Beyond fission and fusion-Diving into the mysteries of mitochondrial shape.

Mitochondrial shape and network formation have been primarily associated with the well-established processes of fission and fusion. However, recent research has unveiled an intricate and multifaceted landscape of mitochondrial morphology that extends far beyond the conventional fission-fusion paradigm. These less-explored dimensions harbor numerous unresolved mysteries. This review navigates through diverse processes influencing mitochondrial shape and network formation, highlighting the intriguing complexities and gaps in our understanding of mitochondrial architecture. The exploration encompasses various scales, from biophysical principles governing membrane dynamics to molecular machineries shaping mitochondria, presenting a roadmap for future research in this evolving field.

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.

Deletion of the Mitochondrial Membrane Protein Fam210b Is Associated with the Development of Systemic Lupus Erythematosus.

Mitochondrial dysfunction has been increasingly recognized as a trigger for systemic lupus erythematosus (SLE). Recent bioinformatics studies have suggested Fam210b as a significant candidate for the classification and therapeutic targeting of SLE. To experimentally prove the role of Fam210b in SLE, we constructed Fam210b knockout (Fam210b-/-) mice using the CRISPR-Cas9 method. We found that approximately 15.68% of Fam210b-/- mice spontaneously developed lupus-like autoimmunity, which was characterized by skin ulcerations, splenomegaly, and an increase in anti-double-stranded DNA (anti-dsDNA) IgG antibodies and anti-nuclear antibodies(ANA). Single-cell sequencing showed that Fam210b was mainly expressed in erythroid cells. Critically, the knockout of Fam210b resulted in abnormal erythrocyte differentiation and development in the spleens of mice. Concurrently, the spleens exhibited an increased number of CD71+ erythroid cells, along with elevated levels of reactive oxygen species (ROS) in the erythrocytes. The co-culture of CD71+ erythroid cells and lymphocytes resulted in lymphocyte activation and promoted dsDNA and IgG production. In summary, Fam210b knockout leads to a low probability of lupus-like symptoms in mice through the overproduction of ROS in CD71+ erythroid cells. Thus, Fam210b reduction may serve as a novel key marker that triggers the development of SLE.

Drp1 Aggravates Copper Nanoparticle-Induced ER-Phagy by Disturbing Mitochondria-Associated Membranes in Chicken Hepatocytes.

As an efficient alternative copper (Cu) source, copper nanoparticles (nano-Cu) have been widely supplemented into animal-producing food. Therefore, it is necessary to assess the effect of nano-Cu exposure on the biological health risk. Recently, the toxic effects of nano-Cu have been confirmed but the underlying mechanism remains unclear. This study reveals the impact of nano-Cu on endoplasmic reticulum autophagy (ER-phagy) in chicken hepatocytes and further identifies Drp1 and its downstream gene FAM134B as crucial regulators of nano-Cu-induced hepatotoxicity. Nano-Cu exposure can induce Cu ion overaccumulation and pathological injury in the liver, trigger excessive mitochondrial fission and mitochondria-associated membrane (MAM) integrity damage, and activate ER-phagy in vivo and in vitro. Interestingly, the knockdown of Drp1 markedly decreases the expression of FAM134B induced by nano-Cu. Furthermore, the expression levels of ATL3, CCPG1, SEC62, TEX264, and LC3II/LC3I induced by nano-Cu exposure are decreased by inhibiting the expression of Drp1. Simultaneously, the inhibition of FAM134B effectively alleviates nano-Cu-induced ER-phagy by downregulating the expression of ATL3, CCPG1, SEC62, TEX264, and LC3II/LC3I. Overall, these results suggest that Drp1-mediated impairment of MAM integrity leads to ER-phagy as a novel molecular mechanism involved in the regulation of nano-Cu-induced hepatotoxicity. These findings provide new ideas for future research on the mechanism of nano-Cu-induced hepatotoxicity.

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.

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.

Mfn2-dependent fusion pathway of PE-enriched micron-sized vesicles.

Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.

Mitochondrial biology: Unique membrane remodeling from the matrix.

A new study reports the identification of a fission yeast dynamin superfamily protein, Mmc1, that self-assembles on the matrix side of the inner mitochondrial membrane and interacts with subunits of the mitochondrial contact site and cristae organizing system to maintain cristae architecture.

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.

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.

Twin Strep-Tag Modified CPT1A Mitochondrial Membrane Chromatography in Screening Lipid Metabolism Regulators.

Mitochondrial Membrane Chromatography (MMC) is a bioaffinity chromatography technique developed to study the interaction between target proteins embedded in the mitochondrial membrane and their ligand compounds. However, the MMC stationary phases (MMSP) prepared by chemical immobilization are prone to nonspecific binding in candidate agent screening inevitably. To address these challenges, Twin Strep-Tag/Strep Tactin was employed to establish a specific affinity system in the present study. We prepared a carnitine palmitoyltransferase 1A (CPT1A) MMSP by specifically linking Strep-tactin-modified silica gel with the Twin Strep-Tag on the CPT1A-oriented mitochondrial membrane. This Twin Strep-Tag/Strep Tactin modified CPT1A/MMC method exhibited remarkably better retention behavior, longer stationary phase lifespan, and higher screening specificity compared with previous MMC systems with glutaraldehyde immobilization. We adopted the CPT1A-specific MMC system in screening CPT1A ligands from traditional Chinese medicines, and successfully identified novel candidate ligands: ononin, isoliquiritigenin, and aloe-emodin, from Glycyrrhiza uralensis Fisch and Senna tora (L.) Roxb extracts. Biological assessments illustrated that the compounds screened promote CPT1A enzyme activity without affecting CPT1A protein expression, as well as effectively reduce the lipid droplets and triglyceride levels in the high fat induction HepG2 cells. The results suggest that we have developed an MMC system, which is promising for studying the bioaffinity of mitochondrial membrane proteins to candidate compounds. This system provides a platform for a key step in mitochondrial medicine discovery, especially for bioactive molecule screening from complex herbal extracts.

TRABD modulates mitochondrial homeostasis and tissue integrity.

High TRABD expression is associated with tau pathology in patients with Alzheimer's disease; however, the function of TRABD is unknown. Human TRABD encodes a mitochondrial outer-membrane protein. The loss of TRABD resulted in mitochondrial fragmentation, and TRABD overexpression led to mitochondrial clustering and fusion. The C-terminal tail of the TRABD anchored to the mitochondrial outer membrane and the TraB domain could form homocomplexes. Additionally, TRABD forms complexes with MFN2, MIGA2, and PLD6 to facilitate mitochondrial fusion. Flies lacking dTRABD are viable and have normal lifespans. However, aging flies exhibit reduced climbing ability and abnormal mitochondrial morphology in their muscles. The expression of dTRABD is increased in aged flies. dTRABD overexpression leads to neurodegeneration and enhances tau toxicity in fly eyes. The overexpression of dTRABD also increased reactive oxygen species (ROS), ATP production, and protein turnover in the mitochondria. This study suggested that TRABD-induced mitochondrial malfunctions contribute to age-related neurodegeneration.

Implications of mitochondrial membrane potential gradients on signaling and ATP production analyzed by correlative multi-parameter microscopy.

The complex architecture and biochemistry of the inner mitochondrial membrane generate ultra-structures with different phospholipid and protein compositions, shapes, characteristics, and functions. The crista junction (CJ) serves as an important barrier separating the cristae (CM) and inner boundary membranes (IBM). Thereby CJ regulates the movement of ions and ensures distinct electrical potentials across the cristae (ΔΨC) and inner boundary (ΔΨIBM) membranes. We have developed a robust and flexible approach to visualize the CJ permeability with super-resolution microscopy as a readout of local mitochondrial membrane potential (ΔΨmito) fluctuations. This method involves analyzing the distribution of TMRM fluorescence intensity in a model that is restricted to the mitochondrial geometry. We show that mitochondrial Ca2+ elevation hyperpolarizes the CM most likely caused by Ca2+ sensitive increase of mitochondrial tricarboxylic acid cycle (TCA) and subsequent oxidative phosphorylation (OXPHOS) activity in the cristae. Dynamic multi-parameter correlation measurements of spatial mitochondrial membrane potential gradients, ATP levels, and mitochondrial morphometrics revealed a CJ-based membrane potential overflow valve mechanism protecting the mitochondrial integrity during excessive cristae hyperpolarization.

Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.

Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival.

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.

Reduced Protein Import via TIM23 SORT Drives Disease Pathology in TIMM50-Associated Mitochondrial Disease.

TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of pre-sequence-containing precursors into the mitochondrial matrix and inner membrane. Here we describe a mitochondrial disease patient who is homozygous for a novel variant in TIMM50 and establish the first proteomic map of mitochondrial disease associated with TIMM50 dysfunction. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure. Using proteomic data sets from TIMM50 patient fibroblasts and a TIMM50 HEK293 cell model of disease, we reveal that laterally released substrates imported via the TIM23SORT complex pathway are most sensitive to loss of TIMM50. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated mitochondrial disease patients. These results highlight the power of using proteomics to elucidate molecular mechanisms of disease and uncovering novel features of fundamental biology, with the implication that human TIMM50 may have a more pronounced role in lateral insertion than previously understood.

MTFP1 controls mitochondrial fusion to regulate inner membrane quality control and maintain mtDNA levels.

Mitochondrial dynamics play a critical role in cell fate decisions and in controlling mtDNA levels and distribution. However, the molecular mechanisms linking mitochondrial membrane remodeling and quality control to mtDNA copy number (CN) regulation remain elusive. Here, we demonstrate that the inner mitochondrial membrane (IMM) protein mitochondrial fission process 1 (MTFP1) negatively regulates IMM fusion. Moreover, manipulation of mitochondrial fusion through the regulation of MTFP1 levels results in mtDNA CN modulation. Mechanistically, we found that MTFP1 inhibits mitochondrial fusion to isolate and exclude damaged IMM subdomains from the rest of the network. Subsequently, peripheral fission ensures their segregation into small MTFP1-enriched mitochondria (SMEM) that are targeted for degradation in an autophagic-dependent manner. Remarkably, MTFP1-dependent IMM quality control is essential for basal nucleoid recycling and therefore to maintain adequate mtDNA levels within the cell.

Exploring the multifaceted role of adenosine nucleotide translocase 2 in cellular and disease processes: A comprehensive review.

Adenosine nucleotide translocases (ANTs) are a family of proteins abundant in the inner mitochondrial membrane, primarily responsible for shuttling ADP and ATP across the mitochondrial membrane. Additionally, ANTs are key players in balancing mitochondrial energy metabolism and regulating cell death. ANT2 isoform, highly expressed in undifferentiated and proliferating cells, is implicated in the development and drug resistance of various tumors. We conduct a detailed analysis of the potential mechanisms by which ANT2 may influence tumorigenesis and drug resistance. Notably, the significance of ANT2 extends beyond oncology, with roles in non-tumor cell processes including blood cell development, gastrointestinal motility, airway hydration, nonalcoholic fatty liver disease, obesity, chronic kidney disease, and myocardial development, making it a promising therapeutic target for multiple pathologies. To better understand the molecular mechanisms of ANT2, this review summarizes the structural properties, expression patterns, and basic functions of the ANT2 protein. In particular, we review and analyze the controversy surrounding ANT2, focusing on its role in transporting ADP/ATP across the inner mitochondrial membrane, its involvement in the composition of the mitochondrial permeability transition pore, and its participation in apoptosis.

Fluorescence Lifetime Super-Resolution Imaging Unveil the Dynamic Relationship between Mitochondrial Membrane Potential and Cristae Structure Using the Förster Resonance Energy Transfer Strategy.

Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.

Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.