A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis.

BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

TOM5 regulates the mitochondrial membrane potential of alveolar epithelial cells in organizing pneumonia.

Deficiency of TOM5, a mitochondrial protein, causes organizing pneumonia (OP) in mice. The clinical significance and mechanisms of TOM5 in the pathogenesis of OP remain elusive. We demonstrated that TOM5 was significantly increased in the lung tissues of OP patients, which was positively correlated with the collagen deposition. In a bleomycin-induced murine model of chronic OP, increased TOM5 was in line with lung fibrosis. In vitro, TOM5 regulated the mitochondrial membrane potential in alveolar epithelial cells. TOM5 reduced the proportion of early apoptotic cells and promoted cell proliferation. Our study shed light on the roles of TOM5 in OP.

Long-read sequencing for 29 immune cell subsets reveals disease-linked isoforms.

Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer's disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms, providing insight into the human genome evolution. In addition, some of the isoforms are expressed in a cell-type specific manner, whose alternative 3'-UTRs usage contributes to their specificity. Further, we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing.

TIMM17A overexpression in lung adenocarcinoma and its association with prognosis.

Lung adenocarcinoma (LUAD), a leading cause of cancer-related mortality worldwide, demands a deeper understanding of its molecular mechanisms and the identification of reliable biomarkers for better diagnosis and targeted therapy. Leveraging data from the Cancer Genome Atlas (TCGA), the Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA), we investigated the mRNA and protein expression profiles of TIMM17A and assessed its prognostic significance through Kaplan-Meier survival curves and Cox regression analysis. Through Gene Set Enrichment Analysis, we explored the regulatory mechanisms of TIMM17A in LUAD progression and demonstrated its role in modulating the proliferative capacity of A549 cells, a type of LUAD cell, via in vitro experiments. Our results indicate that TIMM17A is significantly upregulated in LUAD tissues, correlating with clinical staging, lymph node metastasis, overall survival, and progression-free survival, thereby establishing it as a critical independent prognostic factor. The construction of a nomogram model further enhances our ability to predict patient outcomes. Knockdown of TIMM17A inhibited the growth of LUAD cells. The potential of TIMM17A as a biomarker and therapeutic target for LUAD presents a promising pathway for improving patient diagnosis and treatment strategies.

OMA1 clears traffic jam in TOM tunnel in mammals.

Using an engineered mitochondrial clogger, Krakowczyk et al. (https://doi.org/10.1083/jcb.202306051) identified the OMA1 protease as a critical component that eliminates import failure at the TOM translocase in mammalian cells, providing a novel quality control mechanism that is distinct from those described in yeast.

Exploring Genetic Associations of 3 Types of Risk Factors With Ischemic Stroke: An Integrated Bioinformatics Study.

BACKGROUND: Ischemic stroke (IS) is a major cause of disability and mortality worldwide. Increasing evidence suggests a strong association between blood pressure, blood glucose, circulating lipids, and IS. Nonetheless, the genetic association of these 3 risk factors with IS remains elusive. METHODS: We screened genetic instruments related to blood pressure, blood glucose, and circulating lipids and paired them with IS genome-wide association study data to conduct Mendelian randomization analysis. Positive Mendelian randomization findings were then subjected to colocalization analysis. Subsequently, we utilized the Gene Expression Omnibus data set to perform differential expression analysis, aiming to identify differentially expressed associated genes. We determined the importance scores of these differentially expressed associated genes through 4 machine learning models and constructed a nomogram based on these findings. RESULTS: The combined results of the Mendelian randomization analysis indicate that blood pressure (systolic blood pressure: odds ratio [OR], 1.02 [95% CI, 1.01-1.02]; diastolic blood pressure: OR, 1.03 [95% CI, 1.03-1.04]) and some circulating lipids (low-density lipoprotein cholesterol: OR, 1.06 [95% CI, 1.01-1.12]; apoA1: OR, 0.95 [95% CI, 0.92-0.98]; apoB: OR, 1.05 [95% CI, 1.01-1.09]; eicosapentaenoic acid: OR, 2.36 [95% CI, 1.41-3.96]) have causal relationships with the risk of IS onset. We identified 73 genes that are linked to blood pressure and circulating lipids in the context of IS, and 16 are differentially expressed associated genes. FURIN, MAN2A2, HDDC3, ALDH2, and TOMM40 were identified as feature genes for constructing the nomogram that provides a quantitative prediction of the risk of IS onset. CONCLUSIONS: This study indicates that there are causal links between blood pressure, certain circulating lipids, and the development of IS. The potential mechanisms underlying these causal relationships involve the regulation of lipid metabolism, blood pressure, DNA repair and methylation, cell apoptosis and autophagy, immune inflammation, and neuronal protection, among others.

