What causes cardiac mitochondrial failure at high environmental temperatures?

Although a mechanism accounting for hyperthermic death at critical temperatures remains elusive, the mitochondria of crucial active excitable tissues (i.e. heart and brain) may well be key to this process. Mitochondria produce ∼90% of the ATP required by cells to maintain cellular integrity and function. They also integrate into biosynthetic pathways that support metabolism as a whole, allow communication within the cell, and regulate cellular health and death pathways. We have previously shown that cardiac and brain mitochondria demonstrate decreases in the efficiency of, and absolute capacity for ATP synthesis as temperatures rise, until ultimately there is too little ATP to support cellular demands, and organ failure follows. Importantly, substantial decreases in ATP synthesis occur at temperatures immediately below the temperature of heart failure, and this suggests a causal role of mitochondria in hyperthermic death. However, what causes mitochondria to fail? Here, we consider the answers to this question. Mitochondrial dysfunction at high temperature has classically been attributed to elevated leak respiration suspected to result from increased movement of protons (H+) through the inner mitochondrial membrane (IMM), thereby bypassing the ATP synthases. In this Commentary, we introduce some alternative explanations for elevated leak respiration. We first consider respiratory complex I and then propose that a loss of IMM structure occurs as temperatures rise. The loss of the cristae folds of the IMM may affect the efficiency of H+ transport, increasing H+ conductance either through the IMM or into the bulk water phases of mitochondria. In either case, O2 consumption increases while ATP synthesis decreases.

The Novel Anticancer Aryl-Ureido Fatty Acid CTU Increases Reactive Oxygen Species Production That Impairs Mitochondrial Fusion Mechanisms and Promotes MDA-MB-231 Cell Death.

Cancer cell mitochondria are functionally different from those in normal cells and could be targeted to develop novel anticancer agents. The aryl-ureido fatty acid CTU (16({[4-chloro-3-(trifluoromethyl)phenyl]-carbamoyl}amino)hexadecanoic acid) is the prototype of a new class of targeted agents that enhance the production of reactive oxygen species (ROS) that disrupt the outer mitochondrial membrane (OMM) and kill cancer cells. However, the mechanism by which CTU disrupts the inner mitochondrial membrane (IMM) and activates apoptosis is not clear. Here, we show that CTU-mediated ROS selectively dysregulated the OMA1/OPA1 fusion regulatory system located in the IMM. The essential role of ROS was confirmed in experiments with the lipid peroxyl scavenger α-tocopherol, which prevented the dysregulation of OMA1/OPA1 and CTU-mediated MDA-MB-231 cell killing. The disruption of OMA1/OPA1 and IMM fusion by CTU-mediated ROS accounted for the release of cytochrome c from the mitochondria and the activation of apoptosis. Taken together, these findings demonstrate that CTU depolarises the mitochondrial membrane, activates ROS production, and disrupts both the IMM and OMM, which releases cytochrome c and activates apoptosis. Mitochondrial-targeting agents like CTU offer a novel approach to the development of new therapeutics with anticancer activity.

In situ structure and rotary states of mitochondrial ATP synthase in whole Polytomella cells.

Cells depend on a continuous supply of adenosine triphosphate (ATP), the universal energy currency. In mitochondria, ATP is produced by a series of redox reactions, whereby an electrochemical gradient is established across the inner mitochondrial membrane. The ATP synthase harnesses the energy of the gradient to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. We determined the structure of ATP synthase within mitochondria of the unicellular flagellate Polytomella by electron cryo-tomography and subtomogram averaging at up to 4.2-angstrom resolution, revealing six rotary positions of the central stalk, subclassified into 21 substates of the F1 head. The Polytomella ATP synthase forms helical arrays with multiple adjacent rows defining the cristae ridges. The structure of ATP synthase under native operating conditions in the presence of a membrane potential represents a pivotal step toward the analysis of membrane protein complexes in situ.

Cytotoxic and Apoptotic Effects of Green Synthesized Silver Nanoparticles via Reactive Oxygen Species-Mediated Mitochondrial Pathway in Human Breast Cancer Cells.

