Primary cilia formation requires the Leigh syndrome-associated mitochondrial protein NDUFAF2.

Mitochondria-related neurodegenerative diseases have been implicated in the disruption of primary cilia function. Mutation in an intrinsic mitochondrial complex I component NDUFAF2 has been identified in Leigh syndrome, a severe inherited mitochondriopathy. Mutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome, a ciliopathy with defects in the brain, kidney, and eye. Here, we report a mechanistic link between mitochondria metabolism and primary cilia signaling. We discovered that loss of NDUFAF2 caused both mitochondrial and ciliary defects in vitro and in vivo and identified NDUFAF2 as a binding partner for ARMC9. We also found that NDUFAF2 was both necessary and sufficient for cilia formation and that exogenous expression of NDUFAF2 rescued the ciliary and mitochondrial defects observed in cells from patients with known ARMC9 deficiency. NAD+ supplementation restored mitochondrial and ciliary dysfunction in ARMC9-deficient cells and zebrafish and ameliorated the ocular motility and motor deficits of a patient with ARMC9 deficiency. The present results provide a compelling mechanistic link, supported by evidence from human studies, between primary cilia and mitochondrial signaling. Importantly, our findings have significant implications for the development of therapeutic approaches targeting ciliopathies.

FUNDC1 alleviates doxorubicin-induced cardiotoxicity by restoring mitochondrial-endoplasmic reticulum contacts and blocked autophagic flux.

Rationale: Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a tethering protein of MERCs. We aim to investigate the effect of DOX on MERCs in cardiomyocytes and explore whether it is involved in the dysregulated autophagy in DIC. Methods: We employed confocal microscopy and transmission electron microscopy to assess MERCs structure. Autophagic flux was analyzed using the mCherry-EGFP-LC3B fluorescence assay and western blotting for LC3BII. Mitophagy was studied through the mCherry-EGFP-FIS1 fluorescence assay and colocalization analysis between LC3B and mitochondria. A total dose of 18 mg/kg of doxorubicin was administrated in mice to construct a DIC model in vivo. Additionally, we used adeno-associated virus (AAV) to cardiac-specifically overexpress FUNDC1. Cardiac function and remodeling were evaluated by echocardiography and Masson's trichrome staining, respectively. Results: DOX blocked autophagic flux by inhibiting autophagosome biogenesis, which could be attributed to the downregulation of FUNDC1 and disruption of MERCs structures. FUNDC1 overexpression restored the blocked autophagosome biogenesis by maintaining MERCs structure and facilitating ATG5-ATG12/ATG16L1 complex formation without altering mitophagy. Furthermore, FUNDC1 alleviated DOX-induced oxidative stress and cardiomyocytes deaths in an autophagy-dependent manner. Notably, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. Conclusions: In summary, our study identified that FUNDC1-meditated MERCs exerted a cardioprotective effect against DIC by restoring the blocked autophagosome biogenesis. Importantly, this research reveals a novel role of FUNDC1 in enhancing macroautophagy via restoring MERCs structure and autophagosome biogenesis in the DIC model, beyond its previously known regulatory role as an mitophagy receptor.

Polydatin: a potential NAFLD therapeutic drug that regulates mitochondrial autophagy through SIRT3-FOXO3-BNIP3 and PINK1-PRKN mechanisms - a network pharmacology and experimental investigation.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disorder that is linked to metabolic syndrome, mitochondrial dysfunction and impaired autophagy. Polydatin (PD), a natural polyphenol from Polygonum cuspidatum, exhibits various pharmacological effects and protects against NAFLD. The aim of this study was to reveal the molecular mechanisms and therapeutic potential of PD for NAFLD, with a focus on the role of mitochondrial autophagy mediated by sirtuin 3 (SIRT3), fork-head box O3 (FOXO3) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), and by PTEN-induced putative kinase 1 (PINK1) and parkin (PRKN). We combined network pharmacology analysis, animal models and cell culture experiments to show that PD could regulate the mitochondrial autophagy pathway by modulating several key genes related to mitochondrial function, and ameliorate the liver function, histopathology and mitochondrial biogenesis of NAFLD mice and hepatocytes by activating the SIRT3-FOXO3-BNIP3 axis and the PINK1-PRKN-dependent mechanism of mitochondrial autophagy. We also identified the core targets of PD, including SIRT3, FOXO3A, CASP3, PARKIN, EGFR, STAT3, MMP9 and PINK, and confirmed that silencing SIRT3 could significantly attenuate the beneficial effect of PD. This study provided novel theoretical and experimental support for PD as a promising candidate for NAFLD treatment, and also suggested new avenues and methods for investigating the role of mitochondrial autophagy in the pathogenesis and intervention of NAFLD.