[Genetic basis of postoperative cognitive dysfunction]. / Geneticheskie osnovy postoperatsionnoi kognitivnoi disfunktsii.

This review highlights literature data on potential genetic markers that potentially influence the development of postoperative cognitive dysfunction, such as TOMM40, APOE, TREM2, METTL3, PGC1a, HMGB1 and ERMN. The main pathogenetic mechanisms triggered by these genes and leading to the development of cognitive impairment after anesthesia are described. The paper systematizes previously published works that provide evidence of the impact of specific genetic variants on the development of postoperative cognitive dysfunction.

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria.

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC-MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

On the effect heterogeneity of established disease susceptibility loci for Alzheimer's disease across different genetic ancestries.

INTRODUCTION: Genome-wide association studies have identified numerous disease susceptibility loci (DSLs) for Alzheimer's disease (AD). However, only a limited number of studies have investigated the dependence of the genetic effect size of established DSLs on genetic ancestry. METHODS: We utilized the whole genome sequencing data from the Alzheimer's Disease Sequencing Project (ADSP) including 35,569 participants. A total of 25,459 subjects in four distinct populations (African ancestry, non-Hispanic White, admixed Hispanic, and Asian) were analyzed. RESULTS: We found that nine DSLs showed significant heterogeneity across populations. Single nucleotide polymorphism (SNP) rs2075650 in translocase of outer mitochondrial membrane 40 (TOMM40) showed the largest heterogeneity (Cochran's Q = 0.00, I2 = 90.08), followed by other SNPs in apolipoprotein C1 (APOC1) and apolipoprotein E (APOE). Two additional loci, signal-induced proliferation-associated 1 like 2 (SIPA1L2) and solute carrier 24 member 4 (SLC24A4), showed significant heterogeneity across populations. DISCUSSION: We observed substantial heterogeneity for the APOE-harboring 19q13.32 region with TOMM40/APOE/APOC1 genes. The largest risk effect was seen among African Americans, while Asians showed a surprisingly small risk effect.

New insights into the structure and dynamics of the TOM complex in mitochondria.

To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.

Identification of factors limiting the allotopic production of the Cox2 subunit of yeast cytochrome c oxidase.

Mitochondrial genes can be artificially relocalized in the nuclear genome in a process known as allotopic expression, such is the case of the mitochondrial cox2 gene, encoding subunit II of cytochrome c oxidase (CcO). In yeast, cox2 can be allotopically expressed and is able to restore respiratory growth of a cox2-null mutant if the Cox2 subunit carries the W56R substitution within the first transmembrane stretch. However, the COX2W56R strain exhibits reduced growth rates and lower steady-state CcO levels when compared to wild-type yeast. Here, we investigated the impact of overexpressing selected candidate genes predicted to enhance internalization of the allotopic Cox2W56R precursor into mitochondria. The overproduction of Cox20, Oxa1, and Pse1 facilitated Cox2W56R precursor internalization, improving the respiratory growth of the COX2W56R strain. Overproducing TIM22 components had a limited effect on Cox2W56R import, while overproducing TIM23-related components showed a negative effect. We further explored the role of the Mgr2 subunit within the TIM23 translocator in the import process by deleting and overexpressing the MGR2 gene. Our findings indicate that Mgr2 is instrumental in modulating the TIM23 translocon to correctly sort Cox2W56R. We propose a biogenesis pathway followed by the allotopically produced Cox2 subunit based on the participation of the 2 different structural/functional forms of the TIM23 translocon, TIM23MOTOR and TIM23SORT, that must follow a concerted and sequential mode of action to insert Cox2W56R into the inner mitochondrial membrane in the correct Nout-Cout topology.

Neobractatin induces pyroptosis of esophageal cancer cells by TOM20/BAX signaling pathway.

