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GTPase Era from Staphylococcus aureus belongs to the TRAFAC superfamily of the TrmE-Era-EngA-EngB-Septin-like GTPases class and plays a significant role in the vital activity of this pathogenic microorganism as a maturation factor of the 30S ribosome subunit. However, the functions of this protein are not fully understood, making it a promising object for further study. Here, the 2.76 Å resolution crystal structure of Staphylococcus aureus Era in complex with GDP is presented. Structural comparison with other GTP-bound and GDP-bound homologous proteins, GTPase domain and the KH domain revealed a mutual orientation in S. aureus which has not been described before. The GDP-bound Era structure presented here will facilitate efforts to elucidate its interactions with its regulators and lay the foundation for a structure-based search for specific inhibitors., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Konstantin Usachev reports financial support was provided by Russian Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dysregulated mitochondrial fusion and fission has been implicated in the pathogenesis of numerous diseases. We have identified a novel function of the p53 family protein TAp73 in regulating mitochondrial dynamics. TAp73 regulates the expression of Optic Atrophy 1 (OPA1), a protein responsible for controlling mitochondrial fusion, cristae biogenesis and electron transport chain function. Disruption of this axis results in a fragmented mitochondrial network and an impaired capacity for energy production via oxidative phosphorylation. Owing to the role of OPA1 in modulating cytochrome c release, TAp73 -/- cells display an increased sensitivity to apoptotic cell death, e.g., via BH3-mimetics. We additionally show that the TAp73/OPA1 axis has functional relevance in the upper airway, where TAp73 expression is essential for multiciliated cell differentiation and function. Consistently, ciliated epithelial cells of Trp73 -/- (global p73 knock-out) mice display decreased expression of OPA1 and perturbations of the mitochondrial network, which may drive multiciliated cell loss. In support of this, Trp73 and OPA1 gene expression is decreased in chronic obstructive pulmonary disease (COPD) patients, a disease characterised by alterations in mitochondrial dynamics. We therefore highlight a potential mechanism involving the loss of p73 in COPD pathogenesis. Our findings also add to the growing body of evidence for growth-promoting roles of TAp73 isoforms., (© 2024. The Author(s).)

Sequence-selective G-quadruplex ligands are valuable for controlling gene expression. Here, we established a new fluorescence displacement assay using a NRAS G-quadruplex selective fluorescent probe to identify sequence-selective DNA G-quadruplex ligands. These sequence-selective NRAS G-quadruplex ligands retained their binding affinity even in the presence of excessive human telomeric DNA G-quadruplex and regulated enzymatic activities in a sequence-selective manner.

Outer mitochondrial membrane fusion, a vital cellular process, is mediated by mitofusins. However, the underlying molecular mechanism remains elusive. We have performed extensive multiscale molecular dynamics simulations to predict a model of the transmembrane (TM) domain of the yeast mitofusin Fzo1. Coarse-grained simulations of the two TM domain helices, TM1 and TM2, reveal a stable interface, which is controlled by the charge status of residue Lys716. Atomistic replica-exchange simulations further tune our model, which is confirmed by a remarkable agreement with an independent AlphaFold2 (AF2) prediction of Fzo1 in complex with its fusion partner Ugo1. Furthermore, the presence of the TM domain destabilizes the membrane, even more if Lys716 is charged, which can be an asset for initiating fusion. The functional role of Lys716 was confirmed with yeast experiments, which show that mutating Lys716 to a hydrophobic residue prevents mitochondrial fusion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In the budding yeast Saccharomyces cerevisiae , exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cells. This coupling occurs through a GTPase signaling cascade known as the mitotic exit network (MEN). The MEN senses spindle position via a Ras-like GTPase Tem1 which localizes to the spindle pole bodies (SPBs, yeast equivalent of centrosomes) during anaphase and signals to its effector protein kinase Cdc15. How Tem1 couples the status of spindle position to MEN activation is not fully understood. Here, we show that Cdc15 has a relatively weak preference for Tem1 GTP and Tem1's nucleotide state does not change upon MEN activation. Instead, we find that Tem1's nucleotide cycle establishes a localization-based concentration difference in the cell where only Tem1 GTP is recruited to the SPB, and spindle position regulates the MEN by controlling Tem1 localization to the SPB. SPB localization of Tem1 primarily functions to promote Tem1-Cdc15 interaction for MEN activation by increasing the effective concentration of Tem1. Consistent with this model, we demonstrate that artificially tethering Tem1 to the SPB or concentrating Tem1 in the cytoplasm with genetically encoded multimeric nanoparticles could bypass the requirement of Tem1 GTP and correct spindle position for MEN activation. This localization/concentration-based GTPase signaling mechanism for Tem1 differs from the canonical Ras-like GTPase signaling paradigm and is likely relevant to other localization-based signaling scenarios., Competing Interests: Competing interests statement:The authors declare no competing interest.

Mitochondrial dynamics and mitophagy are intimately linked physiological processes that are essential for cardiac homeostasis. Here we show that cardiac Krüppel-like factor 9 (Klf9) is dysregulated in human and rodent cardiomyopathy. Both global and cardiac-specific Klf9-deficient mice displayed hypertrophic cardiomyopathy. Klf9 knockout led to mitochondrial disarray and fragmentation, impairing mitochondrial respiratory function in cardiomyocytes. Furthermore, cardiac Klf9 deficiency inhibited mitophagy, thereby causing accumulation of dysfunctional mitochondria and acceleration of heart failure in response to angiotensin II treatment. In contrast, cardiac-specific Klf9 transgene improved cardiac systolic function. Mechanistically, Klf9 knockout decreased the expression of PGC-1α and its target genes involved in mitochondrial energy metabolism. Moreover, Klf9 controlled the expression of Mfn2, thereby regulating mitochondrial dynamics and mitophagy. Finally, adeno-associated virus-mediated Mfn2 rescue in Klf9-CKO hearts improved cardiac mitochondrial and systolic function. Thus, Klf9 integrates cardiac energy metabolism, mitochondrial dynamics and mitophagy. Modulating Klf9 activity may have therapeutic potential in the treatment of heart failure., Competing Interests: Competing interests All authors declare no competing interests. There are no conflicts of interest in the submission of this manuscript, which was approved by all authors for publication. We also confirm that this work is original. It has not been published elsewhere and is not currently under consideration for publication elsewhere., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite increasing evidences has highlighted the importance of mitochondria-lipid droplet (LD) coupling in maintaining lipid homeostasis, little progress in unraveling the role of mitochondria-LD coupling in hepatic lipid metabolism has been made. Additionally, diallyl disulfide (DADS), a garlic organosulfur compound, has been proposed to prevent hepatic steatosis; however, no studies have focused on the molecular mechanism to date. To address these gaps, this study investigated the systemic control mechanisms of mitochondria-LD coupling regulating hepatic lipid metabolism, and also explored their function in the process of DADS alleviating hepatic steatosis. To this end, an animal model of lipid metabolism, yellow catfish Pelteobagrus fulvidraco were fed four different diets (control, high-fat, DADS and high-fat + DADS diet) in vivo for 8 weeks; in vitro experiments were conducted to inhibit Mfn2/Atgl-mediated mitochondria-LD coupling in isolated hepatocytes. The key findings are: (1) the activations of hepatic LDs lipolysis and mitochondrial β-oxidation are likely the major drivers for DADS alleviating hepatic steatosis; (2) the underlying mechanism is that DADS enhances mitochondria-LD coupling by promoting the interaction between mitochondrion-localized Mfn2 with LD-localized Atgl, which facilitates the hepatic LDs lipolysis and the transfer of fatty acids (FAs) from LDs to mitochondria for subsequent β-oxidation; (3) Mfn2-mediated mitochondrial fusion facilitates mitochondria to form more PDM, which possess higher β-oxidation capacity in hepatocytes. Significantly, the present research unveils a previously undisclosed mechanism by which Mfn2/Atgl-mitochondria-LD coupling relieves hepatic LDs accumulation, which is a conserved strategy from fish to tetrapod. This study provides another dimension for mitochondria-LD coupling and opens up new avenues for the therapeutic interventions in hepatic steatosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Frailty increases the incidence of geriatric syndromes and even the risk of death in old adults. However, the diagnostic criteria for frailty are inconsistent because of complex pathological processes and diverse clinical manifestations. To determine the effective biomarker and recognize frail status early, we investigated the correlation of mitochondrial morphology and function of human peripheral blood mononuclear cells (PBMCs) with frailty status in older adults., Methods: This Cross-sectional study followed 393 participants (aged 25-100 years, female 31.04 %) from the First Affiliated Hospital of Nanjing Medical University. The frailty status of subjects was assessed by the physical frailty phenotype (PFP) scale. We analyzed mitochondria functions including mitochondria copy number (mtDNAcn), the mRNA expressions of mitochondrial dynamics-related genes mitofusin 1(MFN1), mitofusin 2(MFN2), optic atrophy protein-1(OPA1), fission protein-1(FIS1) and dynamin-related protein 1(DRP1), mitochondrial oxidative respiration and reactive oxygen species(ROS) levels in PBMCs. Mitochondria morphology, size, and number were observed by transmission electron microscopy (TEM)., Results: After adjustment for sex and BMI, mtDNAcn, the mRNA expression of FIS1, mitochondrial respiratory function (proton leak, maximum oxygen consumption, and respiratory reserve) and ROS level were significantly correlated with age (P = 0.031, 0.030, 0.042, 0.003, 0.002, 0.022, respectively). After correcting for age, sex, and BMI, mtDNAcn and the mRNA expression of OPA1 were correlated with 4 m gait speed respectively (P = 0.003, 0.028, respectively). Compared with non-frail people, mtDNAcn, the mRNA expression of MFN1, mitochondrial basal respiration, proton leak, maximum oxygen consumption, ATP production and space capacity were significantly decreased in frail older adults (P = 0.013, 0.036, 0.026, 0.024, 0.012, 0.032, 0.020, respectively). ROS levels were significantly increased in the frail group (P = 0.016). Compared with non-frail people, the number, length, and perimeter, area of mitochondria were reduced in frail group under TEM (all P < 0.001)., Conclusion: Mitochondrial dysfunctions (decreased mtDNAcn, impaired mitochondrial morphology, imbalanced mitochondrial dynamic, impaired mitochondrial respiratory function, and increased ROS levels) were significantly correlated with frail status., Competing Interests: Declaration of competing interest We declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation and the mitochondrial dynamics regulated by fusion-related genes MFN1, MFN2, and OPA1 and fission-related genes DNM1L and MFF. An analysis of previously published gene expression datasets showed that high expression of MFF was significantly associated with poor prognosis in patients with AML. Based on this finding, we investigated the impact of mitochondrial dynamics in AML. Transduction of shRNA against fission-related genes, DNM1L and MFF, inhibited growth and increased the mitochondrial area in AML cell lines. Extracellular flux analysis showed that deletion of mitochondrial dynamic regulators reduced mitochondrial respiration without significantly affecting glycolysis, except in shDNM1L-transfected cells. Immunodeficient NOG mice transplanted with DNM1L- or MFF-knockdown AML cells survived significantly longer than controls. Treatment of AML cell lines with Mdivi-1, which inhibits the DRP1 encoded by DNM1L, inhibited cell proliferation and oxidative phosphorylation. Our results show that mitochondrial dynamics play an important role in AML, and provide novel biological insights. The inhibition of mitochondrial dynamics induces unique mitochondrial alterations, which may be explored as a potential therapeutic target in AML., (© 2024. Japanese Society of Hematology.)

