Dissociation of mitochondrial HK-II elicits mitophagy and confers cardioprotection against ischemia

Abstract Preservation of mitochondrial integrity is critical for maintaining cellular homeostasis. Mitophagy is a mitochondria-specific type of autophagy which eliminates damaged mitochondria thereby contributing to mitochondrial quality control. Depolarization of the mitochondrial membrane potential is an established mechanism for inducing mitophagy, mediated through PINK1 stabilization and Parkin recruitment to mitochondria. Hexokinase-II (HK-II) which catalyzes the first step in glucose metabolism, also functions as a signaling molecule to regulate cell survival, and a significant fraction of cellular HK-II is associated with mitochondria (mitoHK-II). We demonstrate here that pharmacological interventions and adenoviral expression of a mitoHK-II dissociating peptide which reduce mitoHK-II levels lead to robust increases in mitochondrial Parkin and ubiquitination of mitochondrial proteins in cardiomyocytes and in a human glioblastoma cell line 1321N1, independent of mitochondrial membrane depolarization or PINK1 accumulation. MitoHK-II dissociation-induced mitophagy was demonstrated using Mito-Keima in cardiomyocytes and in 1321N1 cells. Subjecting cardiomyocytes or the in vivo heart to ischemia leads to modest dissociation of mitoHK-II. This response is potentiated by expression of the mitoHK-II dissociating peptide, which increases Parkin recruitment to mitochondria and, importantly, provides cardioprotection against ischemic stress. These results suggest that mitoHK-II dissociation is a physiologically relevant cellular event that is induced by ischemic stress, the enhancement of which protects against ischemic damage. The mechanism which underlies the effects of mitoHK-II dissociation can be attributed to the ability of Bcl2-associated athanogene 5 (BAG5), an inhibitor of Parkin, to localize to mitochondria and form a molecular complex with HK-II. Overexpression of BAG5 attenuates while knockdown of BAG5 sensitizes the effect of mitoHK-II dissociation on mitophagy. We suggest that HK-II, a glycolytic molecule, can function as a sensor for metabolic derangements at mitochondria to trigger mitophagy, and modulating the intracellular localization of HK-II could be a novel way of regulating mitophagy to prevent cell death induced by ischemic stress.

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Abstract 16870: Dissociation of Mitochondrial HK-II Triggers Mitochondria Specific Autophagy

Circulation ◽

10.1161/circ.130.suppl_2.16870 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Shigeki Miyamoto ◽

David J Roberts ◽

Valerie P Tan-Sah

Keyword(s):

Energy Production ◽

Ventricular Myocytes ◽

Neonatal Rat ◽

Mouse Heart ◽

Hexokinase Ii ◽

Survival Pathway ◽

Mitochondrial Membrane Depolarization ◽

Neonatal Rat Ventricular Myocytes

Introduction: There is emerging evidence that the metabolic pathway interplays with the survival pathway to preserve cellular homeostasis. Hexokinases (HKs) catalyze the first step of glucose metabolism and hexokinase-II (HK-II) is the predominant isoform in the heart. Our recent study revealed that HK-II positively regulates general autophagy in the absence of glucose. Mitochondrial HK-II (mitoHK-II) is regulated by Akt and provides cardioprotection while it is decreased in the ischemic heart. Hypothesis: We tested the hypothesis that mitoHK-II dissociation triggers mitochondria specific autophagy (mitophagy). Results: As previously reported, mitoHK-II levels were decreased by ~40% in the perfused mouse heart subjected to global ischemia and in neonatal rat ventricular myocytes (NRVMs) subjected to simulated ischemia. To assess the role of mitoHK-II dissociation, mitoHK-II dissociating peptide (15NG) was expressed in NRVMs. MitoHK-II was decreased by 40% in NRVMs expressing 15NG which was accompanied with Parkin translocation to mitochondria and ubiquitination of mitochondrial proteins. This response was attenuated by Parkin knockdown and reversed by the recovery of mitoHK-II by co-expression of HK-II but not by that of mitochondria binding deficient mutant. 15NG expression did not induce mitochondrial membrane depolarization nor PINK1 stabilization at mitochondria, suggesting that the effects of mitoHK-II dissociation is not dependent on the previously established mitochondria depolarization/PINK1 pathway. This was confirmed by the experiments using PINK1 siRNA. Modest dissociation of mitoHK-II (by 20%) did not induce mitophagic responses but remarkably enhanced FCCP induced mitophagy, indicating that these two pathways are synergetic. We will be analyzing 15NG transgenic mice generated in our lab to determine the mitophagic role of mitoHK-II dissociation in vivo. Conclusions: These results suggest that mitoHK-II dissociation can regulate Parkin dependent mitophagy, in conjunction with depolarization dependent mechanisms and that HK-II could confer cardioprotection by switching the cell from an energy production to an energy conservation mode under ischemia.