BACKGROUND: Emerging evidence suggests that pyroptosis, a form of programmed cell death, has been implicated in cancer progression. The involvement of specific proteins in pyroptosis is an area of growing interest. TOM20, an outer mitochondrial membrane protein, has recently garnered attention for its potential role in pyroptosis. Our previous study found that NBT could induce pyroptosis by ROS/JNK pathway in esophageal cancer cells. PURPOSE: This study aims to investigate whether NBT induces pyroptosis and verify whether such effects are involved in up-regulation of TOM20 in esophageal cancer cells. METHODS: The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) was used to analyze the clinical significance of GSDME in esophageal cancer. MTT assay, morphological observation and Western blot were performed to verify the roles of TOM20 and BAX in NBT-induced pyroptosis after CRISPR-Cas9-mediated knockout. Immunofluorescence was used to determine the subcellular locations of BAX and cytochrome c. MitoSOX Red was employed to assess the mitochondrial reactive oxygen species (ROS) level. KYSE450 and TOM20 knockout KYSE450-/- xenograft models were established to elucidate the mechanisms involved in NBT-induced cell death. RESULTS: In this study, NBT effectively upregulated the expression of TOM20 and facilitated the translocation of BAX to mitochondria, which promoted the release of cytochrome c from mitochondria to the cytoplasm, leading to the activation of caspase-9 and caspase-3, and finally induced pyroptosis. Knocking out TOM20 by CRISPR-Cas9 significantly inhibited the expression of BAX and the downstream BAX/caspase-3/GSDME pathway, which attenuated NBT-induced pyroptosis. The elevated mitochondrial ROS level was observed after NBT treatment. Remarkably, the inhibition of ROS by N-acetylcysteine (NAC) effectively suppressed the activation of TOM20/BAX pathway. Moreover, in vivo experiments demonstrated that NBT exhibited potent antitumor effects in both KYSE450 and TOM20 knockout KYSE450-/- xenograft models. Notably, the attenuated antitumor effects and reduced cleavage of GSDME were observed in the TOM20 knockout model. CONCLUSION: These findings reveal that NBT induces pyroptosis through ROS/TOM20/BAX/GSDME pathway, which highlight the therapeutic potential of targeting TOM20 and GSDME, providing promising prospects for the development of innovative and effective treatment approaches for esophageal cancer.

A first-in-class TIMM44 blocker inhibits bladder cancer cell growth.

Mitochondria play a multifaceted role in supporting bladder cancer progression. Translocase of inner mitochondrial membrane 44 (TIMM44) is essential for maintaining function and integrity of mitochondria. We here tested the potential effect of MB-10 (MitoBloCK-10), a first-in-class TIMM44 blocker, against bladder cancer cells. TIMM44 mRNA and protein expression is significantly elevated in both human bladder cancer tissues and cells. In both patient-derived primary bladder cancer cells and immortalized (T24) cell line, MB-10 exerted potent anti-cancer activity and inhibited cell viability, proliferation and motility. The TIMM44 blocker induced apoptosis and cell cycle arrest in bladder cancer cells, but failed to provoke cytotoxicity in primary bladder epithelial cells. MB-10 disrupted mitochondrial functions in bladder cancer cells, causing mitochondrial depolarization, oxidative stress and ATP reduction. Whereas exogenously-added ATP and the antioxidant N-Acetyl Cysteine mitigated MB-10-induced cytotoxicity of bladder cancer cells. Genetic depletion of TIMM44 through CRISPR-Cas9 method also induced robust anti-bladder cancer cell activity and MB-10 had no effect in TIMM44-depleted cancer cells. Contrarily, ectopic overexpression of TIMM44 using a lentiviral construct augmented proliferation and motility of primary bladder cancer cells. TIMM44 is important for Akt-mammalian target of rapamycin (mTOR) activation. In primary bladder cancer cells, Akt-S6K1 phosphorylation was decreased by MB-10 treatment or TIMM44 depletion, but enhanced after ectopic TIMM44 overexpression. In vivo, intraperitoneal injection of MB-10 impeded bladder cancer xenograft growth in nude mice. Oxidative stress, ATP reduction, Akt-S6K1 inhibition and apoptosis were detected in MB-10-treated xenograft tissues. Moreover, genetic depletion of TIMM44 also arrested bladder cancer xenograft growth in nude mice, leading to oxidative stress, ATP reduction and Akt-S6K1 inhibition in xenograft tissues. Together, targeting overexpressed TIMM44 by MB-10 significantly inhibits bladder cancer cell growth in vitro and in vivo.

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation.

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.

Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early-onset Parkinson's disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin (Ub) ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane forming a high-molecular-weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates Ub, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 N-terminal-C-terminal extension module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery.

The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

The E3 ligase HUWE1 increases the sensitivity of CRC to oxaliplatin through TOMM20 degradation.