Mitoferrin2 is a synthetic lethal target for chromosome 8p deleted cancers.

BACKGROUND: Somatic copy number alterations are a hallmark of cancer that offer unique opportunities for therapeutic exploitation. Here, we focused on the identification of specific vulnerabilities for tumors harboring chromosome 8p deletions. METHODS: We developed and applied an integrative analysis of The Cancer Genome Atlas (TCGA), the Cancer Dependency Map (DepMap), and the Cancer Cell Line Encyclopedia to identify chromosome 8p-specific vulnerabilities. We employ orthogonal gene targeting strategies, both in vitro and in vivo, including short hairpin RNA-mediated gene knockdown and CRISPR/Cas9-mediated gene knockout to validate vulnerabilities. RESULTS: We identified SLC25A28 (also known as MFRN2), as a specific vulnerability for tumors harboring chromosome 8p deletions. We demonstrate that vulnerability towards MFRN2 loss is dictated by the expression of its paralog, SLC25A37 (also known as MFRN1), which resides on chromosome 8p. In line with their function as mitochondrial iron transporters, MFRN1/2 paralog protein deficiency profoundly impaired mitochondrial respiration, induced global depletion of iron-sulfur cluster proteins, and resulted in DNA-damage and cell death. MFRN2 depletion in MFRN1-deficient tumors led to impaired growth and even tumor eradication in preclinical mouse xenograft experiments, highlighting its therapeutic potential. CONCLUSIONS: Our data reveal MFRN2 as a therapeutic target of chromosome 8p deleted cancers and nominate MFNR1 as the complimentary biomarker for MFRN2-directed therapies.

Peripheral blood mononuclear cells show markers of mitochondrial dysfunction in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. Metabolic and mitochondrial dysregulation are critical causal factors in the pathogenesis and progression of RA. Mitochondrial dysfunction include abnormal energy metabolism, and excessive production of reactive oxygen species (ROS). This study aimed to investigate the adenosine triphosphate (ATP), mitochondrial membrane potential (ΔΨm), ROS, and mRNA expression level of ROMO1 (as ROS modulator) and OMA1 (as regulator mitochondrial dynamics) of peripheral blood mononuclear cells (PBMC) in RA patients. The study participants were 50 patients with RA and 50 sex- and age-matched healthy volunteers. PBMC of all participant were isolated by Ficoll-Paque. Alteration in ΔΨm and cellular ROS were measured using flow cytometry, ATP level was also assessed via luminometry, and ROMO1 and OMA1 mRNA expression via qRT-PCR assay. A significant decrease in ATP (p = .005) and ΔΨm (p < .001) was observed in the PBMC of RA compared to control. The ROS levels were significantly higher in the PBMC of RA compared to the control (p < .001). ROMO1 and OMA1 mRNA expression was also significantly increased in RA patients compared to control (p < .001). The decrease in ATP is strongly associated with ROS increasing in PBMC of RA patients, denoting an inverse and negative relationship between ATP and ROS production. Also, a decrease in ΔΨm was observed. It seems that in line with mitochondrial dysfunction in PBMC, increased expression of ROMO1 and OMA1 genes could also be involved in the development of RA.

Hypoxia-inducible factor prolyl hydroxylase inhibitor alleviates heatstroke-induced acute kidney injury by activating BNIP3-mediated mitophagy.