Obg, a GTPase, binds to the premature 50S ribosomal subunit and facilitates recruitment of rproteins and rRNA processing to form the mature 50S subunit. This binding depends on nucleotide-induced conformational changes (GDP/GTP). However, the mechanism by which Obg undergoes conformational changes to associate with the premature 50S subunit is unknown. Therefore, 1000 ns molecular dynamics simulations were conducted to investigate this mechanism. Visualization of the simulated trajectory showed that in GDP and GTP-bound states, the C-domain moved towards the SwI region, while in GTP-Mg 2+ and ppGpp-bound states, the C-domain shifted towards the N-tails. Further, positioning these conformations of Obg on the 50S subunit suggests possible mechanisms by which the GTP-Mg 2+ bound state is responsible for recruiting rprotein, as well as the impact of the absence of Mg 2+ in the GTP-bound state. Furthermore, the study provides insights into the conformational changes that may lead to the dissociation of the GDP-bound state from the 50S subunit and explores the potential role of the ppGpp-bound state in inhibiting 70S ribosome formation. Additionally, RMSF and community network analyses reveal how internal dynamics and intricate connections within Obg affect C-domain motion., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hypertrophic scar (HS) formation is a cutaneous fibroproliferative disease that occurs after skin injuries and results in severe functional and esthetic disability. To date, few drugs have shown satisfactory outcomes for the treatment of HS formation. Transforming growth factor-beta (TGF-β)/Notch interaction via small mothers against decapentaplegic 3 (Smad3) could facilitate HS formation; therefore, targeting TGF-β/ Notch interaction via Smad3 is a potential therapeutic strategy to attenuate HS formation. In addition, optic atrophy 1 (OPA1)-mediated mitochondrial fusion contributes to fibroblast proliferation, and TGF-β/Smad3 axis and the Notch1 pathway facilitate OPA1-mediated mitochondrial fusion. Thus, the aim of this study was to investigate whether drugs targeting TGF-β/Notch interaction via Smad3 suppressed fibroblast proliferation to attenuate HS formation through OPA1-mediated mitochondrial fusion. We found that the TGF-β pathway, Notch pathway, and TGF-β/Notch interaction via Smad3 were inhibited by pirfenidone, the gamma- secretase inhibitor DAPT, and SIS3 in human keloid fibroblasts (HKF) and an HS rat model, respectively. Protein interaction was detected by co-immunoprecipitation, and mitochondrial morphology was determined by electron microscopy. Our results indicated that pirfenidone, DAPT, and SIS3 suppressed the proliferation of HKFs and attenuated HS formation in the HS rat model by inhibiting TGF-β/Notch interaction via Smad3. Moreover, pirfenidone, DAPT, and SIS3 hindered OPA1-mediated mitochondrial fusion through inhibiting TGF-β/Notch interaction, thereby suppressing the proliferation of HS fibroblasts and HS formation. In summary, these findings investigating the effects of drugs targeting TGF-β/Notch interaction on HS formation might lead to novel drugs for the treatment of HS formation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Neuregulin 4 (Nrg4) is an adipokine that belongs to the epidermal growth factor family and binds to ErbB4 tyrosine kinase receptors. In 3T3-L1 adipocytes, the downregulation of Nrg4 expression enhances inflammation and autophagy, resulting in insulin resistance. Here, we searched for the causes of this phenotype. Nrg4 knockdown (Nrg4 KD) adipocytes showed a significant reduction in mitochondrial content and elongation, along with a lower content of the mitochondria fusion protein mitofusin 2 (MFN2), and increased H 2 O 2 production compared to the control scrambled cells (Scr). The antioxidant N-acetylcysteine reversed the oxidative stress and reduced the gene expression of the pro-inflammatory cytokine tumor necrosis factor α (TNFα). Nrg4 KD adipocytes showed enhanced lipolysis and reduced lipogenesis, in addition to a significant reduction in several intermediates of the Krebs cycle. In summary, Nrg4 downregulation in adipocytes affects mitochondrial content and functioning, causing impaired cellular metabolism, which in turn results in oxidative stress, inflammation, and insulin resistance.

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations., Competing Interests: Competing interests statement:The authors declare no competing interest.

Osteoarthritis (OA) is an age-related cartilage-degenerating joint disease. Mitochondrial dysfunction has been reported to promote the development of OA. Poly (ADP-ribose) polymerase family member 12 (PARP12) is a key regulator of mitochondrial function, protein translation, and inflammation. However, the role of PARP12 in OA-based cartilage degradation and the underlying mechanisms are relatively unknown. Here, we first demonstrated that PARP12 inhibits mitophagy and promotes OA progression in human OA cartilage and a monosodium iodoacetate-induced rat OA model. Using mass spectrometry and co-immunoprecipitation assay, PARP12 was shown to interact with ISG15, upregulate mitofusin 1 and 2 (MFN1/2) ISGylation, which downregulated MFN1/2 ubiquitination and SUMOylation, thereby inhibiting PINK1/Parkin-dependent chondrocyte mitophagy and promoting cartilage degradation. Moreover, inflammatory cytokine-induced interferon regulatory factor 1 (IRF1) activation was required for the upregulation of PARP12 expression, and it directly bound to the PARP12 promoter to activate transcription. XAV-939 inhibited PARP12 expression and suppressed OA pathogenesis in vitro and in vivo. Clinically, PARP12 can be used to predict the severity of OA; thus, it represents a new target for the study of mitophagy and OA progression. In brief, the IRF1-mediated upregulation of PARP12 promoted cartilage degradation by inhibiting PINK1/Parkin-dependent mitophagy via ISG15-based attenuation of MFN1/2 ubiquitylation and SUMOylation. Our data provide new insights into the molecular mechanisms underlying PARP12-based regulation of mitophagy and can facilitate the development of therapeutic strategies for the treatment of OA., (© 2024. The Author(s).)