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Decavanadate Toxicology and Pharmacological Activities: V10or V1, Both or None?

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/6103457 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 38

Author(s):

M. Aureliano

Keyword(s):

Atpase Activity ◽

Mitochondrial Membrane ◽

Actin Polymerization ◽

Calcium Pump ◽

Mitochondrial Depolarization ◽

Pharmacological Activities ◽

Oxidative Stress Parameters ◽

Mitochondrial Membrane Depolarization

This review covers recent advances in the understanding of decavanadate toxicology and pharmacological applications. Toxicologicalin vivostudies point out that V10induces several changes in several oxidative stress parameters, different from the ones observed for vanadate (V1). Inin vitrostudies with mitochondria, a particularly potent V10effect, in comparison with V1, was observed in the mitochondrial depolarization (IC50= 40 nM) and oxygen consumption (99 nM). It is suggested that mitochondrial membrane depolarization is a key event in decavanadate induction of necrotic cardiomyocytes death. Furthermore, only decavanadate species and not V1potently inhibited myosin ATPase activity stimulated by actin (IC50= 0.75μM) whereas exhibiting lower inhibition activities for Ca2+-ATPase activity (15 μM) and actin polymerization (17 μM). Because both calcium pump and actin decavanadate interactions lead to its stabilization, it is likely that V10interacts at specific locations with these proteins that protect against hydrolysis but, on the other hand, it may induce V10reduction to oxidovanadium(IV). Putting it all together, it is suggested that the pharmacological applications of V10species and compounds whose mechanism of action is still to be clarified might involve besides V10and V1also vanadium(IV) species.

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Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA

10.1101/2021.08.06.455397 ◽

2021 ◽

Author(s):

David Pla-Martin ◽

Ayesha Sen ◽

Sebastian Kallabis ◽

Julian Nuechel ◽

Kanjanamas Maliphol ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Dominant Negative ◽

Control Mechanisms ◽

Mtdna Copy Number ◽

Mitochondrial Quality Control ◽

Mtdna Damage ◽

Potential Benefits ◽

Normal Ageing ◽

Different Levels

Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria together with mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here we show that mtDNA depletion after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, is due to an exacerbated mtDNA turnover. Targeting of nucleoids is controlled by Twinkle which, together with the mitochondrial transmembrane proteins ATAD3 and SAMM50, orchestrate mitochondrial membrane remodeling to form extrusions. mtDNA removal depends on autophagy and requires the vesicular trafficking protein VPS35 which binds to Twinkle-enriched mitochondrial subcompartments upon mtDNA damage. Stimulation of autophagy by rapamycin selectively removes mtDNA deletions which accumulated during muscle regeneration in vivo, but without affecting mtDNA copy number. With these results we unveil a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network. We reveal the molecular targets involved in a process with multiple potential benefits against human mtDNA related diseases, either inherited, acquired or due to normal ageing.

