Metal-binding propensity in the mitochondrial dynamin-related protein 1

Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Unlike that seen with the native mitochondrial lipid cardiolipin, metal-bound chelator lipids recruit Drp1 to the membrane but is rendered functionally inactive in membrane fission. Metal-bound chelator beads also recruit Drp1 and represents a potential strategy to deplete or purify the protein from native tissue lysates.

Pathways shaping the mitochondrial inner membrane

Mitochondria are complex organelles with two membranes. Their architecture is determined by characteristic folds of the inner membrane, termed cristae. Recent studies in yeast and other organisms led to the identification of four major pathways that cooperate to shape cristae membranes. These include dimer formation of the mitochondrial ATP synthase, assembly of the mitochondrial contact site and cristae organizing system (MICOS), inner membrane remodelling by a dynamin-related GTPase (Mgm1/OPA1), and modulation of the mitochondrial lipid composition. In this review, we describe the function of the evolutionarily conserved machineries involved in mitochondrial cristae biogenesis with a focus on yeast and present current models to explain how their coordinated activities establish mitochondrial membrane architecture.

Evaluation of the hepatoprotective and antioxidant properties of an aqueous extract of plant polyphenols (helichrysum maracandicum)

This study investigated in vivo and in vitro the effects of helmar 2 polyphenol extracts isolated from the plant Helichrysum maracandicum in the conditions of toxic hepatitis poisoned by carbon dioxide (CCl4) in rats. The experiments were performed on healthy male rats and grouped hepatitis model animals with CCl4. In toxic hepatitis, helmar 2 polyphenol extracts at a dose of 20 mg/kg showed an inhibitory effect on hepatic mitochondrial lipid peroxidation. Evidently, the inhibitory effect of polyphenol extracts on the peroxidation of hepatic mitochondrial lipids was very close to that of the hepatoprotective drug silymarin.

Differential and Synergistic Effects of Low Birth Weight and Western Diet on Skeletal Muscle Vasculature, Mitochondrial Lipid Metabolism and Insulin Signaling in Male Guinea Pigs

Low birth weight (LBW) offspring are at increased risk for developing insulin resistance, a key precursor in metabolic syndrome and type 2 diabetes mellitus. Altered skeletal muscle vasculature, extracellular matrix, amino acid and mitochondrial lipid metabolism, and insulin signaling are implicated in this pathogenesis. Using uteroplacental insufficiency (UPI) to induce intrauterine growth restriction (IUGR) and LBW in the guinea pig, we investigated the relationship between UPI-induced IUGR/LBW and later life skeletal muscle arteriole density, fibrosis, amino acid and mitochondrial lipid metabolism, markers of insulin signaling and glucose uptake, and how a postnatal high-fat, high-sugar “Western” diet (WD) modulates these changes. Muscle of 145-day-old male LBW glucose-tolerant offspring displayed diminished vessel density and altered acylcarnitine levels. Disrupted muscle insulin signaling despite maintained whole-body glucose homeostasis also occurred in both LBW and WD-fed male “lean” offspring. Additionally, postnatal WD unmasked LBW-induced impairment of mitochondrial lipid metabolism, as reflected by increased acylcarnitine accumulation. This study provides evidence that early markers of skeletal muscle metabolic dysfunction appear to be influenced by the in utero environment and interact with a high-fat/high-sugar postnatal environment to exacerbate altered mitochondrial lipid metabolism, promoting mitochondrial overload.

Therapeutic Targeting of Ferroptosis Pathway in Combination with Mitochondrial Oxidative Stress Induction in Acute Myeloid Leukemia