In cancer research, RAS biology has been focused on only a handful of tumor types. While RAS genes have long been suspected as common contributors to a wide spectrum of cancer types, robust evidence is required to firmly establish their critical oncogenic significance. We present a data mining study using DepMap genome-wide CRISPR screening data, which provide substantial evidence to support the prominent pervasive oncogenic role and tissue-specific permissiveness of RAS gene mutations. Differential analysis of CRISPR effect scores identifies K- or N-RAS genes as the most differential gene in contrasts of (K-, N-, combined) RAS mutant versus wild-type cell lines across multiple tissue types. The distinguished tissue-specific pattern of KRAS vs. NRAS as top differential genes in subsets of tissue types and evidence from genome data supported the idea of KRAS- and NRAS-engaged tissue types. To our knowledge, this is the first report of prominent pervasive oncogenic role of RAS mutations revealed by gene dependency data that is beyond the current understanding of the oncogenic role of RAS genes and their well-known involved tissue types. Our findings strongly support RAS mutations as primary oncogenic drivers beyond traditionally recognized cancer types and offer insights into their tissue-specific permissiveness., (© 2024. The Author(s).)

Huntington's Disease (HD) is an inheritable neurodegenerative condition caused by an expanded CAG trinucleotide repeat in the HTT gene with a direct correlation between CAG repeats expansion and disease severity with earlier onset-of- disease. Previously we have shown that primary skin fibroblasts from HD patients exhibit unique phenotype disease features, including distinct nuclear morphology and perturbed actin cap linked with cell motility, that are correlated with the HD patient disease severity. Here we provide further evidence that mitochondrial fission-fusion morphology balance dynamics, classified using a custom image-based high-content analysis (HCA) machine learning tool, that improved correlation with HD severity status. This mitochondrial phenotype is supported by appropriate changes in fission-fusion biomarkers (Drp1, MFN1, MFN2, VAT1) levels in the HD patients' fibroblasts. These findings collectively point towards a dysregulation in mitochondrial dynamics, where both fission and fusion processes may be disrupted in HD cells compared to healthy controls. This study shows for the first time a methodology that enables identification of HD phenotype before patient's disease onset (Premanifest). Therefore, we believe that this tool holds a potential for improving precision in HD patient's diagnostics bearing the potential to evaluate alterations in mitochondrial dynamics throughout the progression of HD, offering valuable insights into the molecular mechanisms and drug therapy evaluation underlying biological differences in any disease stage., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

NRAS belongs to the RAS family of GTPases. In colorectal cancer (CRC), NRAS mutations are rare compared to KRAS, but may lead to worse outcomes. We report the functional characterization of the novel NRAS mutants-G48C, Q43K, and E37K-identified in Filipino young-onset CRC patients. Unlike previously characterized NRAS mutants with no apparent effects on cell proliferation, these mutants enhanced proliferation of both HCT116 and NIH3T3 cells. This was confirmed in 3D spheroid assays to mimic the spatial organization of cells. G48C and E37K showed apoptosis resistance in both cell lines, and Q43K showed resistance in HCT116 cells. All three showed no effect on cellular migration in NIH3T3, but G48C enhanced the migration rate of HCT116 cells. Actin staining of NIH3T3 cells expressing the mutants showed a shrunken cytoplasm and transient structures associated with motility and invasiveness. Docking simulations show that GDP is only able to bind fully within the binding pocket of wild-type NRAS, but not in the mutants. Further, G48C, Q43K, and E37K all have less negative ΔG values, indicating a weaker GDP-binding affinity compared to wild-type NRAS. Taken together, the results suggest that oncogenic readouts of NRAS mutants are codon- and mutation-specific, with potential repercussions on the aggressiveness, resistance, and therapeutic response.

Mitochondrial functions can be regulated by membrane contact sites with the endoplasmic reticulum (ER). These mitochondria-ER contact sites (MERCs) are functionally heterogeneous and maintained by various tethers. Here, we found that REEP5, an ER tubule-shaping protein, interacts with Mitofusins 1/2 to mediate mitochondrial distribution throughout the cytosol by a new transport mechanism, mitochondrial "hitchhiking" with tubular ER on microtubules. REEP5 depletion led to reduced tethering and increased perinuclear localization of mitochondria. Conversely, increasing REEP5 expression facilitated mitochondrial distribution throughout the cytoplasm. Rapamycin-induced irreversible REEP5-MFN1/2 interaction led to mitochondrial hyperfusion, implying that the dynamic release of mitochondria from tethering is necessary for normal mitochondrial distribution and dynamics. Functionally, disruption of MFN2-REEP5 interaction dynamics by forced dimerization or silencing REEP5 modulated the production of mitochondrial reactive oxygen species (ROS). Overall, our results indicate that dynamic REEP5-MFN1/2 interaction mediates cytosolic distribution and connectivity of the mitochondrial network by "hitchhiking" and this process regulates mitochondrial ROS, which is vital for multiple physiological functions., (© 2024 Chen et al.)

Following the Goldilocks principle, mitochondria size must be "just right." Mitochondria balance division and fusion to avoid becoming too big or too small. Defects in this balance produce dysfunctional mitochondria in human diseases. Mitochondrial safeguard (MitoSafe) is a defense mechanism that protects mitochondria against extreme enlarging by suppressing fusion in mammalian cells. In MitoSafe, hyperfused mitochondria elicit flickering-short pulses of mitochondrial depolarization. Flickering activates an inner membrane protease, Oma1, which in turn proteolytically inactivates a mitochondrial fusion protein, Opa1. The mechanisms underlying flickering are unknown. Using a live-imaging screen, we identified Slc25a3 (a mitochondrial carrier transporting phosphate and copper) as necessary for flickering and Opa1 cleavage. Remarkably, copper, but not phosphate, is critical for flickering. Furthermore, we found that two copper-containing mitochondrial enzymes, superoxide dismutase 1 and cytochrome c oxidase, regulate flickering. Our data identify an unforeseen mechanism linking copper, redox homeostasis, and membrane flickering in mitochondrial defense against deleterious fusion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Induction of resistin-like molecule β (Relm-β) and mitofusin 2 (MFN2) mediated aberrant mitochondrial fission have been found to be involved in the pathogenesis of pulmonary arterial hypertension (PAH). However, the molecular mechanisms underlying Relm-β regulation of MFN2 therefore mitochondrial fission remain unclear. This study aims to address these issues. Primary cultured PASMCs and monocrotaline (MCT)-induced PAH rats were applied in this study. The results showed that Relm-β promoted cells proliferation in PASMCs, this was accompanied with the upregulation of USP18, Twist1 and miR-214, and downregulation of MFN2. We found that Relm-β increased USP18 expression which in turn raised Twist1 by suppressing its proteasome degradation. Elevation of Twist1 increased miR-214 expression and then reduced MFN2 expression and mitochondrial fragmentation leading to PASMCs proliferation. In vivo study, we confirmed that Relm-β was elevated in MCT-induced PAH rat model, and USP18/Twist1/miR-214/MFN2 axis was altered similar as in vitro. Targeting this cascade by Relm-β receptor inhibitor Calhex231, proteasome inhibitor MG-132, Twist1 inhibitor Harmine or miR-214 antagomiR prevented the development of pulmonary vascular remodeling and therefore PAH in MCT-treated rats. In conclusion, we demonstrate that Relm-β promotes PASMCs proliferation and vascular remodeling by activating USP18/Twist1/miR-214 dependent MFN2 reduction and mitochondrial fission, suggesting that this signaling pathway might be a promising target for management of PAH., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Bladder cancer (BCa) is a common malignancy in the urinary tract. It has high recurrence rates and often requires microscopic examination, which presents significant challenges in clinical treatment. Previous research has shown that circular TAF4B (circTAF4B) is significantly upregulated in BCa and is associated with a poor prognosis. However, the specific targets and molecular mechanisms by which circTAF4B functions in BCa are still not well - understood., Methods: In this study, an RNA pull - down assay and mass spectrometry were utilized to identify MFN2 as a binding protein of circTAF4B. Additionally, siRNA was used to silence MFN2 to observe the amplification of the inhibitory effects of circTAF4B overexpression on cell growth and migration in BCa cells. Moreover, circTAF4B shRNA lentiviral particles were employed to study their impact on BCa progression by examining the regulation of p27 and the blocking of AKT signaling., Results: It was found that MFN2 is a binding protein of circTAF4B. Silencing MFN2 with siRNA enhanced the inhibitory effects of circTAF4B overexpression on cell growth and migration in BCa cells. Also, circTAF4B shRNA lentiviral particles inhibited BCa progression by upregulating p27 and blocking AKT signaling., Discussion: In conclusion, the physical binding of circTAF4B to MFN2 is a crucial process in the tumorigenesis and progression of BCa. Targeting circTAF4B or its complexes may have potential as a therapeutic strategy for BCa diagnosis and treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Xu, Zhuang, Wang, Li and Zhu.)