Hypoxia-induced inflammation and apoptosis are important pathophysiological features of heat stroke-induced acute kidney injury (HS-AKI). Hypoxia-inducible factor (HIF) is a key protein that regulates cell adaptation to hypoxia. HIF-prolyl hydroxylase inhibitor (HIF-PHI) stabilizes HIF to increase cell adaptation to hypoxia. Herein, we reported that HIF-PHI pretreatment significantly improved renal function, enhanced thermotolerance, and increased the survival rate of mice in the context of HS. Moreover, HIF-PHI could alleviate HS-induced mitochondrial damage, inflammation, and apoptosis in renal tubular epithelial cells (RTECs) by enhancing mitophagy in vitro and in vivo. By contrast, mitophagy inhibitors Mdivi-1, 3-MA, and Baf-A1 reversed the renoprotective effects of HIF-PHI. Mechanistically, HIF-PHI protects RTECs from inflammation and apoptosis by enhancing Bcl-2 adenovirus E18 19-kDa-interacting protein 3 (BNIP3)-mediated mitophagy, while genetic ablation of BNIP3 attenuated HIF-PHI-induced mitophagy and abolished HIF-PHI-mediated renal protection. Thus, our results indicated that HIF-PHI protects renal function by upregulating BNIP3-mediated mitophagy to improve HS-induced inflammation and apoptosis of RTECs, suggesting HIF-PHI as a promising therapeutic agent to treat HS-AKI.

In vivo and in vitro studies on the role of regulating Smac expression in the occurrence and development of colon cancer.

We aimed to explore the role of regulating Smac expression levels in the occurrence and development of colon cancer through in vitro and in vivo experiments. Colon cancer cells HT-29 were cultured and transfected into different groups. qRT-PCR was used to detect the expression level of Smac in cells; Flow cytometry was used to detect the apoptotic ability of each group of cells; Western blot was used to detect the protein expression of Smac and apoptosis-related factors Survivin and Caspase-3; The nude mouse tumorigenesis experiment was conducted to detect the regulatory effect of regulating Smac expression levels on the growth of colon cancer transplanted tumors in vivo. In comparison to the FHC group, the HT-29 group exhibited a decrease in Smac expression. The si-Smac group, when compared with the si-NC group, showed significant reductions in Smac mRNA and protein levels, weaker cell apoptosis, increased Survivin, and decreased Caspase-3 expression. Contrarily, the oe-Smac group, against the oe-NC group, displayed increased Smac mRNA and protein levels, enhanced apoptosis, reduced Survivin, and elevated Caspase-3 expression. In nude mice tumor transplantation experiments, the LV-sh-Smac group, as opposed to the LV-sh-NC group, had tumors with greater volume and weight, reduced Smac and Caspase-3, and increased Survivin expression. In contrast, the LV-oe-Smac group, compared with the LV-oe-NC group, showed tumors with decreased volume and mass, increased expressions of Smac and Caspase-3, and decreased Survivin. Smac is lowly expressed in colon cancer. Upregulation of Smac expression can inhibit the occurrence and development of colon cancer, possibly by inhibiting Survivin expression and promoting Caspase-3 expression, thereby enhancing the pro-apoptotic function.

PJA1-mediated suppression of pyroptosis as a driver of docetaxel resistance in nasopharyngeal carcinoma.

Chemoresistance is a main reason for treatment failure in patients with nasopharyngeal carcinoma, but the exact regulatory mechanism underlying chemoresistance in nasopharyngeal carcinoma remains to be elucidated. Here, we identify PJA1 as a key E3 ubiquitin ligase involved in nasopharyngeal carcinoma chemoresistance that is highly expressed in nasopharyngeal carcinoma patients with nonresponse to docetaxel-cisplatin-5-fluorouracil induction chemotherapy. We find that PJA1 facilitates docetaxel resistance by inhibiting GSDME-mediated pyroptosis in nasopharyngeal carcinoma cells. Mechanistically, PJA1 promotes the degradation of the mitochondrial protein PGAM5 by increasing its K48-linked ubiquitination at K88, which further facilitates DRP1 phosphorylation at S637 and reduced mitochondrial reactive oxygen species production, resulting in suppression of GSDME-mediated pyroptosis and the antitumour immune response. PGAM5 knockdown fully restores the docetaxel sensitization effect of PJA1 knockdown. Moreover, pharmacological targeting of PJA1 with the small molecule inhibitor RTA402 enhances the docetaxel sensitivity of nasopharyngeal carcinoma in vitro and in vivo. Clinically, high PJA1 expression indicates inferior survival and poor clinical efficacy of TPF IC in nasopharyngeal carcinoma patients. Our study emphasizes the essential role of E3 ligases in regulating chemoresistance and provides therapeutic strategies for nasopharyngeal carcinoma based on targeting the ubiquitin-proteasome system.