Due to their exceptional physicochemical features, green synthesized silver nanoparticles (AgNPs) have been of considerable interest in cancer treatment. In the present study, for the first time, we aimed to green synthesize AgNPs from Euphorbia retusa and explore their anticancer potential on human breast cancer (MCF-7) cells. First, the green synthesized AgNPs (EU-AgNPs) were well characterized by UV-visible spectroscopy, Fourier transmission infrared (FTIR) spectrum, XRD, scanning and transmission electron microscopy (SEM and TEM), and EDX techniques. The characterization data exhibited that EU-AgNPs were spherical in shape and crystalline in nature with an average size of 17.8 nm. FTIR results established the presence of active metabolites in EU-AgNPs. Second, the anticancer effect of EU-AgNPs was evaluated against MCF-7 cells by MTT and neutral red uptake (NRU) assays. Moreover, morphological changes, ROS production, MMP, and apoptotic marker genes were also studied upon exposure to cytotoxic doses of EU-AgNPs. Our results showed that EU-AgNPs induce cytotoxicity in a concentration-dependent manner, with an IC50 value of 40 µg/mL. Morphological changes in MCF-7 cells exposed to EU-AgNPs also confirm their cytotoxic effects. Increased ROS and decreased MMP levels revealed that EU-AgNPs induced oxidative stress and mitochondrial membrane dysfunction. Moreover, ROS-mediated apoptosis was confirmed by elevated levels of proapoptotic marker genes (p53, Bax, caspase-3, and caspase-9) and reduced levels of an antiapoptotic gene (Bcl-2). Altogether, these findings suggested that EU-AgNPs could induce potential anticancer effects through ROS-mediated apoptosis in MCF-7 cells.

MAVS Cys508 palmitoylation promotes its aggregation on the mitochondrial outer membrane and antiviral innate immunity.

Cysteine palmitoylation or S-palmitoylation catalyzed by the ZDHHC family of acyltransferases regulates the biological function of numerous mammalian proteins as well as viral proteins. However, understanding of the role of S-palmitoylation in antiviral immunity against RNA viruses remains very limited. The adaptor protein MAVS forms functionally essential prion-like aggregates upon activation by viral RNA-sensing RIG-I-like receptors. Here, we identify that MAVS, a C-terminal tail-anchored mitochondrial outer membrane protein, is S-palmitoylated by ZDHHC7 at Cys508, a residue adjacent to the tail-anchor transmembrane helix. Using superresolution microscopy and other biochemical techniques, we found that the mitochondrial localization of MAVS at resting state mainly depends on its transmembrane tail-anchor, without regulation by Cys508 S-palmitoylation. However, upon viral infection, MAVS S-palmitoylation stabilizes its aggregation on the mitochondrial outer membrane and thus promotes subsequent propagation of antiviral signaling. We further show that inhibition of MAVS S-palmitoylation increases the host susceptibility to RNA virus infection, highlighting the importance of S-palmitoylation in the antiviral innate immunity. Also, our results indicate ZDHHC7 as a potential therapeutic target for MAVS-related autoimmune diseases.

A promising anti-tumor targeting on ERMMDs mediated abnormal lipid metabolism in tumor cells.

The investigation of aberrations in lipid metabolism within tumor has become a burgeoning field of study that has garnered significant attention in recent years. Lipids can serve as a potent source of highly energetic fuel to support the rapid growth of neoplasia, in where the ER-mitochondrial membrane domains (ERMMDs) provide an interactive network for facilitating communication between ER and mitochondria as well as their intermembrane space and adjunctive proteins. In this review, we discuss fatty acids (FAs) anabolic and catabolic metabolism, as well as how CPT1A-VDAC-ACSL clusters on ERMMDs participate in FAs transport, with a major focus on ERMMDs mediated collaborative loop of FAO, Ca2+ transmission in TCA cycle and OXPHOS process. Here, we present a comprehensive perspective on the regulation of aberrant lipid metabolism through ERMMDs conducted tumor physiology might be a promising and potential target for tumor starvation therapy.

SBDS Gene Mutation Increases ROS Production and Causes DNA Damage as Well as Oxidation of Mitochondrial Membranes in the Murine Myeloid Cell Line 32Dcl3.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutation in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SDS has a variety of clinical features, including exocrine pancreatic insufficiency and hematological dysfunction. Neutropenia is the most common symptom in patients with SDS. SDS is also associated with an elevated risk of developing myelodysplastic syndromes and acute myeloid leukemia. The SBDS protein is involved in ribosome biogenesis, ribosomal RNA metabolism, stabilization of mitotic spindles and cellular stress responses, yet the function of SBDS in detail is still incompletely understood. Considering the diverse function of SBDS, the effect of SBDS seems to be different in different cells and tissues. In this study, we established myeloid cell line 32Dcl3 with a common pathogenic SBDS variant on both alleles in intron 2, 258 + 2T > C, and examined the cellular damage that resulted. We found that the protein synthesis was markedly decreased in the mutant cells. Furthermore, reactive oxygen species (ROS) production was increased, and oxidation of the mitochondrial membrane lipids and DNA damage were induced. These findings provide new insights into the cellular and molecular pathology caused by SBDS deficiency in myeloid cells.