Continuous administration of oxaliplatin, the most widely used first-line chemotherapy drug for colorectal cancer (CRC), eventually leads to drug resistance. Increasing the sensitivity of CRC cells to oxaliplatin is a key strategy to overcome this issue. Impairment of mitochondrial function is a pivotal mechanism determining the sensitivity of CRC to oxaliplatin. We discovered an inverse correlation between Translocase of Outer Mitochondrial Membrane 20 (TOMM20) and oxaliplatin sensitivity as well as an inverse relationship between TOMM20 and HECT, UBA, and WWE domain containing E3 ligase 1 (HUWE1) expression in CRC. For the first time, we demonstrated that HUWE1 ubiquitinates TOMM20 directly and also regulates TOMM20 degradation via the PARKIN-mediated pathway. Furthermore, we showed that overexpression of HUWE1 in CRC cells has a negative effect on mitochondrial function, including the generation of ATP and maintenance of mitochondrial membrane potential, leading to increased production of ROS and apoptosis. This effect was amplified when cells were treated simultaneously with oxaliplatin. Our study conclusively shows that TOMM20 is a novel target of HUWE1. Our findings indicate that HUWE1 plays a critical role in regulating oxaliplatin sensitivity by degrading TOMM20 and inducing mitochondrial damage in CRC.

TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.

Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.

New insights into the role of mitochondrial metabolic dysregulation and immune infiltration in septic cardiomyopathy by integrated bioinformatics analysis and experimental validation.

BACKGROUND: Septic cardiomyopathy (SCM), a common cardiovascular comorbidity of sepsis, has emerged among the leading causes of death in patients with sepsis. SCM's pathogenesis is strongly affected by mitochondrial metabolic dysregulation and immune infiltration disorder. However, the specific mechanisms and their intricate interactions in SCM remain unclear. This study employed bioinformatics analysis and drug discovery approaches to identify the regulatory molecules, distinct functions, and underlying interactions of mitochondrial metabolism and immune microenvironment, along with potential interventional strategies in SCM. METHODS: GSE79962, GSE171546, and GSE167363 datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and module genes were identified using Limma and Weighted Correlation Network Analysis (WGCNA), followed by functional enrichment analysis. Machine learning algorithms, including support vector machine-recursive feature elimination (SVM-RFE), least absolute shrinkage and selection operator (LASSO) regression, and random forest, were used to screen mitochondria-related hub genes for early diagnosis of SCM. Subsequently, a nomogram was developed based on six hub genes. The immunological landscape was evaluated by single-sample gene set enrichment analysis (ssGSEA). We also explored the expression pattern of hub genes and distribution of mitochondria/inflammation-related pathways in UMAP plots of single-cell dataset. Potential drugs were explored using the Drug Signatures Database (DSigDB). In vivo and in vitro experiments were performed to validate the pathogenetic mechanism of SCM and the therapeutic efficacy of candidate drugs. RESULTS: Six hub mitochondria-related DEGs [MitoDEGs; translocase of inner mitochondrial membrane domain-containing 1 (TIMMDC1), mitochondrial ribosomal protein S31 (MRPS31), F-box only protein 7 (FBXO7), phosphatidylglycerophosphate synthase 1 (PGS1), LYR motif containing 7 (LYRM7), and mitochondrial chaperone BCS1 (BCS1L)] were identified. The diagnostic nomogram model based on the six hub genes demonstrated high reliability and validity in both the training and validation sets. The immunological microenvironment differed between SCM and control groups. The Spearman correlation analysis revealed that hub MitoDEGs were significantly associated with the infiltration of immune cells. Upregulated hub genes showed remarkably high expression in the naive/memory B cell, CD14+ monocyte, and plasma cell subgroup, evidenced by the feature plot. The distribution of mitochondria/inflammation-related pathways varied across subgroups among control and SCM individuals. Metformin was predicted to be the most promising drug with the highest combined score. Its efficacy in restoring mitochondrial function and suppressing inflammatory responses has also been validated. CONCLUSIONS: This study presents a comprehensive mitochondrial metabolism and immune infiltration landscape in SCM, providing a potential novel direction for the pathogenesis and medical intervention of SCM.

Mitochondrial entry gate as regulatory hub.

Mitochondria import 1000-1300 different precursor proteins from the cytosol. The main mitochondrial entry gate is formed by the translocase of the outer membrane (TOM complex). Molecular coupling and modification of TOM subunits control and modulate protein import in response to cellular signaling. The TOM complex functions as regulatory hub to integrate mitochondrial protein biogenesis and quality control into the cellular proteostasis network.