Objective: Acute respiratory distress syndrome (ARDS) patients currently have relatively high mortality, which is associated with early lung fibrosis. This study aimed to investigate whether miR-17 suppression could alleviate ARDS-associated lung fibrosis by regulating Mfn2., Methods: A mouse model of ARDS-related lung fibrosis was constructed via intratracheal instillation of bleomycin. The expression level of miR-17 in lung tissues was detected via quantitative real time polymerase chain reaction (qRT-PCR). In the ARDS mouse model of lung fibrosis, the mitigating effects of miR-17 interference were evaluated via tail vein injection of the miR negative control or the miR-17 antagomir. The pathological changes in the lung tissue were examined via HE staining and Masson's trichrome staining, and the underlying molecular mechanism was investigated via ELISA, qRT-PCR and Western blotting., Results: Bleomycin-induced pulmonary fibrosis significantly increased collagen deposition and the levels of hydroxyproline (HYP) and miR-17. Interfering with miR-17 significantly reduced the levels of HYP and miR-17 and upregulated the expression of Mfn2. The intravenous injection of the miR-17 antagomir alleviated lung inflammation and reduced collagen deposition. In addition, interference with miR-17 could upregulate LC3B expression, downregulate p62 expression, and improve mitochondrial structure., Conclusion: Interfering with miR-17 can improve pulmonary fibrosis in mice by promoting mitochondrial autophagy via Mfn2., (© 2024. Huazhong University of Science and Technology.)

Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy most frequently associated with OPA1 mutations. Most variants result in haploinsufficiency, and patient cells express roughly half of the normal levels of OPA1 protein. OPA1 is a mitochondrial GTPase that is essential for normal mitochondrial function. We identified and characterized STK-002, an antisense oligonucleotide (ASO) designed to prevent the incorporation of a naturally occurring alternatively spliced nonproductive exon in OPA1 . STK-002 dose dependently reduced the inclusion of this exon, and increased OPA1 protein in human cells, including ADOA patient-derived fibroblasts. ADOA patient cells manifest reduced mitochondrial respiration, and treatment with STK-002 improved the parameters of mitochondrial respiratory function in these cells. Since STK-002 increases OPA1 through the wild-type allele, we assessed retinal OPA1 in wild-type cynomolgus monkeys and rabbits after intravitreal administration of STK-002 or a rabbit-specific surrogate. Increased OPA1 protein was produced in retinal tissue in both species at 4 weeks after ASO injection and persisted in monkeys at 8 weeks. STK-002 and enhanced OPA1 immunofluorescence were visualized in retinal ganglion cells of cynomolgus monkeys treated with the ASO. Cumulatively, these data support the progression of STK-002 toward the clinic as the first potential disease-modifying treatment for ADOA.

Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier family 25 member (SLC25A46) interacts with both the outer and inner membrane dynamin family GTPases mitofusin 1/2 and optic atrophy 1 (Opa1). While SLC25A46 levels are known to affect mitochondrial morphology, how SLC25A46 interacts with mitofusin 1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass spectrometry and AlphaFold 2 modeling to identify interfaces mediating an SLC25A46 interaction with Opa1 and Mfn2. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and present evidence of an Mfn2 interaction involving the SLC25A46 cytosolic face. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance., Competing Interests: Conflict of interest L. H. C. is an advisor for Stealth Biotherapeutics. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Alveolar macrophages (AM) are key effectors of the immune response and are essential for host responses to S. pneumoniae. Mitochondria are highly dynamic organelles whose function aids in regulating the cell cycle, innate immunity, autophagy, redox signaling, calcium homeostasis, and mitochondrial quality control in AM. In response to cellular stress, mitochondria can engage in stress-induced mitochondrial hyperfusion (SIMH). The current study aimed to investigate the role of Mfn1 on mitochondrial control of reactive oxygen species (ROS) in AMs and the role of Mfn1 deficiency on immune responses to S. pneumoniae. Compared to Mfn1 FloxCre- controls, there were distinct histological differences in lung tissue collected from Mfn1 Floxed; CreLysM mice, with less injury and inflammation observed in mice with Mfn1 deficient myeloid cells. There was a significant decrease in lipid peroxidation and ROS production in Mfn1 deficient AM that was associated with increased superoxide dismutase (SOD) and antioxidant activity. Our findings demonstrate that Mfn1 deficiency in myeloid cells decreased inflammation and lung tissue injury during S. pneumoniae infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aberrant mitochondrial fission/fusion dynamics are frequently associated with pathologies, including cancer. We show that alternative splice variants of the fission protein Drp1 (DNM1L) contribute to the complexity of mitochondrial fission/fusion regulation in tumor cells. High tumor expression of the Drp1 alternative splice variant lacking exon 16 relative to other transcripts is associated with poor outcome in ovarian cancer patients. Lack of exon 16 results in Drp1 localization to microtubules and decreased association with mitochondrial fission sites, culminating in fused mitochondrial networks, enhanced respiration, changes in metabolism, and enhanced pro-tumorigenic phenotypes in vitro and in vivo. These effects are inhibited by siRNAs designed to specifically target the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the pathophysiological importance of Drp1 alternative splicing, highlight the divergent functions and consequences of changing the relative expression of Drp1 splice variants in tumor cells, and strongly warrant consideration of alternative splicing in future studies focused on Drp1., (© 2024. The Author(s).)

Introduction: Molecular Dynamics (MD) simulations can support mechanism-based drug design. Indeed, MD simulations by capturing biomolecule motions at finite temperatures can reveal hidden binding sites, accurately predict drug-binding poses, and estimate the thermodynamics and kinetics, crucial information for drug discovery campaigns. Small-Guanosine Triphosphate Phosphohydrolases (GTPases) regulate a cascade of signaling events, that affect most cellular processes. Their deregulation is linked to several diseases, making them appealing drug targets. The broad roles of small-GTPases in cellular processes and the recent approval of a covalent KRas inhibitor as an anticancer agent renewed the interest in targeting small-GTPase with small molecules., Area Covered: This review emphasizes the role of MD simulations in elucidating small-GTPase mechanisms, assessing the impact of cancer-related variants, and discovering novel inhibitors., Expert Opinion: The application of MD simulations to small-GTPases exemplifies the role of MD simulations in the structure-based drug design process for challenging biomolecular targets. Furthermore, AI and machine learning-enhanced MD simulations, coupled with the upcoming power of quantum computing, are promising instruments to target elusive small-GTPases mutations and splice variants. This powerful synergy will aid in developing innovative therapeutic strategies associated to small-GTPases deregulation, which could potentially be used for personalized therapies and in a tissue-agnostic manner to treat tumors with mutations in small-GTPases.

