth phase, washed free of culture medium and then incubated with selenite, were used to obtain extracellular Se NPs. Their sizes depended on the initial selenite concentration (∼25–80 nm in diameter at 50–10 mM selenite, respectively). The Se NPs obtained were characterised by transmission electron microscopy (TEM), dynamic light scattering, as well as Raman and UV–vis spectroscopies. Their zeta potential was found to be negative (ca. minus 21–24 mV). Bacterial selenite reduction was also studied in the presence of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). In this case, TEM indicated the formation only of intracellular selenium crystallites. The data show that the formation of extracellular Se NPs requires normal bacterial metabolic activity, while CCCP evidently blocks the membrane export of Se0 nuclei.

Mitophagy is involved in chromium (VI)-induced mitochondria damage in DF-1cells.

Cr (VI), which is a common heavy metal pollutant with strong oxidizing property, exists widely in nature. Organisms can be exposed to Cr (VI) through various means. Cr (VI) causes mitochondrial dysfunction after being absorbed by cells. Whether Cr (VI) induces the selective autophagic degradation of mitochondria, which is a biological process called mitophagy, remains unclear. Mitophagy not only recycles intracellularly damaged mitochondria to compensate for nutrient deprivation but also is involved in mitochondria quality control. Thus, this study investigated whether Cr (VI) could induce mitophagy in DF-1cells. Carbonyl cyanide m-chlorophenylhydrazone, which is a mitochondrial-uncoupling reagent that induces mitophagy, was used. DF-1cells were incubated with different doses of Cr (VI) for varying durations. The autophagy-related proteins LC3-II and p62 levels decreased after 6h of Cr (VI) treatment but recovered within 24h. The mitochondrial membrane potential, which is an indicator of mitochondrial damage, was detected by flow cytometry. We found that different durations of Cr (VI) treatment induced mitochondrial mass decrease and depolarization. Furthermore, the expression of the protein translocase of outer mitochondrial membrane 20 (TOMM20), which is a mitochondrial outer membrane protein, was decreased significantly in the presence of Cr (VI). Our findings indicate that Cr (VI) may contribute to the mitochondrial morphology and function damage and may therefore lead to the autophagic clearance of mitochondria.

Molecular Basis of Growth Inhibition by Acetate of an Adenylate Cyclase-Deficient Mutant of Corynebacterium glutamicum.

In Corynebacterium glutamicum, cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a ΔcyaB mutant. While no growth defect of the ΔcyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media resulted in a severe growth inhibition, which could be reversed by plasmid-based cyaB expression or by supplementation of the medium with cAMP. The effect was concentration- and pH-dependent, suggesting a link to the uncoupling activity of acetate. In agreement, the ΔcyaB mutant had an increased sensitivity to the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The increased uncoupler sensitivity correlated with a lowered membrane potential of acetate-grown ΔcyaB cells compared to wild-type cells. A reduced membrane potential affects major cellular processes, such as ATP synthesis by F1FO-ATP synthase and numerous transport processes. The impaired membrane potential of the ΔcyaB mutant could be due to a decreased expression of the cytochrome bc1-aa3 supercomplex, which is the major contributor of proton-motive force in C. glutamicum. Expression of the supercomplex genes was previously reported to be activated by GlxR-cAMP. A suppressor mutant of the ΔcyaB strain with improved growth on acetate was isolated, which carried a single mutation in the genome leading to an Ala131Thr exchange in GlxR. Introduction of this point mutation into the original ΔcyaB mutant restored the growth defect on acetate. This supported the importance of GlxR for the phenotype of the ΔcyaB mutant and, more generally, of the cAMP-GlxR system for the control of energy metabolism in C. glutamicum.

Membrane Potential is Vital for Rapid Permeabilization of Plasma Membranes and Lipid Bilayers by the Antimicrobial Peptide Lactoferricin B

Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m-chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity.

Quantitative Mitochondrial Proteomics Reveals ANXA7 as a Crucial Factor in Mitophagy.

Mitochondria are involved in many crucial cellular processes. Maintaining healthy mitochondria is essential for cellular homeostasis. Parkin-dependent mitophagy plays an important role in selectively eliminating damaged mitochondria in mammalian cells. However, mechanisms of Parkin-dependent mitophagy remain elusive. In this research, we performed data-independent acquisition-based quantitative mitochondrial proteomics to study the proteomic alterations of carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced Parkin-mediated mitophagy. We identified 222 differentially expressed proteins, with 76 upregulations and 146 downregulations, which were potentially involved in mitophagy. We then demonstrated that annexin A7 (ANXA7), a calcium-dependent phospholipid-binding protein, can translocate to impaired mitochondria upon CCCP treatment, where it played a pivotal part in the process of Parkin-dependent mitophagy via interacting with BASP1. As a mitochondrial uncoupling agent, CCCP indirectly regulated ANXA7 and BASP1 to induce Parkin-dependent mitophagy.