ATAD1 prevents clogging of TOM and damage caused by un-imported mitochondrial proteins.

Mitochondria require the constant import of nuclear-encoded proteins for proper functioning. Impaired protein import not only depletes mitochondria of essential factors but also leads to toxic accumulation of un-imported proteins outside the organelle. Here, we investigate the consequences of impaired mitochondrial protein import in human cells. We demonstrate that un-imported proteins can clog the mitochondrial translocase of the outer membrane (TOM). ATAD1, a mitochondrial ATPase, removes clogged proteins from TOM to clear the entry gate into the mitochondria. ATAD1 interacts with both TOM and stalled proteins, and its knockout results in extensive accumulation of mitochondrial precursors as well as decreased protein import. Increased ATAD1 expression contributes to improved fitness of cells with inefficient mitochondrial protein import. Overall, we demonstrate the importance of the ATAD1 quality control pathway in surveilling protein import and its contribution to cellular health.

Cobra Venom Cytotoxins as a Tool for Probing Mechanisms of Mitochondrial Energetics and Understanding Mitochondrial Membrane Structure.

In this paper, we provide an overview of mitochondrial bioenergetics and specific conditions that lead to the formation of non-bilayer structures in mitochondria. Secondly, we provide a brief overview on the structure/function of cytotoxins and how snake venom cytotoxins have contributed to increasing our understanding of ATP synthesis via oxidative phosphorylation in mitochondria, to reconcile some controversial aspects of the chemiosmotic theory. Specifically, we provide an emphasis on the biochemical contribution of delocalized and localized proton movement, involving direct transport of protons though the Fo unit of ATP synthase or via the hydrophobic environment at the center of the inner mitochondrial membrane (proton circuit) on oxidative phosphorylation, and how this influences the rate of ATP synthesis. Importantly, we provide new insights on the molecular mechanisms through which cobra venom cytotoxins affect mitochondrial ATP synthesis, mitochondrial structure, and dynamics. Finally, we provide a perspective for the use of cytotoxins as novel pharmacological tools to study membrane bioenergetics and mitochondrial biology, how they can be used in translational research, and their potential therapeutic applications.

Pore-Forming VDAC Proteins of the Outer Mitochondrial Membrane: Regulation and Pathophysiological Role.

Voltage-dependent anion channels (VDAC1-3) of the outer mitochondrial membrane are a family of pore-forming ß-barrel proteins that carry out controlled "filtration" of small molecules and ions between the cytoplasm and mitochondria. Due to the conformational transitions between the closed and open states and interaction with cytoplasmic and mitochondrial proteins, VDACs not only regulate the mitochondrial membrane permeability for major metabolites and ions, but also participate in the control of essential intracellular processes and pathological conditions. This review discusses novel data on the molecular structure, regulatory mechanisms, and pathophysiological role of VDAC proteins, as well as future directions in this area of research.

Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX.

PPTC7 is a mitochondrial-localized phosphatase that suppresses BNIP3- and NIX-mediated mitophagy, but the mechanisms underlying this regulation remain ill-defined. Here, we demonstrate that loss of PPTC7 upregulates BNIP3 and NIX post-transcriptionally and independent of HIF-1α stabilization. Loss of PPTC7 prolongs the half-life of BNIP3 and NIX while blunting their accumulation in response to proteasomal inhibition, suggesting that PPTC7 promotes the ubiquitin-mediated turnover of BNIP3 and NIX. Consistently, overexpression of PPTC7 limits the accumulation of BNIP3 and NIX protein levels, which requires an intact catalytic motif but is surprisingly independent of its targeting to mitochondria. Consistently, we find that PPTC7 is dual-localized to the outer mitochondrial membrane and the matrix. Importantly, anchoring PPTC7 to the outer mitochondrial membrane is sufficient to blunt BNIP3 and NIX accumulation, and proximity labeling and fluorescence co-localization experiments demonstrate that PPTC7 dynamically associates with BNIP3 and NIX within the native cellular environment. Collectively, these data reveal that a fraction of PPTC7 localizes to the outer mitochondrial membrane to promote the proteasomal turnover of BNIP3 and NIX, limiting basal mitophagy.

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4.