Artemisinins ameliorate polycystic ovarian syndrome by mediating LONP1-CYP11A1 interaction.

Polycystic ovary syndrome (PCOS), a prevalent reproductive disorder in women of reproductive age, features androgen excess, ovulatory dysfunction, and polycystic ovaries. Despite its high prevalence, specific pharmacologic intervention for PCOS is challenging. In this study, we identified artemisinins as anti-PCOS agents. Our finding demonstrated the efficacy of artemisinin derivatives in alleviating PCOS symptoms in both rodent models and human patients, curbing hyperandrogenemia through suppression of ovarian androgen synthesis. Artemisinins promoted cytochrome P450 family 11 subfamily A member 1 (CYP11A1) protein degradation to block androgen overproduction. Mechanistically, artemisinins directly targeted lon peptidase 1 (LONP1), enhanced LONP1-CYP11A1 interaction, and facilitated LONP1-catalyzed CYP11A1 degradation. Overexpression of LONP1 replicated the androgen-lowering effect of artemisinins. Our data suggest that artemisinin application is a promising approach for treating PCOS and highlight the crucial role of the LONP1-CYP11A1 interaction in controlling hyperandrogenism and PCOS occurrence.

The mitochondrial protease ClpP is a druggable target that controls VSMC phenotype by a SIRT1-dependent mechanism.

Vascular smooth muscle cells (VSMCs), known for their remarkable lifelong phenotypic plasticity, play a pivotal role in vascular pathologies through their ability to transition between different phenotypes. Our group discovered that the deficiency of the mitochondrial protein Poldip2 induces VSMC differentiation both in vivo and in vitro. Further comprehensive biochemical investigations revealed Poldip2's specific interaction with the mitochondrial ATPase caseinolytic protease chaperone subunit X (CLPX), which is the regulatory subunit for the caseinolytic protease proteolytic subunit (ClpP) that forms part of the ClpXP complex - a proteasome-like protease evolutionarily conserved from bacteria to humans. This interaction limits the protease's activity, and reduced Poldip2 levels lead to ClpXP complex activation. This finding prompted the hypothesis that ClpXP complex activity within the mitochondria may regulate the VSMC phenotype. Employing gain-of-function and loss-of-function strategies, we demonstrated that ClpXP activity significantly influences the VSMC phenotype. Notably, both genetic and pharmacological activation of ClpXP inhibits VSMC plasticity and fosters a quiescent, differentiated, and anti-inflammatory VSMC phenotype. The pharmacological activation of ClpP using TIC10, currently in phase III clinical trials for cancer, successfully replicates this phenotype both in vitro and in vivo and markedly reduces aneurysm development in a mouse model of elastase-induced aortic aneurysms. Our mechanistic exploration indicates that ClpP activation regulates the VSMC phenotype by modifying the cellular NAD+/NADH ratio and activating Sirtuin 1. Our findings reveal the crucial role of mitochondrial proteostasis in the regulation of the VSMC phenotype and propose the ClpP protease as a novel, actionable target for manipulating the VSMC phenotype.

Suppressing mitochondrial inner membrane protein (IMMT) inhibits the proliferation of breast cancer cells through mitochondrial remodeling and metabolic regulation.