Background: Drp1 (dynamin-related protein 1), a large GTPase, mediates the increased mitochondrial fission, which contributes to hyperproliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension (PAH). We developed a potent Drp1 GTPase inhibitor, Drpitor1a, but its specificity, pharmacokinetics, and efficacy in PAH are unknown., Methods: Drpitor1a's ability to inhibit recombinant and endogenous Drp1 GTPase was assessed. Drpitor1a's effects on fission were studied in control and PAH human pulmonary artery smooth muscle cells (hPASMC) and blood outgrowth endothelial cells (BOEC). Cell proliferation and apoptosis were studied in hPASMC. Pharmacokinetics and tissue concentrations were measured following intravenous and oral drug administration. Drpitor1a's efficacy in regressing monocrotaline-PAH was assessed in rats. In a pilot study, Drpitor1a reduced PA remodeling only in females. Subsequently, we compared Drpitor1a to vehicles in control and monocrotaline-PAH females., Results: Drp1 GTPase activity was increased in PAH hPASMC. Drpitor1a inhibited the GTPase activity of recombinant and endogenous Drp1 and reversed the increased fission, seen in PAH hPASMC and PAH BOEC. Drpitor1a inhibited proliferation and induced apoptosis in PAH hPASMC without affecting electron transport chain activity, respiration, fission/fusion mediator expression, or mitochondrial Drp1 translocation. Drpitor1a did not inhibit proliferation or alter mitochondrial dynamics in normal hPASMC. Drpitor1a regressed monocrotaline-PAH without systemic vascular effects or toxicity., Conclusions: Drpitor1a is a specific Drp1 GTPase inhibitor that reduces mitochondrial fission in PAH hPASMC and PAH BOEC. Drpitor1a reduces proliferation and induces apoptosis in PAH hPASMC and regresses monocrotaline-PAH. Drp1 is a therapeutic target in PAH, and Drpitor1a is a potential therapy with an interesting therapeutic sexual dimorphism., Competing Interests: The authors acknowledge that they hold a patent for the use of Drpitor1a in diseases including pulmonary hypertension and cancer: Wu D, Wells M, and Archer SL. Inhibitors of Mitochondrial Fission. Patent Application No. 62/826,247.

Charcot-Marie-Tooth type 2A (CMT2A) is a single-gene motor sensory neuropathy caused by Mfn2 mutation. It is generally believed that CMT2A involves mitochondrial fusion disruption. However, how Mfn2 mutation mediates the mitochondrial membrane fusion loss and its further pathogenic mechanisms remain unclear. Here, in vivo and in vitro mouse models harboring the Mfn2 R364W , Mfn2 G176S and Mfn2 H165R mutations were constructed. Mitochondrial membrane fusion and fission proteins analysis showed that Mfn2 R364W , Mfn2 G176S , and Mfn2 H165R/+ mutations maintain the expression of Mfn2, but promote Drp1 upregulation and Opa1 hydrolytic cleavage. In Mfn2 H165R/H165R mutation, Mfn2, Drp1, and Opa1 all play a role in inducing mitochondrial fragmentation, and the mitochondrial aggregation is affected by Mfn2 loss. Further research into the pathogenesis of CMT2A showed these three mutations all induce mitochondria-mediated apoptosis, and mitochondrial oxidative phosphorylation damage. Overall, loss of overall fusion activity affects mitochondrial DNA (mtDNA) stability and causes mitochondrial loss and dysfunction, ultimately leading to CMT2A disease. Interestingly, the differences in the pathogenesis of CMT2A between Mfn2 R364W , Mfn2 G176S , Mfn2 H165R/+ and Mfn2 H165R/H165R mutations, including the distribution of Mfn2 and mitochondria, the expression of mitochondrial outer membrane-associated proteins (Bax, VDAC1 and AIF), and the enzyme activity of mitochondrial complex I, are related to the expression of Mfn2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mitochondria undergo fission and fusion, and their coordinated balance is crucial for maintaining mitochondrial homeostasis. In yeast, the dynamin-related protein Dnm1 is a mitochondrial fission factor acting from outside the mitochondria. We recently reported the mitochondrial intermembrane space protein Atg44/mitofissin/Mdi1/Mco8 as a novel fission factor, but the relationship between Atg44 and Dnm1 remains elusive. Here, we show that Atg44 is required to complete Dnm1-mediated mitochondrial fission under homeostatic conditions. Atg44-deficient cells often exhibit enlarged mitochondria with accumulated Dnm1 and rosary-like mitochondria with Dnm1 foci at constriction sites. These mitochondrial constriction sites retain the continuity of both the outer and inner membranes within an extremely confined space, indicating that Dnm1 is unable to complete mitochondrial fission without Atg44. Moreover, accumulated Atg44 proteins are observed at mitochondrial constriction sites. These findings suggest that Atg44 and Dnm1 cooperatively execute mitochondrial fission from inside and outside the mitochondria, respectively. Abbreviation: ATG: autophagy related; CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; ERMES: endoplasmic reticulum-mitochondria encounter structure; GA: glutaraldehyde; GFP: green fluorescent protein; GTP: guanosine triphosphate: IMM: inner mitochondrial membrane; IMS: intermembrane space; OMM: outer mitochondrial membrane; PB: phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RFP: red fluorescent protein; WT: wild type.

Background: The dynamics of mitochondrial respiratory cristae (MRC) and its impact on oxidative phosphorylation (OXPHOS) play a crucial role in driving the progression of high-grade glioma (HGG). However, the underlying mechanism remains unclear., Methods: In the present study, we employed machine learning-based transmission electron microscopy analysis of 7141 mitochondria from 54 resected glioma patients. Additionally, we conducted bioinformatics analysis and multiplex immunohistochemical (mIHC) staining of clinical glioma microarrays to identify key molecules involved in glioma. Subsequently, we modulated the expression levels of mitochondrial dynamic-1-like protein (DNM1L/DRP1), and its two receptors, mitochondrial fission protein 1 (FIS1) and mitochondrial fission factor (MFF), via lentiviral transfection to further investigate the central role of these molecules in the dynamics of glioblastoma (GBM) cells and glioma stem cells (GSCs). We then evaluated the potential impact of DNM1L/DRP1, FIS1, and MFF on the proliferation and progression of GBM cells and GSCs using a combination of CCK-8 assay, Transwell assay, Wound Healing assay, tumor spheroid formation assay and cell derived xenograft assay employing NOD/ShiLtJGpt-Prkdc em26Cd52 Il2rg em26Cd22 /Gpt (NCG) mouse model. Subsequently, we validated the ability of the DNM1L/DRP1-FIS1 axis to remodel MRC structure through mitophagy by utilizing Seahorse XF analysis technology, mitochondrial function detection, MRC abundance detection and monitoring dynamic changes in mitophagy., Results: Our findings revealed that compared to low-grade glioma (LGG), HGG exhibited more integrated MRC structures. Further research revealed that DNM1L/DRP1, FIS1, and MFF played pivotal roles in governing mitochondrial fission and remodeling MRC in HGG. The subsequent validation demonstrated that DNM1L/DRP1 exerts a positive regulatory effect on FIS1, whereas the interaction between MFF and FIS1 demonstrates a competitive inhibition relationship. The down-regulation of the DNM1L/DRP1-FIS1 axis significantly impaired mitophagy, thereby hindering the remodeling of MRC and inhibiting OXPHOS function in glioma, ultimately leading to the inhibition of its aggressive progression. In contrast, MFF exerts a contrasting effect on MRC integrity, OXPHOS activity, and glioma progression., Conclusions: This study highlights that the DNM1L/DRP1-FIS1 axis stabilizes MRC structures through mitophagy in HGG cells while driving their OXPHOS activity ultimately leading to robust disease progression. The inhibition of the DNM1L/DRP1-FIS1 axis hinders MRC remodeling and suppresses GBM progression. We propose that down-regulation of the DNM1L/DRP1-FIS1 axis could be a potential therapeutic strategy for treating HGG., (© 2024. The Author(s).)