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Abstract 208: Augmented Cerebrovascular Responses to Diazoxide, an Opener of MitokAtp Channels, Following Middle Cerebral Artery (MCA) Occlusion in Rats

Hypertension ◽

10.1161/hyp.62.suppl_1.a208 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Ibolya Rutkai ◽

Somhrita Dutta ◽

Dana Liu ◽

Prasad V Katakam ◽

David W Busija

Keyword(s):

Mitochondrial Membrane ◽

Major Effect ◽

Mitochondrial Proteins ◽

Mitochondrial Morphology ◽

Western Blots ◽

Vascular Responses ◽

Cox Inhibitor ◽

Mitochondrial Membrane Depolarization ◽

Nos Inhibitor ◽

And Function

Previous research has not examined the effects of ischemia on cerebral vascular responses to mitochondrial activation in experimental strokes caused by occlusion of the MCA (MCAO). We investigated the role and mechanisms of mitochondrial derived vasoreactivity in the MCA of male SD rats following 90 min ischemia/48 h reperfusion injury. Ischemia was induced ipsilaterally (I) and the contralateral (C) side was non-ischemic. Electron microscopy showed disrupted mitochondrial morphology on the I side. Western blots for expression of mitochondrial proteins (Mean±SEM of immunoband intensity normalized to β-actin and as C vs. I): DRP-1 (1±0.1 vs. 3.1±0.2); VDAC (0.4±0.1 vs. 0.8±0.2); and complex-V (1.3±0.3 vs.2.0±0.3) as well as the non-mitochondrial proteins: phosphorylated (ph) ph-nNOS (0.4±0.1 vs. 0.7±0.1); ph-eNOS (0.2±0.06 vs. 0.7±0.2); COX-1 (0.5±0.1 vs. 0.8±0.1); and COX-2 (0.16±0.04 vs. 0.3±0.03) were elevated on I compared with C. The I mitochondrial membrane potential was greater (165±7 %) compared with C using TMRE fluorescence. Mitochondrial membrane depolarization decreased the TMRE intensity in both groups (100 to 80±3 vs. 165±7 to 70±4). Vascular responses of the MCA were characterized using the isolated, pressurized artery technique. Vasodilation in response to Ach (12±2 vs. 3±1), BK (42±9 vs. 32±6), SNP (75±7 vs. 38±4), and vasoconstriction to serotonin (65±8 vs. 33±7), were significantly decreased in I compared to C MCAs. On the other hand, 50 μM DZ induced vasodilation was enhanced in I arteries compared with C (5±1 vs. 17±2). Diazoxide induced dilation was decreased in the presence of the NOS inhibitor L-NAME (5±1 to 1±2 vs. 17±2 to 3±2) and the non-selective COX inhibitor indomethacin (5±1 to 1±2 vs.17±2 to 6±3). Our results indicate that experimental stoke has an major effect on mitochondria via inducing mitochondrial biogenesis leading to altered morphology, protein expression and function. Furthermore, the nitric-oxide and prostanoid pathways appear to be involved in enhanced diazoxide mediated vasodilation after MCAO. We speculate that targeting mitochondria may be useful therapy for improving outcome in stroke patients.

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The mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress

10.1101/2020.09.14.296194 ◽

2020 ◽

Author(s):

Sandra Backes ◽

Yury S. Bykov ◽

Markus Räschle ◽

Jialin Zhou ◽

Svenja Lenhard ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Surface Protein ◽

Mitochondrial Proteins ◽

Outer Mitochondrial Membrane ◽

Induced Stress ◽

Precursor Proteins ◽

Surface Receptor ◽

Chaperone Binding

SummaryMost mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high content screens, we revisited the question of Tom70 function and considerably expanded the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, in particular of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondria-specifying targeting receptor.

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Pegylated recombinant human arginase 1 induces autophagy and apoptosis via the ROS-activated AKT/mTOR pathway in bladder cancer cells

10.21203/rs.3.rs-33231/v1 ◽

2020 ◽

Author(s):

Zhuyun Zhao ◽

Peng Zhang ◽

Wei Li ◽

Dengchuan Wang ◽

Changneng Ke ◽

...