Metabolic reprogramming is widely recognized as a hallmark of malignant tumors, and the targeting of metabolism has emerged as an appealing approach for cancer treatment. Mitochondria, as pivotal organelles, play a crucial role in the metabolic regulation of tumor cells, and their morphological and functional alterations are intricately linked to the biological characteristics of tumors. As a key regulatory subunit of mitochondria, mitochondrial inner membrane protein (IMMT), plays a vital role in degenerative diseases, but its role in tumor is almost unknown. The objective of this research was to investigate the roles that IMMT play in the development and progression of breast cancer (BC), as well as to elucidate the underlying biological mechanisms that drive these effects. In this study, it was confirmed that the expression of IMMT in BC tissues was significantly higher than that in normal tissues. The analysis of The Cancer Genome Atlas (TCGA) database revealed that IMMT can serve as an independent prognostic factor for BC patients. Additionally, verification in clinical specimens of BC demonstrated a positive association between high IMMT expression and larger tumor size (> 2 cm), Ki-67 expression (> 15%), and HER-2 status. Furthermore, in vitro experiments have substantiated that the suppression of IMMT expression resulted in a reduction in cell proliferation and alterations in mitochondrial cristae, concomitant with the liberation of cytochrome c, but it did not elicit mitochondrial apoptosis. Through Gene Set Enrichment Analysis (GSEA) analysis, we have predicted the associated metabolic genes and discovered that IMMT potentially modulates the advancement of BC through its interaction with 16 metabolic-related genes, and the changes in glycolysis related pathways have been validated in BC cell lines after IMMT inhibition. Consequently, this investigation furnishes compelling evidence supporting the classification of IMMT as prognostic marker in BC, and underscoring its prospective utility as a novel target for metabolic therapy.

Increased risk of hearing loss associated with MT-RNR1 gene mutations: a real-world investigation among Han Taiwanese Population.

BACKGROUND: Previous studies have implicated inherited mutations in mitochondrial DNA (mtDNA) in sensorineural hearing loss (SNHL). However, the definitive association between mitochondrial 12S rRNA (MT-RNR1) variants and hearing loss in the population has not been well established, particularly in Asia. The objective of this retrospective cohort study was to assess the association between MT-RNR1 variants and the risk of SNHL in patients in Taiwan. METHODS: The cohort included 306,068 participants from Taiwan between January 2003 and December 2020. Participants were classified based on genetic variants, particularly mitochondrial mutations (rs267606618, rs267606619, rs267606617). MT-RNR1 variant cases were matched 1:10 with non-mutant patients by age, gender, and visit year, excluding those with pre-existing hearing loss. The primary endpoint was SNHL, identified using specific ICD-TM codes with a 90% positive predictive value. Medication exposure history was determined via self-report or electronic medical records in the hospital. Cox proportional hazard regression models were used to assess the association between MT-RNR1 variants and hearing loss, adjusting for various covariates. Kaplan-Meier survival curves and log-rank tests compared hearing loss incidence between groups. RESULTS: The mean age of the mtDNA variants group is 32.4 years, with a standard deviation of 19.2 years. The incidence density of hearing loss for the mutation group was 36.42 per 10,000 person-years (95% Confidence Interval [CI], 27.21-47.73), which was higher than the 23.77per 10,000 person-years (95% CI, 21.32-26.42) in the wild-type group (p = 0.0036). Additionally, diabetes mellitus was associated with an increased risk of developing SNHL in individuals with MT-RNR1 variants (adjusted hazard ratio = 1.76 [95% CI, 1.00-3.09], p < 0.05). CONCLUSION: This study highlights the increased risk of hearing loss in patients carrying MT-RNR1 variants, particularly those with diabetes mellitus. Future research that integrates genetic and clinical data is crucial for developing more precise interventions to monitor and treat hearing loss in this vulnerable population.

Biochemical and functional characterization of the p.A165T missense variant of mitochondrial amidoxime-reducing component 1.

Recent genome-wide association studies have identified a missense variant p.A165T in mitochondrial amidoxime-reducing component 1 (mARC1) that is strongly associated with protection from all-cause cirrhosis and improved prognosis in nonalcoholic steatohepatitis. The precise mechanism of this protective effect is unknown. Substitution of alanine 165 with threonine is predicted to affect mARC1 protein stability and to have deleterious effects on its function. To investigate the mechanism, we have generated a knock-in mutant mARC1 A165T and a catalytically dead mutant C273A (as a control) in human hepatoma HepG2 cells, enabling characterization of protein subcellular distribution, stability, and biochemical functions of the mARC1 mutant protein expressed from its endogenous locus. Compared to WT mARC1, we found that the A165T mutant exhibits significant mislocalization outside of its traditional location anchored in the mitochondrial outer membrane and reduces protein stability, resulting in lower basal levels. We evaluated the involvement of the ubiquitin proteasome system in mARC1 A165T degradation and observed increased ubiquitination and faster degradation of the A165T variant. In addition, we have shown that HepG2 cells carrying the MTARC1 p.A165T variant exhibit lower N-reductive activity on exogenously added amidoxime substrates in vitro. The data from these biochemical and functional assays suggest a mechanism by which the MTARC1 p.A165T variant abrogates enzyme function which may contribute to its protective effect in liver disease.