Mitochondrial form and function are regulated by the opposing forces of mitochondrial dynamics: fission and fusion. Mitochondrial dynamics are highly active and consequential during neuronal ischemia/reperfusion (I/R) injury. Mitochondrial fusion is executed at the mitochondrial inner membrane by Opa1. The balance of long (L-Opa1) and proteolytically cleaved short (S-Opa1) isoforms is critical for efficient fusion. Oma1 is the predominant stress-responsive protease for Opa1 processing. In neuronal cell models, we assessed Oma1 and Opa1 regulation during mitochondrial stress. In an immortalized mouse hippocampal neuron line (HT22), Oma1 was sensitive to mitochondrial membrane potential depolarization (rotenone, FCCP) and hyperpolarization (oligomycin). Further, oxidative stress was sufficient to increase Oma1 activity and necessary for depolarization-induced proteolysis. We generated Oma1 knockout (KO) HT22 cells that displayed normal mitochondrial morphology and fusion capabilities. FCCP-induced mitochondrial fragmentation was exacerbated in Oma1 KO cells. However, Oma1 KO cells were better equipped to perform restorative fusion after fragmentation, presumably due to preserved L-Opa1. We extended our investigations to a combinatorial stress of neuronal oxygen-glucose deprivation and reoxygenation (OGD/R), where we found that Opa1 processing and Oma1 activation were initiated during OGD in an ROS-dependent manner. These findings highlight a novel dependence of Oma1 on oxidative stress in response to depolarization. Further, we demonstrate contrasting fission/fusion roles for Oma1 in the acute response and recovery stages of mitochondrial stress. Collectively, our results add intersectionality and nuance to the previously proposed models of Oma1 activity., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the post-translational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the co-translational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for post-translational transport, while a ribosome-associated pool coordinates its co-translational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in post-translational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Introduction: Reducing Optic Atrophy 1 (OPA1) expression in skeletal muscle in male mice induces Activation Transcription Factor 4 (ATF4) and the integrated stress response (ISR). Additionally, skeletal muscle secretion of Fibroblast Growth Factor 21 (FGF21) is increased, which mediates metabolic adaptations including resistance to diet-induced obesity (DIO) and glucose intolerance in these mice. Although FGF21 induction in this model can be reversed with pharmacological attenuation of ER stress, it remains to be determined if ATF4 is responsible for FGF21 induction and its metabolic benefits in this model., Methods: We generated mice with homozygous floxed Opa1 and Atf4 alleles and a tamoxifen-inducible Cre transgene controlled by the human skeletal actin promoter to enable simultaneous depletion of OPA1 and ATF4 in skeletal muscle (mAO DKO). Mice were fed high fat (HFD) or control diet and evaluated for ISR activation, body mass, fat mass, glucose tolerance, insulin tolerance and circulating concentrations of FGF21 and growth differentiation factor 15 (GDF15)., Results: In mAO DKO mice, ATF4 induction is absent. Other indices of ISR activation, including XBP1s, ATF6, and CHOP were induced in mAO DKO males, but not in mOPA1 or mAO DKO females. Resistance to diet-induced obesity was not reversed in mAO DKO mice of both sexes. Circulating FGF21 and GDF15 illustrated sexually dimorphic patterns. Loss of OPA1 in skeletal muscle increases circulating FGF21 in mOPA1 males, but not in mOPA1 females. Additional loss of ATF4 decreased circulating FGF21 in mAO DKO male mice, but increased circulating FGF21 in female mAO DKO mice. Conversely, circulating GDF15 was increased in mAO DKO males and mOPA1 females, but not in mAO DKO females., Conclusion: Sex differences exist in the transcriptional outputs of the ISR following OPA deletion in skeletal muscle. Deletion of ATF4 in male and female OPA1 KO mice does not reverse the resistance to DIO. Induction of circulating FGF21 is ATF4 dependent in males, whereas induction of circulating GDF15 is ATF4 dependent in females. Elevated GDF15 in males and FGF21 in females could reflect activation by other transcriptional outputs of the ISR, that maintain mitokine-dependent metabolic protection in an ATF4-independent manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Streeter, Persaud, Gao, Manika, Fairman, García-Peña, Marti, Manika, Gaddi, Schickling, Pereira and Abel.)

SynDLP, a dynamin-like protein (DLP) encoded in the cyanobacterium Synechocystis sp. PCC 6803, has recently been identified to be structurally highly similar to eukaryotic dynamins. To elucidate structural changes during guanosine triphosphate (GTP) hydrolysis, we solved the cryoelectron microscopy (cryo-EM) structures of oligomeric full-length SynDLP after addition of guanosine diphosphate (GDP) at 4.1 Å and GTP at 3.6-Å resolution as well as a GMPPNP-bound dimer structure of a minimal G-domain construct of SynDLP at 3.8-Å resolution. In comparison with what has been seen in the previously resolved apo structure, we found that the G-domain is tilted upward relative to the stalk upon GTP hydrolysis and that the G-domain dimerizes via an additional extended dimerization domain not present in canonical G-domains. When incubated with lipid vesicles, we observed formation of irregular tubular SynDLP assemblies that interact with negatively charged lipids. Here, we provide the structural framework of a series of different functional SynDLP assembly states during GTP turnover., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

RUNX1::RUNX1T1 (R::RT1) acute myeloid leukaemia (AML) remains a clinical challenge, and further research is required to model and understand leukaemogenesis. Previous zebrafish R::RT1 models were hampered by embryonic lethality and low penetrance of the malignant phenotype. Here, we overcome this by developing an adult zebrafish model in which the human R::RT1 isoform 9a is co-expressed with the frequently co-occurring oncogenic NRASG12D mutation in haematopoietic stem and progenitor cells (HSPCs), using the Runx1+23 enhancer. Approximately 50% of F0 9a+NRASG12D transgenic zebrafish developed signs of haematological disease between 5 and 14 months, with 27% exhibiting AML-like pathology: myeloid precursor expansion, erythrocyte reduction, kidney marrow hypercellularity and the presence of blasts. Moreover, only 9a+NRASG12D transplant recipients developed leukaemia with high rates of mortality within 40 days, inferring the presence of leukaemia stem cells. These leukaemic features were rare or not observed in animals expressing either the NRAS or 9a oncogenes alone, suggesting 9a and NRAS cooperation drives leukaemogenesis. This novel adult AML zebrafish model provides a powerful new tool for investigating the basis of R::RT1 - NRAS cooperativity with the potential to uncover new therapeutic targets., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.

Lidamycin (LDM) has been confirmed to have a strong anti-pancreatic cancer effect and can affect the mitochondrial function of pancreatic cancer cells. Mitofusin-2 (Mfn2) is located in the outer membrane of mitochondria, and Mfn2 is currently believed to play a role in cancer inhibition in pancreatic cancer. In order to explore whether the anti-pancreatic cancer effect of LDM is related to Mfn2-mediated mitophagy, Bioinformatics and in vitro cell experiments are used for experimental research. The experimental results demonstrated that Mfn2 is correlated with mitochondrial autophagy in pancreatic cancer. Lidamycin can increase the expression of Mfn2 in pancreatic cancer and affect the process of EMT, affect the level of reactive oxygen species and mitochondrial membrane potential, and increase the expression of mitochondrial autophagy marker proteins BNIP3L and Beclin1. These results demonstrate that Mfn2 affects mitophagy in pancreatic cancer cells by regulating the expression of Mfn2., (© 2024. The Author(s).)

Background: The interplay between intrauterine and early postnatal environments has been associated with an increased risk of cardiovascular diseases in adulthood, including pulmonary arterial hypertension (PAH). While emerging evidence highlights the crucial role of mitochondrial pathology in PAH, the specific mechanisms driving fetal-originated PAH remain elusive., Methods and Results: To elucidate the role of mitochondrial dynamics in the pathogenesis of fetal-originated PAH, we established a rat model of postnatal catch-up growth following intrauterine growth restriction (IUGR) to induce pulmonary arterial hypertension (PAH). RNA-seq analysis of pulmonary artery samples from the rats revealed dysregulated mitochondrial metabolic genes and pathways associated with increased pulmonary arterial pressure and pulmonary arterial remodeling in the RC group (postnatal catch-up growth following IUGR). In vitro experiments using pulmonary arterial smooth muscle cells (PASMCs) from the RC group demonstrated elevated proliferation, migration, and impaired mitochondrial functions. Notably, reduced expression of Mitofusion 2 (Mfn2), a mitochondrial outer membrane protein involved in mitochondrial fusion, was observed in the RC group. Reconstitution of Mfn2 resulted in enhanced mitochondrial fusion and improved mitochondrial functions in PASMCs of RC group, effectively reversing the Warburg effect. Importantly, Mfn2 reconstitution alleviated the PAH phenotype in the RC group rats., Conclusions: Imbalanced mitochondrial dynamics, characterized by reduced Mfn2 expression, plays a critical role in the development of fetal-originated PAH following postnatal catch-up growth after IUGR. Mfn2 emerges as a promising therapeutic strategy for managing IUGR-catch-up growth induced PAH., (© 2024. The Author(s).)