Keyword(s):

Bladder Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Mitochondrial Membrane ◽

Time Dependent ◽

Anticancer Effect ◽

Mitochondrial Membrane Depolarization ◽

Bladder Cancer Cells

Abstract Background Bladder cancer is one of the dominant cancers worldwide, especially for male. Currently, the therapeutical regimen of bladder cancer is based on surgery, radiation therapy, chemotherapy and immunotherapy, but the clinical outcome is still needed to improve. Recombinant human arginase (rhArg, BCT-100) is a novel agent to show great anticancer effect on arginine auxotrophic tumor. However, the effect of rhArg on bladder cancer still remains unclear. Methods A panel of six bladder cancer cell lines (BIU-87, EJ-1, J82, SCaBER, T24 and 5637) was employed to assess the anticancer effect of BCT-100 in vitro by MTT assay. T24 nude mice xenograft models were established to evaluate the anticancer effect of BCT-100 in vivo. Protein level (argininosuccinate synthetase 1 (ASS1), ornithine transcarbamylase, cleaved-PARP, PEG, Survivin, p62, Beclin-1, LC3B, p-AKT, p-mTOR) was detected by Western blot. Intracellular, serum and intratumoral arginine concentrations were examined by ELISA. Apoptotic rate, H2O2 and mitochondrial membrane depolarization were tested by flow cytometer. Immunoflorescence on ki67 and TUNEL assay were applied to identify cellular and tumoral apoptotic events. Results BCT-100 displayed anticancer effects on bladder cancer cells in vitro and in vivo. The expression of ASS1 varies in different bladder cancer cell lines, and ornithine transcarbamylase is almost deficient except weakly expressed in SCaBER cell line. Knockdown ASS1 in BIU-87 cells could enhance the cytotoxicity induced by BCT-100. Intracellular arginine level was sharply decreased followed by apoptotic events. Futhermore, BCT-100 induced H2O2 production and mitochondrial membrane depolarization, leading to cytochrome c and smac released from mitochondria to cytosol. The expression of LC3B and Becllin-1 was up-regulated, while p62 was down-regulated in a time dependent manner. Autophagic flux was also observed upon BCT-100 treatment. Besides, the phosphorylation of AKT/mTOR pathway was suppressed in a time dependent fashion in BCT-100-treated T24 cells. N-Acetyl-L-cystein reduced the apoptosis and autophagy induced by BCT-100, while CQ, MK-2206 and rapamycin potentiated the apoptosis triggered by BCT-100. Conclusions The present study demonstrated that BCT-100 induced autophagy and apoptosis via ROS mediated AKT/mTOR signaling pathway in bladder cancer cells.

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Low-Dose IL-2 Reduces Mitochondrial Priming and Increases Bcl2 Expression in CD4 Memory Regulatory T Cells

Blood ◽

10.1182/blood.v124.21.1414.1414 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1414-1414

Author(s):

Kazuyuki Murase ◽

Haesook T. Kim ◽

Masahiro Hirakawa ◽

Tiago Reis Matos ◽

Yutaka Kawano ◽

...

Keyword(s):

T Cells ◽

Mitochondrial Membrane ◽

Low Dose ◽

Chronic Gvhd ◽

Peak Effect ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Mitochondrial Membrane Depolarization ◽