Background: Targeting apoptosis pathways in cancer has been extensively studied including the recent breakthrough therapy using venetoclax in acute myeloid leukemia (AML). However, certain therapy-resistant subsets of AML still pose a clinical challenge, leading us to investigate therapeutic strategies to bypass apoptosis pathways. Recently, non-apoptotic regulated cell death modes have attracted attention. Ferroptosis is a form of regulated cell death characterized by reactive oxygen species (ROS) induction and iron-dependent lipid peroxidation. Leukemia cells exhibit increased oxidative stress, which is further enhanced by iron overload, but only limited studies have explored therapeutic potential of targeting ferroptosis in AML. With mitochondria being the center for ROS production and iron metabolism containing 20-50% of cellular iron, mitochondrial (mito-) regulation of ferroptosis remains unclear. Incorporating our recent knowledge on mito-protease ClpP, hyperactivation of which selectively kills cancer cells through mito-proteolysis and oxidative stress (Ishizawa et al. 2019 Cancer Cell), we here aim to study the therapeutic targeting of the ferroptosis pathway and potential combinatorial targeting of mitochondria in AML. Results: We first utilized the TCGA dataset to analyze gene expression of key components in one of the major anti-ferroptosis pathways cysteine-glutathione-GPX4 axis. AML patients with higher mRNA expression of SLC7A11 (cystine importer), GCLM (a subunit of rate-limiting glutathione synthetase) or GPX4 have significantly shorter overall survival (p < 0.05), suggesting potential prognostic impact of this pathway. Here we focused on GPX4, the most downstream molecule that reduces lipid hydroperoxide to block ferroptosis. Although GPX4 is essential for embryonic development in mice, acquired depletion with conditional knock out was reported to have no significant effect on the number and function of hematopoietic stem cells, suggesting the tolerability of GPX4-targeted therapy. We demonstrate that GPX4 inhibition with sub-micromolar concentrations of ML210, a covalent GPX4 inhibitor with proteome-wide specificity, or with shRNA-mediated knockdown induces prominent cell death in various AML cell lines. The anti-leukemia effects were associated with lipid peroxidation and were almost completely abrogated by a lipophilic antioxidant Liproxstatin-1 (Lip1). The effect of ML210 was also blocked by the iron chelator deferoxamine, indicating ferroptosis. We also demonstrate that GPX4 knockdown increases ROS prominently in mitochondria before cell death is induced, suggesting that mitochondria is involved in the anti-leukemia effects. Meanwhile, GPX4 is among the top 15 sensitization hits in a previously published genome-wide CRISPR screening of leukemia cells treated with potent ClpP agonists ONC201 and ONC212 (Jacques et al. 2020 Genetics), indicating the protective function of GPX4 against mito-oxidative stress. We then found that genetic or pharmacologic hyperactivation of ClpP upregulated GPX4 protein expression predominantly in mitochondria in AML cells, collectively suggesting that upregulation of mito-GPX4 is cell-protective against ClpP-mediated cell death. Indeed, combinatorial ClpP hyperactivation enhanced the anti-leukemia effects of GPX4 inhibition through increased induction of mito-ROS and lipid peroxidation. We further demonstrate that the synergistic cell killing is inhibited by Lip1, indicating that the dual targeting of ClpP and GPX4 induces lipid peroxidation-mediated cell death. Furthermore, the inhibition of lipid peroxidation simultaneously resulted in over 80% reduction of mito-ROS, unexpectedly. This suggests that most of the increased mito-ROS by this combination is associated with lipid peroxidation. Mechanisms of induction of mitochondrial lipid peroxides jointly engaged by ClpP and mito-GPX4, as well as its involvement in the induced oxidative cell death, have yet to be explored. Conclusion: Our data suggests the potential involvement of mitochondrial lipid peroxidation in the anti-leukemia effects of GPX4 inhibition, along with its therapeutic potential in conjunction with mito-oxidative stress induction though instability in mito-proteome. Further investigations are in progress to assess the molecular mechanisms and the in vivo efficacy of the combinatorial treatment. Disclosures Andreeff: Oxford Biomedica UK: Research Funding; Aptose: Consultancy; Medicxi: Consultancy; Syndax: Consultancy; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Research Funding; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; AstraZeneca: Research Funding.

Differential and synergistic effects of low birth weight and Western diet on skeletal muscle vasculature, mitochondrial lipid metabolism and insulin signaling in male guinea pigs

Low birth weight (LBW) offspring are at increased risk for developing insulin resistance, a key precursor in metabolic syndrome and type 2 diabetes mellitus. Altered skeletal muscle vasculature, extracellular matrix, amino acid and mitochondrial lipid metabolism, and insulin signaling are implicated in this pathogenesis. Using uteroplacental insufficiency (UPI) to induce intrauterine growth restriction (IUGR) and LBW in the guinea pig, we investigated the relationship between UPI-induced IUGR/LBW and later life skeletal muscle arteriole density, fibrosis, amino acid and mitochondrial lipid metabolism, markers of insulin signaling and glucose uptake, and how a postnatal high-fat, high-sugar Western diet (WD) modulates these changes. Muscle of 145-day-old male LBW glucose tolerant offspring displayed diminished vessel density and altered acylcarnitine levels. Disrupted muscle insulin signaling despite maintained whole-body glucose homeostasis also occurred in both LBW and WD-fed male lean offspring. Additionally, postnatal WD unmasked LBW-induced impairment of mitochondrial lipid metabolism as reflected by in-creased acylcarnitine accumulation. This study provides evidence that early markers of skeletal muscle metabolic dysfunction appear to be influenced by the in utero environment and interact with a high fat-sugar postnatal environment to exacerbate altered mitochondrial lipid metabolism promoting mitochondrial overload.

Mitochondrial regulation of ferroptosis

Ferroptosis is a form of iron-dependent regulated cell death driven by uncontrolled lipid peroxidation. Mitochondria are double-membrane organelles that have essential roles in energy production, cellular metabolism, and cell death regulation. However, their role in ferroptosis has been unclear and somewhat controversial. In this Perspective, I summarize the diverse metabolic processes in mitochondria that actively drive ferroptosis, discuss recently discovered mitochondria-localized defense systems that detoxify mitochondrial lipid peroxides and protect against ferroptosis, present new evidence for the roles of mitochondria in regulating ferroptosis, and outline outstanding questions on this fascinating topic for future investigations. An in-depth understanding of mitochondria functions in ferroptosis will have important implications for both fundamental cell biology and disease treatment.