Background: Interferon-alpha (IFNα) is a common treatment for chronic hepatitis B virus (HBV) infection, but its efficacy varies widely among patients. GTPASE, an interferon-stimulated gene (ISG), has recently been identified as a factor in antiviral immunity, though its role in HBV infection is not fully understood., Objective: This study investigates the role of GTPASE in enhancing the antiviral effects of IFNα against HBV and elucidates its mechanism of action., Methods: We analyzed the impact of GTPASE overexpression and silencing on HBV replication and clearance in HBV-infected cells. Molecular docking studies assessed the interaction between GTPASE and HBV surface antigens (HBs). Clinical samples from HBV patients undergoing Peg-IFNα treatment were also evaluated for GTPASE expression and its correlation with treatment efficacy., Results: Overexpression of GTPASE led to significant inhibition of HBV replication, increased HBeAg seroconversion, and enhanced HBsAg clearance. GTPASE directly bound to HBs proteins, reducing their levels and affecting viral particle formation. Silencing GTPASE reduced these effects, while combined treatment with Peg-IFNα and GTPASE overexpression further improved antiviral outcomes. Mutational analysis revealed that specific sites in GTPASE are crucial for its antiviral activity., Conclusions: GTPASE acts as a positive regulator in IFNα-induced antiviral immunity against HBV. It enhances the therapeutic efficacy of IFNα by targeting HBs and modulating viral replication. GTPASE levels may serve as a predictive biomarker for response to Peg-IFNα therapy, highlighting its potential for improving individualized treatment strategies for chronic HBV infection., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The arrest of neural crest-derived sympathoadrenal neuroblast differentiation contributes to neuroblastoma formation, and overriding this blocked differentiation is a clear strategy for treating high-risk neuroblastoma. A better understanding of neuroblast or neuroblastoma differentiation is essential for developing new therapeutic approaches. It has been proposed that Krueppel-like factor 7 (KLF7) is a neuroblastoma super-enhancer-associated transcription factor gene. Moreover, KLF7 was found to be intensely active in postmitotic neuroblasts of the developing nervous system during embryogenesis. However, the role of KLF7 in the differentiation of neuroblast or neuroblastoma is unknown. Here, we find a strong association between high KLF7 expression and favorable clinical outcomes in neuroblastoma. KLF7 induces differentiation of neuroblastoma cells independently of the retinoic acid (RA) pathway and acts cooperatively with RA to induce neuroblastoma differentiation. KLF7 alters the GTPase activity and multiple differentiation-related genes by binding directly to the promoters of neuroblast differentiation-associated protein (AHNAK and AHNAK2) and glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) and regulating their expression. Furthermore, we also observe that silencing KLF7 in neuroblastoma cells promotes the adrenergic-to-mesenchymal transition accompanied by changes in enhancer-mediated gene expression. Our results reveal that KLF7 is an inducer of neuroblast or neuroblastoma differentiation with prognostic significance and potential therapeutic value., (© 2024 Federation of European Biochemical Societies.)

Keratin intermediate filaments form dynamic filamentous networks, which provide mechanical stability, scaffolding, and protection against stress to epithelial cells. Keratins and other intermediate filaments have been increasingly linked to the regulation of mitochondrial function and homeostasis in different tissues and cell types. While deletion of keratin 8 (K8 -/- ) in mouse colon elicits a colitis-like phenotype, epithelial hyperproliferation, and blunted mitochondrial ketogenesis, the role of K8 in colonocyte mitochondrial function and energy metabolism is unknown. We used two K8 knockout mouse models and CRISPR/Cas9 K8 -/- colorectal adenocarcinoma Caco-2 cells to answer this question. The results show that K8 -/- colonocyte mitochondria in vivo are smaller and rounder and that mitochondrial motility is increased in K8 -/- Caco-2 cells. Furthermore, K8 -/- Caco-2 cells displayed diminished mitochondrial respiration and decreased mitochondrial membrane potential compared with controls, whereas glycolysis was not affected. The levels of mitochondrial respiratory chain complex proteins and mitochondrial regulatory proteins mitofusin-2 and prohibitin were decreased both in vitro in K8 -/- Caco-2 cells and in vivo in K8 -/- mouse colonocytes, and reexpression of K8 into K8 -/- Caco-2 cells normalizes the mitofusin-2 levels. Mitochondrial Ca 2+ is an important regulator of mitochondrial energy metabolism and homeostasis, and Caco-2 cells lacking K8 displayed decreased levels and altered dynamics of mitochondrial matrix and cytoplasmic Ca 2+ . In summary, these novel findings attribute an important role for colonocyte K8 in stabilizing mitochondrial shape and movement and maintaining mitochondrial respiration and Ca 2+ signaling. Further, how these metabolically compromised colonocytes are capable of hyperproliferating presents an intriguing question for future studies. NEW & NOTEWORTHY In this study, we show that colonocyte intermediate filament protein keratin 8 is important for stabilizing mitochondria and maintaining mitochondrial energy metabolism, as keratin 8-deficient colonocytes display smaller, rounder, and more motile mitochondria, diminished mitochondrial respiration, and altered Ca 2+ dynamics. Changes in fusion-regulating proteins are rescued with reexpression of keratin 8. These alterations in colonocyte mitochondrial homeostasis contribute to keratin 8-associated colitis pathophysiology.