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

The C-terminal sequences of Bcl-2 family proteins mediate interactions that regulate cell death.

Programmed cell death via the both intrinsic and extrinsic pathways is regulated by interactions of the Bcl-2 family protein members that determine whether the cell commits to apoptosis via mitochondrial outer membrane permeabilization (MOMP). Recently the conserved C-terminal sequences (CTSs) that mediate localization of Bcl-2 family proteins to intracellular membranes, have been shown to have additional protein-protein binding functions that contribute to the functions of these proteins in regulating MOMP. Here we review the pivotal role of CTSs in Bcl-2 family interactions including: (1) homotypic interactions between the pro-apoptotic executioner proteins that cause MOMP, (2) heterotypic interactions between pro-apoptotic and anti-apoptotic proteins that prevent MOMP, and (3) heterotypic interactions between the pro-apoptotic executioner proteins and the pro-apoptotic direct activator proteins that promote MOMP.

Advancements in unravelling the fundamental function of the ATAD3 protein in multicellular organisms.

ATPase family AAA domain containing protein 3, commonly known as ATAD3 is a versatile mitochondrial protein that is involved in a large number of pathways. ATAD3 is a transmembrane protein that spans both the inner mitochondrial membrane and outer mitochondrial membrane. It, therefore, functions as a connecting link between the mitochondrial lumen and endoplasmic reticulum facilitating their cross-talk. ATAD3 contains an N-terminal domain which is amphipathic in nature and is inserted into the membranous space of the mitochondria, while the C-terminal domain is present towards the lumen of the mitochondria and contains the ATPase domain. ATAD3 is known to be involved in mitochondrial biogenesis, cholesterol transport, hormone synthesis, apoptosis and several other pathways. It has also been implicated to be involved in cancer and many neurological disorders making it an interesting target for extensive studies. This review aims to provide an updated comprehensive account of the role of ATAD3 in the mitochondria especially in lipid transport, mitochondrial-endoplasmic reticulum interactions, cancer and inhibition of mitophagy.

Lipid unsaturation promotes BAX and BAK pore activity during apoptosis.

BAX and BAK are proapoptotic members of the BCL2 family that directly mediate mitochondrial outer membrane permeabilition (MOMP), a central step in apoptosis execution. However, the molecular architecture of the mitochondrial apoptotic pore remains a key open question and especially little is known about the contribution of lipids to MOMP. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.

Understanding mitochondrial potassium channels: 33 years after discovery.

Mitochondrial investigations have extended beyond their traditional functions, covering areas such as ATP synthesis and metabolism. Mitochondria are now implicated in new functional areas such as cytoprotection, cellular senescence, tumor function and inflammation. The basis of these new areas still relies on fundamental biochemical/biophysical mitochondrial functions such as synthesis of reactive oxygen species, mitochondrial membrane potential, and the integrity of the inner mitochondrial membrane i.e., the passage of various molecules through the mitochondrial membranes. In this view transport of potassium cations, known as the potassium cycle, plays an important role. It is believed that K+ influx is mediated by various potassium channels present in the inner mitochondrial membrane. In this article, we present an overview of the key findings and characteristics of mitochondrial potassium channels derived from research of many groups conducted over the past 33 years. We propose a list of six fundamental observations and most important ideas dealing with mitochondrial potassium channels. We also discuss the contemporary challenges and future prospects associated with research on mitochondrial potassium channels.

The regulation of the apoptotic pore-An immunological tightrope walk.

Apoptotic pore formation in mitochondria is the pivotal point for cell death during mitochondrial apoptosis. It is regulated by BCL-2 family proteins in response to various cellular stress triggers and mediates mitochondrial outer membrane permeabilization (MOMP). This allows the release of mitochondrial contents into the cytosol, which triggers rapid cell death and clearance through the activation of caspases. However, under conditions of low caspase activity, the mitochondrial contents released into the cytosol through apoptotic pores serve as inflammatory signals and activate various inflammatory responses. In this chapter, we discuss how the formation of the apoptotic pore is regulated by BCL-2 proteins as well as other cellular or mitochondrial proteins and membrane lipids. Moreover, we highlight the importance of sublethal MOMP in the regulation of mitochondrial-activated inflammation and discuss its physiological consequences in the context of pathogen infection and disease and how it can potentially be exploited therapeutically, for example to improve cancer treatment.

Comprehensive pan-cancer analysis of mitochondrial outer membrane permeabilisation activity reveals positive immunomodulation and assists in identifying potential therapeutic targets for immunotherapy resistance.