Exploring the Role of HtrA Family Genes in Cancer: A Systematic Review.

PURPOSE: HtrA1, HtrA2, HtrA3 and HtrA4 appear to be involved in the development of pathologies such as cancer. This systematic review reports the results of a literature search performed to compare the expression of HtrA family genes and proteins in cancer versus non-cancer tissues and cell lines, assess relationships between HtrA expression and cancer clinical features in cancer, and analyse the molecular mechanism, by which HtrA family affects cancer. METHODS: The literature search was conducted according to the PRISMA statement among four databases (PubMed, Web of Science, Embase and Scopus). RESULTS: A total of 38 articles met the inclusion criteria and involved the expression of HtrA family members and concerned the effect of HtrA expression on cancer and metastasis development or on the factor that influences it. Additionally, 31 reports were retrieved manually. Most articles highlighted that HtrA1 and HtrA3 exhibited tumour suppressor activity, while HtrA2 was associated with tumour growth and metastasis. There were too few studies to clearly define the role of the HtrA4 protease in tumours. CONCLUSION: Although the expression of serine proteases of the HtrA family was dependent on tumour type, stage and the presence of metastases, most articles indicated that HtrA1 and HtrA3 expression in tumours was downregulated compared with healthy tissue or cell lines. The expression of HtrA2 was completely study dependent. The limited number of studies on HtrA4 expression made it impossible to draw conclusions about differences in expression between healthy and tumour tissue. The conclusions drawn from the study suggest that HtrA1 and HtrA3 act as tumour suppressors.

MCU inhibition protects against intestinal ischemia‒reperfusion by inhibiting Drp1-dependent mitochondrial fission.

Intestinal ischemia‒reperfusion (IIR) injury is a common complication of surgery, but clear molecular insights and valuable therapeutic targets are lacking. Mitochondrial calcium overload is an early sign of various diseases and is considered a vital factor in ischemia‒reperfusion injury. The mitochondrial calcium uniporter (MCU), which is located on the inner mitochondrial membrane, is the primary mediator of calcium ion entry into the mitochondria. However, the specific mechanism of MCU in IIR injury remains to be clarified. In this study, we generated an IIR model using C57BL/6 mice and Caco-2 cells and found increases in the calcium levels and MCU expression following IIR injury. The specific inhibition of MCU markedly attenuated IIR injury. Moreover, MCU knockdown alleviates mitochondrial dysfunction by reducing oxidative stress and apoptosis. Mechanistically, MCU knockdown substantially reduced the translocation of Drp1 and thus its binding to Fis1 receptors, resulting in decreased mitochondrial fission. Taken together, our findings demonstrated that MCU is a novel upstream regulator of Drp1 in ischemia‒reperfusion and represents a predictive and therapeutic target for IIR.

Lon1 Inactivation Downregulates Autophagic Flux and Brassinosteroid Biogenesis, Modulating Mitochondrial Proportion and Seed Development in Arabidopsis.

Mitochondrial protein homeostasis is crucially regulated by protein degradation processes involving both mitochondrial proteases and cytosolic autophagy. However, it remains unclear how plant cells regulate autophagy in the scenario of lacking a major mitochondrial Lon1 protease. In this study, we observed a notable downregulation of core autophagy proteins in Arabidopsis Lon1 knockout mutant lon1-1 and lon1-2, supporting the alterations in the relative proportions of mitochondrial and vacuolar proteins over total proteins in the plant cells. To delve deeper into understanding the roles of the mitochondrial protease Lon1 and autophagy in maintaining mitochondrial protein homeostasis and plant development, we generated the lon1-2atg5-1 double mutant by incorporating the loss-of-function mutation of the autophagy core protein ATG5, known as atg5-1. The double mutant exhibited a blend of phenotypes, characterized by short plants and early senescence, mirroring those observed in the individual single mutants. Accordingly, distinct transcriptome alterations were evident in each of the single mutants, while the double mutant displayed a unique amalgamation of transcriptional responses. Heightened severity, particularly evident in reduced seed numbers and abnormal embryo development, was observed in the double mutant. Notably, aberrations in protein storage vacuoles (PSVs) and oil bodies were evident in the single and double mutants. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of genes concurrently downregulated in lon1-2, atg5-1, and lon1-2atg5-1 unveiled a significant suppression of genes associated with brassinosteroid (BR) biosynthesis and homeostasis. This downregulation likely contributes to the observed abnormalities in seed and embryo development in the mutants.