It has been demonstrated that circular RNAs (circRNAs) are associated with the development of diabetic retinopathy (DR). Nevertheless, the function of circSLC16A10 in the development of DR remains unclear. In order to investigate the role of circSLC16A10, we employed cell and animal models of DR. An analysis of a public database revealed that hsa&#95;circSLC16A10 was expressed at lower levels in DR patients than in diabetic patients without DR or healthy controls. Additionally, the level of hsa&#95;circSLC16A10 was lower in high glucose (HG)-exposed ARPE-19 cells and diabetic mice. hsa&#95;circSLC16A10 was observed to be mainly distributed in the cytoplasm. Moreover, overexpression of hsa&#95;circSLC16A10 alleviated HG-induced endoplasmic reticulum stress and cell apoptosis in vitro. Furthermore, overexpression of hsa&#95;circSLC16A10 ameliorated HG-induced mitochondrial dysfunction, as evidenced by improvements in mitochondrial structure and function. hsa&#95;circSLC16A10 acted as a hsa-miR-761-5p sponge to increase MFN2 expression. MFN2 knockdown or hsa-miR-761-5p overexpression partially reversed the protective effect of hsa&#95;circSLC16A10 in vitro. The protective effect of mmu&#95;circSLC16A10 against DR was confirmed in an animal model of DR. These findings indicate that circSLC16A10 may regulate DR progression by improving mitochondrial function via the miR-761-5p/MFN2 axis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψ m ) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψ m is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis. Previously, we demonstrated that L-OPA1 of H9c2 cardiomyoblasts is insensitive to loss of Δψ m via challenge with the protonophore carbonyl cyanide chlorophenyl hydrazone (CCCP), but that CCCP-induced OPA1 processing is activated upon differentiation in media with low serum supplemented with all-trans retinoic acid (ATRA). Here, we show that this developmental induction of OPA1 processing in H9c2 cells is independent of ATRA; moreover, pretreatment of undifferentiated H9c2s with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, recapitulates the Δψ m -sensitive OPA1 processing observed in differentiated H9c2s. L6.C11 and C2C12 myoblast lines display the same developmental and CAP-sensitive induction of OPA1 processing, demonstrating a general mechanism of OPA1 regulation in mammalian myoblast cell settings. Restoration of CCCP-induced OPA1 processing correlates with increased apoptotic sensitivity. Moreover, OPA1 knockdown indicates that intact OPA1 is necessary for effective myoblast differentiation. Taken together, our results indicate that a novel developmental mechanism acts to regulate OMA1-mediated OPA1 processing in myoblast cell lines, in which differentiation engages mitochondrial stress sensing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Toxoplasmosis is a common parasitic zoonosis that can be life-threatening in immunocompromised patients. About one-third of the human population is infected with Toxoplasma gondii. Primary infection triggers an innate immune response wherein IFN-γ-induced host cell GTPases, namely IRG and GBP proteins, serve as a vital component for host cell resistance. In the past decades, interest in elucidating the function of these GTPase families in controlling various intracellular pathogens has emerged. Numerous T. gondii effectors were identified to inactivate particular IRG proteins. T. gondii is re-optimizing its effectors to combat IRG function and in this way secures transmission. We discuss the IRG-specific effectors employed by the parasite in murine infections, contributing to a better understanding of T. gondii virulence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ethnopharmacological Relevance: Chimonanthus nitens Oliv. is a traditional Chinese medicine with anti-inflammatory and antioxidant properties that has commonly been used for colds, fevers, and other diseases. However, its role and specific mechanism in sepsis-associated intestinal injury have not been reported., Aim of the Study: C. nitens Oliv. essential oil (CEO), an organic active compound extracted from the traditional Chinese medicine C. nitens Oliv. exhibits notable anti-inflammatory and antioxidant properties. Nevertheless, the therapeutic potential of CEO for septic intestinal injury remains undocumented. This study thus aims to elucidate the anti-inflammatory and antioxidant effects of CEO in the context of acute intestinal injury and to investigate its mechanisms of action in septic rats., Materials and Methods: Cell and animal models were established using LPS to investigate the impact of CEO on LPS-induced intestinal pathological injury and the secretion of inflammatory factor IL-1β. The effects of CEO on the expression of NLRP3, caspase-1, and MFN2, p-p65 protein were also examined, as well as its influence on oxidative stress injury and mitochondrial-associated endoplasmic reticulum membrane (MAM) formation. Generation of an MFN2 knockout IEC-6 cell line allowed comprehensive investigation of the protective mechanism of CEO., Results: In rat models, CEO reduced IL-1β secretion, inhibited caspase-1, ZO-1 expression and NF-κB p65 phosphorylation, while also decreasing malondialdehyde levels and enhancing superoxide dismutase activity in intestinal tissues. Cellular experiments demonstrated its ability to decrease IL-1β secretion; NLRP3, caspase-1, and MFN2 expression; NF-κB p65 phosphorylation; reactive oxygen species (ROS) production, and mitochondrial dysfunction. MFN2 knockdown enhanced these effects synergistically with CEO, indicating potential therapeutic synergy. Further, MFN2 knockdown significantly mitigated LPS-induced NLRP3 and caspase-1 expression, IL-1β secretion, ROS production, NF-κB p65 phosphorylation and MMP reduction in IEC-6 cells, while inhibiting MAM formation and NLRP3 localization on MAMs. Importantly, MFN2 downregulation and CEO synergistically reduced LPS-induced IL-1β secretion and ROS production while inhibiting MAM formation in IEC-6 cells, thus inhibiting NLRP3 inflammasome activation., Conclusion: CEO mitigates inflammation and oxidative stress by inhibiting MAM formation and is thus a promising intervention for septic intestinal injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Saffron is derived from the dried stigmas of Crocus sativus L., which was considered by ancient nations for food and medicinal purposes. In traditional medicine, the therapeutic use of Crocus sativus includes antispasmodic, antitussive and expectorant., Aim of the Study: Mitochondrial fusion, fission, biogenesis, and mitophagy are essential processes for maintaining mitochondrial dynamics in response to cellular stress. The primary objective of this research was to examine how crocin affected the levels of important mitochondrial regulators, including Drp1, Pgc1α, Nrf1, and Mfn2, in the lung tissue of ovalbumin-sensitized mice., Materials and Methods: A total of fifty male BALB/C mice were randomly assigned to five unique groups (n = 10 for each group), including the control group, ovalbumin-sensitized group (OVA), OVA group treated with 30 mg/kg of crocin, OVA group treated with 60 mg/kg of crocin, and OVA group treated with 1 mg/kg of dexamethasone. Post-sensitization and ovalbumin challenge, mice lung tissues were evaluated for the expression of Drp1, Pgc1α, Nrf1, and Mfn2 mRNA levels using real-time PCR as well as histopathological assessments., Results: In the OVA group, there was a significant elevated in inflammatory cells such as eosinophils, neutrophils, macrophages, and lymphocytes; however, crocin (both concentrations) and dexamethasone intervention showed significant inhibitory effects (P < 0.01 to P < 0.001). Moreover, an increase in the expression of Drp1, Pgc1α, and Nrf1 levels was seen in the OVA group, while crocin and dexamethasone showed protective benefits (P < 0.05 to P < 0.001). Furthermore, the levels of Mfn2 were reduced in the lung tissue of mice exposed to ovalbumin, but this decrease was reversed by crocin 60 (P < 0.05) and dexamethasone treatment (P < 0.001)., Conclusion: In mice with OVA sensitization, the balance of mitochondrial dynamics in lung tissue was disrupted, but intervention of crocin identified to have a protective effect., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: As a heterogeneous and life-threatening disease, the pathogenesis of acute liver failure (ALF) is complex. Our previous study has shown that IDH1/MDH1 deacetylation promotes ALF by regulating NETosis (a novel mode of cell death). In this article, we explore the manners of IDH1/MDH1 deacetylation regulates NETosis., Methods: In vitro experiments, the formation of NETs was detected by immunofluorescence staining and Western blotting. LC3 fluorescence staining was used to detect autophagosome formation. To observe mitochondrial morphology, cells were stained by Mito-Tracker Red. Western blotting was used to detect the levels of autophagy protein and mitochondrial dynamin. In vivo experiments, the ALF model in mouse was established with LPS/D-gal, and the formation of NETs was detected by immunofluorescence staining and Western blotting. The autophagy levels were detected by Western blotting in liver samples., Results: In dHL-60 cells, Western blotting results showed that the expression of OPA1 was higher in the IDH1/MDH1 deacetylated group compared with the IDH1/MDH1 WT group. And histone deacetylase inhibitor 6 (HDAC6i, ACY1215) decreased the expression level of OPA1 in IDH1/MDH1 deacetylated group. IDH1/MDH1 deacetylation increased the expression levels of both LC3B-II and Beclin 1, while decreasing the expression level of P62. It was reversed by ACY1215. Combined with our previous experiments, IDH1/MDH1 deacetylation upregulated autophagy concomitant with the increased expression of the markers of NETs formation. In a mouse model of ALF, ACY1215 further decreased the expression levels of LC3B-II and Beclin 1, while increasing the expression level of P62 in IDH1/MDH1 deacetylated mice., Conclusions: IDH1/MDH1 deacetylation promoted NETosis by regulating autophagy and OPA1 in vitro. The regulation of neutrophil autophagy on NETosis during IDH1/MDH1 deacetylation might be masked in mice. ACY1215 might attenuate NETosis by regulating neutrophil autophagy, which alleviated ALF aggravated by IDH1/MDH1 deacetylation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ribosome biogenesis is a highly regulated multistep process aided by energy-consuming auxiliary factors. GTPases form the largest class of auxiliary factors used by bacterial, cytosolic, and mitochondrial ribosomes for their maturation. Mtg3, a circularly permuted YqeH family of GTPase, is implicated in the mitoribosome small subunit biogenesis. However, its precise mechanistic role has yet to be characterized. Mtg3 is likely to bind precursor mitoribosome molecules during subunit maturation in vivo. However, this interaction has yet to be observed with mitoribosomes biochemically. In this study, we delineate the specific conditions necessary for preserving the association of Mtg3 with mitoribosomes on a sucrose density gradient. We show that the C-terminal domain of Mtg3 is required for robust binding to the mitoribosome. Furthermore, point mutants likely to abrogate GTP/GDP binding and GTPase activity compromise protein function in vivo. Surprisingly, the association with the mitoribosome was not compromised in mutants likely to be deficient for nucleotide binding/hydrolysis. Thus, our finding supports a model wherein Mtg3 binds to a precursor mitoribosome through its C-terminus to facilitate a conformational change or validate a folding intermediate driven by the GTP/GDP binding and hydrolysis cycle., Competing Interests: Declaration of competing interest The authors declare no potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and is the most common cause of end-stage renal disease. Tripartite motif-containing (TRIM) proteins are a large family of E3 ubiquitin ligases that contribute to protein quality control by regulating the ubiquitin - proteasome system. However, the detailed mechanisms through which various TRIM proteins regulate downstream events have not yet been fully elucidated. The current research aimed to determine the function and mechanism of TRIM22 in DN., Methods: DN models were established by inducing HK-2 cells using high glucose (HG) and diabetic mice (db/db mice). Cell viability, apoptosis, mitochondrial reactive oxygen species, and mitochondrial membrane potential were detected by Cell Counting Kit-8 and flow cytometry, respectively. Pathological changes were evaluated using hematoxylin and eosin, periodic acid schiff and Masson staining. The binding between TRIM22 and optic atrophy 1 (OPA1) was analyzed using co-immunoprecipitation. The m 6 A level of TRIM22 5'UTR was detected using RNA immunoprecipitation., Results: TRIM22 was highly expressed in patients with DN. TRIM22 silencing inhibited HG-induced apoptosis and mitochondrial dysfunction in HK-2 cells. Promoting mitochondrial fusion alleviated TRIM22 overexpression-induced cell apoptosis, mitochondrial dysfunction in HK-2 cells, and kidney damage in mice. Mechanistically, TRIM22 interacted with OPA1 and induced its ubiquitination. Wilms tumor 1-associating protein (WTAP) promoted m 6 A modification of TRIM22 through the m 6 A reader insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)., Discussion: TRIM22 silencing inhibited the progression of DN by interacting with OPA1 and inducing its ubiquitination. Furthermore, WTAP promoted m 6 A modification of TRIM22 via IGF2BP1.