BACKGROUND: Mitochondrial outer membrane permeabilisation (MOMP) plays a pivotal role in cellular death and immune activation. A deeper understanding of the impact of tumour MOMP on immunity will aid in guiding more effective immunotherapeutic strategies. METHODS: A comprehensive pan-cancer dataset comprising 30 cancer-type transcriptomic cohorts, 20 immunotherapy transcriptomic cohorts and three immunotherapy scRNA-seq datasets was collected and analysed to determine the influence of tumour MOMP activity on clinical prognosis, immune infiltration and immunotherapy effectiveness. Leveraging 65 scRNA-Seq datasets, the MOMP signature (MOMP.Sig) was developed to accurately reflect tumour MOMP activity. The clinical predictive value of MOMP.Sig was explored through machine learning models. Integration of the MOMP.Sig model and a pan-cancer immunotherapy CRISPR screen further investigated potential targets to overcome immunotherapy resistance, which subsequently underwent clinical validation. RESULTS: Our research revealed that elevated MOMP activity reduces mortality risk in cancer patients, drives the formation of an anti-tumour immune environment and enhances the response to immunotherapy. This finding emphasises the potential clinical application value of MOMP activity in immunotherapy. MOMP.Sig, offering a more precise indicator of tumour cell MOMP activity, demonstrated outstanding predictive efficacy in machine-learning models. Moreover, with the assistance of the MOMP.Sig model, FOXO1 was identified as a core modulator that promotes immune resistance. Finally, these findings were successfully validated in clinical immunotherapy cohorts of skin cutaneous melanoma and triple-negative breast cancer patients. CONCLUSIONS: This study enhances our understanding of MOMP activity in immune modulation, providing valuable insights for more effective immunotherapeutic strategies across diverse tumours.

Complex regulation of mitochondrial signaling by human adenovirus minor capsid protein VI.

The controlled release of mitochondrial content into the cytosol has emerged as one of the key steps in mitochondrial signaling. In particular, the release of mitochondrial DNA (mtDNA) into the cytosol has been shown to activate interferon beta (IFN-ß) gene expression to execute the innate immune response. In this report, we show that human adenovirus type 5 (HAdV-C5) infection induces the release of mtDNA into the cytosol. The release of mtDNA is mediated by the viral minor capsid protein VI (pVI), which localizes to mitochondria. The presence of the mitochondrial membrane proteins Bak and Bax are needed for the mtDNA release, whereas the viral E1B-19K protein blocked pVI-mediated mtDNA release. Surprisingly, the pVI-mediated mtDNA release did not increase but inhibited the IFN-ß gene expression. Notably, the pVI expression caused mitochondrial leakage of the HSP60 protein. The latter prevented specific phosphorylation of the interferon regulatory factor 3 (IRF3) needed for IFN-ß gene expression. Overall, we assign a new mitochondria and IFN-ß signaling-modulating function to the HAdV-C5 minor capsid protein VI. IMPORTANCE: Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, including conjunctivitis and the common cold. HAdVs need to interfere with multiple cellular signaling pathways during the infection to gain control over the host cell. In this study, we identified human adenovirus type 5 (HAdV-C5) minor capsid protein VI as a factor modulating mitochondrial membrane integrity and mitochondrial signaling. We show that pVI-altered mitochondrial signaling impedes the cell's innate immune response, which may benefit HAdV growth. Overall, our study provides new detailed insights into the HAdV-mitochondria interactions and signaling. This knowledge is helpful when developing new anti-viral treatments against pathogenic HAdV infections and improving HAdV-based therapeutics.

Mastering Death: The Roles of Viral Bcl-2 in dsDNA Viruses.

Proteins of the Bcl-2 family regulate cellular fate via multiple mechanisms including apoptosis, autophagy, senescence, metabolism, inflammation, redox homeostasis, and calcium flux. There are several regulated cell death (RCD) pathways, including apoptosis and autophagy, that use distinct molecular mechanisms to elicit the death response. However, the same proteins/genes may be deployed in multiple biochemical pathways. In apoptosis, Bcl-2 proteins control the integrity of the mitochondrial outer membrane (MOM) by regulating the formation of pores in the MOM and apoptotic cell death. A number of prosurvival genes populate the genomes of viruses including those of the pro-survival Bcl-2 family. Viral Bcl-2 proteins are sequence and structural homologs of their cellular counterparts and interact with cellular proteins in apoptotic and autophagic pathways, potentially allowing them to modulate these pathways and determine cellular fate.