Genomic analysis and phylogenetic characterization of Himalayan snow trout, Schizothorax esocinus based on mitochondrial protein-coding genes.

BACKGROUND: Mitochondrial DNA (mtDNA) has become a significant tool for exploring genetic diversity and delineating evolutionary links across diverse taxa. Within the group of cold-water fish species that are native to the Indian Himalayan region, Schizothorax esocinus holds particular importance due to its ecological significance and is potentially vulnerable to environmental changes. This research aims to clarify the phylogenetic relationships within the Schizothorax genus by utilizing mitochondrial protein-coding genes. METHODS: Standard protocols were followed for the isolation of DNA from S. esocinus. For the amplification of mtDNA, overlapping primers were used, and then subsequent sequencing was performed. The genetic features were investigated by the application of bioinformatic approaches. These approaches covered the evaluation of nucleotide composition, codon usage, selective pressure using nonsynonymous substitution /synonymous substitution (Ka/Ks) ratios, and phylogenetic analysis. RESULTS: The study specifically examined the 13 protein-coding genes of Schizothorax species which belongs to the Schizothoracinae subfamily. Nucleotide composition analysis showed a bias towards A + T content, consistent with other cyprinid fish species, suggesting evolutionary conservation. Relative Synonymous Codon Usage highlighted leucine as the most frequent (5.18%) and cysteine as the least frequent (0.78%) codon. The positive AT-skew and the predominantly negative GC-skew indicated the abundance of A and C. Comparative analysis revealed significant conservation of amino acids in multiple genes. The majority of amino acids were hydrophobic rather than polar. The purifying selection was revealed by the genetic distance and Ka/Ks ratios. Phylogenetic study revealed a significant genetic divergence between S. esocinus and other Schizothorax species with interspecific K2P distances ranging from 0.00 to 8.87%, with an average of 5.76%. CONCLUSION: The present study provides significant contributions to the understanding of mitochondrial genome diversity and genetic evolution mechanisms in Schizothoracinae, hence offering vital insights for the development of conservation initiatives aimed at protecting freshwater fish species.

Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies.

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

E3 ubiquitin ligase RBCK1 confers ferroptosis resistance in pancreatic cancer by facilitating MFN2 degradation.

Ferroptosis, a novel form of iron-dependent non-apoptotic cell death, plays an active role in the pathogenesis of diverse diseases, including cancer. However, the mechanism through which ferroptosis is regulated in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, our study, via combining bioinformatic analysis with experimental validation, showed that ferroptosis is inhibited in PDAC. Genome-wide sequencing further revealed that the ferroptosis activator imidazole ketone erastin (IKE) induced upregulation of the E3 ubiquitin ligase RBCK1 in PDAC cells at the transcriptional or translational level. RBCK1 depletion or knockdown rendered PDAC cells more vulnerable to IKE-induced ferroptotic death in vitro. In a mouse xenograft model, genetic depletion of RBCK1 increased the killing effects of ferroptosis inducer on PDAC cells. Mechanistically, RBCK1 interacts with and polyubiquitylates mitofusin 2 (MFN2), a key regulator of mitochondrial dynamics, to facilitate its proteasomal degradation under ferroptotic stress, leading to decreased mitochondrial reactive oxygen species (ROS) production and lipid peroxidation. These findings not only provide new insights into the defense mechanisms of PDAC cells against ferroptotic death but also indicate that targeting the RBCK1-MFN2 axis may be a promising option for treating patients with PDAC.