Bcl2 Expression

Abstract CD4+CD25+Foxp3+ regulatory T cells (Treg) play a critical role in establishment of immune tolerance and prevention of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The recovery and maintenance of Treg after HSCT is dependent on homeostatic factors including the generation of naïve Treg from hematopoietic precursor cells, the proliferation and expansion of mature Treg and the survival of Treg in vivo. We previously reported that Treg in healthy donors are more susceptible to mitochondrial apoptotic priming than conventional T cells (Tcon). Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T cell subsets and particularly in patients with chronic GVHD. Interleukin-2 (IL-2) is a critical homeostatic cytokine that is required for Treg development, expansion, activity and survival. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo, clinical improvement of chronic GVHD (Koreth et al. NEJM 2011), increased Treg Bcl2 expression and increased Treg resistance to apoptosis in vitro (Matsuoka et al. SciTM, 2013). These observations led us to undertake a more detailed analysis of apoptotic pathways in functionally and phenotypically distinct Treg subsets including naïve Treg, recent thymic emigrants (RTE) Treg and memory Treg. To examine the mechanisms whereby IL-2 affects susceptibility to apoptosis in each Treg subset, we used a functional flow cytometry-based assay (apoptotic profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assay allowed us to compare "priming", which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization, in naïve, RTE and memory subsets. We also examined cytoplasmic expression of Bcl2 as an additional measure of susceptibility to apoptosis in each subset. In resting blood obtained from healthy donors (n=25), memory Treg were more "primed" than either naïve or RTE subsets when exposed to several BH3 peptides (BID, NOXA, PUMA, BMF and the combination of BAD+NOXA) (Figure 1). Memory Treg were also found to have decreased expression of Bcl2 compared to naïve or RTE subsets. Thus, memory Treg in healthy individuals are more susceptible to apoptosis than other Treg subsets through the mitochondrial pathway (Figure 1). To examine the functional effects of IL-2 on T cell homeostasis, Treg and Tcon were purified by cell sorting and cultured for 5 days with different concentrations of IL-2 (0, 10, 100, 1000 U/ml). Low-concentration IL-2 (10 U/ml) lowered apoptotic priming and increased Bcl2 expression in all Treg subsets. Similar effects were observed in all Tcon subsets, but higher concentrations of IL-2 (≥100 U/ml) were required to decrease apoptotic priming and increase Bcl2 expression in Tcon subsets. The peak effect of IL-2 on Treg priming occurred on day 3 but these effects appeared more rapidly in naïve and RTE Treg subsets than in memory Treg. However, the effect of IL-2 on apoptotic priming persisted for a longer period in memory Treg than in naïve and RTE Treg subsets. The peak effect of IL-2 on Bcl2 expression occurred on day 3 in all Treg subsets. To compare the effect of IL-2 on Treg and Tcon subsets, we examined apoptotic priming and Bcl2 expression after 3 day incubation with different IL-2 concentrations (0.001 - 1000 U/ml). Very low-concentration IL-2 (1 U/ml) lowered priming and increased Bcl2 expression in memory Treg, but higher concentrations of IL-2 (≥10 U/ml) were required to increase Bcl2 expression and decrease priming in naïve and RTE Treg. All Tcon subsets showed a similar pattern of response but higher concentrations of IL-2 (≥100 U/ml) were required. Memory Treg are the predominant regulatory population in patients with cGVHD and the high level of priming in this subset contributes to the inability to maintain adequate numbers of CD4 Treg after HSCT. In healthy donors, low concentrations of IL-2 are sufficient to reduce mitochondrial priming and increase Bcl2 expression in memory Treg. IL-2 has similar effects on Tcon but 10-100 fold higher concentrations of IL-2 are required. These concentration-dependent effects of IL-2 likely contribute to the selective expansion and persistence of Treg in patients with cGVHD receiving daily low-dose IL-2 therapy after HSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.

10.21203/rs.3.rs-629802/v1 ◽

2021 ◽

Author(s):

David Pla-Martin ◽

Ayesha Sen ◽

Sebastian Kallabis ◽

Julian Nüchel ◽

Kanjanamas Maliphol ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Dominant Negative ◽

Control Mechanisms ◽

Mtdna Copy Number ◽

Mitochondrial Quality Control ◽

Mtdna Damage ◽

Potential Benefits ◽

Normal Ageing ◽

Different Levels

Abstract Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria together with mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here we show that mtDNA depletion after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, is due to an exacerbated mtDNA turnover. Targeting of nucleoids is controlled by Twinkle which, together with the mitochondrial transmembrane proteins ATAD3 and SAMM50, orchestrate mitochondrial membrane remodeling to form extrusions. mtDNA removal depends on autophagy and requires the vesicular trafficking protein VPS35 which binds to Twinkle-enriched mitochondrial subcompartments upon mtDNA damage. Stimulation of autophagy by rapamycin selectively removes mtDNA deletions which accumulated during muscle regeneration in vivo, but without affecting mtDNA copy number. With these results we unveil a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network. We reveal the molecular targets involved in a process with multiple potential benefits against human mtDNA related diseases, either inherited, acquired or due to normal ageing.

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Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

Journal of Fungi ◽

10.3390/jof7020130 ◽

2021 ◽

Vol 7 (2) ◽

pp. 130

Author(s):

Nathan P. Wiederhold

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Candida Species ◽

Mammalian Cells ◽

Mitochondrial Membrane ◽

Fungal Infections ◽

Published Data ◽

Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

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