Mitochondrial Inner Membrane Protein Research Articles

#489 UCP1 regulates cardiolipin metabolism and mediates mitochondrial homeostasis maintenance of ANXA1 in diabetic nephropathy

Abstract Background and Aims Diabetic nephropathy (DN) is accounted for a large proportion of chronic kidney disease (CKD) and is the leading cause of end-stage renal disease (ESRD) nowadays. The complex pathogenesis of DN has hindered the development of a single effective medicine, so it is of great significance to seek new therapeutic targets. Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for DN. Recently, we found that ANXA1 could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Method We used kidney samples from patients in our hospital. ANXA1-KO mice with local renal overexpression of UCP1 were constructed, UCP1-KO mice were constructed for STZ/HFD modeling, and db/db mice were treated with CL316243. UCP1-KO mice with renal overexpression of CRLS1 were constructed. Mice with PTECs-specific deletion of ANXA1 were constructed. Cell lines with UCP1/ANXA1/GATA3 knockdown and UCP1/ANXA1/GATA3 overexpression were constructed, and cell reversal tests were performed using Ac2-26, CL316243 and siRNA targeting the above genes. Results Here, we identified uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) through combining with thioredoxin. Moreover, specific overexpression of UCP1 in renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. Conclusion Thus, our data unraveled specific mechanism by which UCP1 mediated mitochondrial homeostasis maintenance via the ANXA1-GATA3-UCP1-ARX-CRLS1 signaling pathway. This work established a novel principle to harness the power of uncouplers for the treatment of DN.

Abstract 5879: Role of the inner mitochondrial membrane protein (Mic60) in MAPKi-resistant melanoma

Abstract Introduction: Metastatic melanoma is one of the most aggressive cancers, despite recent improvements in therapy. The rising melanoma incidence and mortality, along with its high propensity for metastasis highlights the urgency to find more effective therapeutic targets. The efficacy of targeted MAPK signaling pathway inhibitors (MAPKi) in metastatic melanoma therapy is limited by the development of resistance mechanisms that result in disease relapse. This situation necessitates alternative treatments for melanoma patients who develop resistance to targeted therapy. We have recently found out that there exists a group of human tumors that express lower levels of Mic60, a mitochondrial structural protein also known as mitofilin or inner membrane mitochondrial protein (IMMT). In a model of prostate cancer, we showed that Mic60-low tumors have lower mitochondrial fitness, but increased cell motility, invasion, and propensity to metastasize. However, a potential differential expression of Mic60 in melanoma has not been investigated and whether reduced mitochondrial fitness in Mic60-low tumors can expose new therapeutic vulnerabilities in combination with targeted therapies has not been determined. Experimental Procedures: We analyzed melanoma-specific TMA for differential expression of Mic60 in tumor vs. adjacent normal tissue by IHC. Differential mRNA expression levels were examined by single-cell RNA-Seq and quantified by bioinformatics analysis. Differential therapeutic vulnerabilities of Mic60-low melanoma cell lines to mitochondrial-targeted small molecule Hsp90 inhibitor, Gamitrinib, were assessed for cell death induction and mitochondrial dysfunction. Results: We identified subsets of melanoma patients with constitutively reduced or outright undetectable expression of Mic60, in vivo. By single-cell RNA-Seq, Mic60-low melanomas were characterized by reduced oxidative phosphorylation and decreased ATP production, slower cell cycle progression but paradoxically increased cell motility and invasion. Loss of Mic60 exposed new, actionable therapeutic vulnerabilities, and the combination of Gamitrinib plus MAPKi potently induced mitochondrial cell death in MAPKi-resistant melanoma cell lines. Conclusion: Mic60 is heterogeneously expressed and often reduced in melanoma. Although this is associated with increased cell invasion and metastatic propensity, the combination of Gamitrinib plus MAPKi restored mitochondrial cell death in therapy-resistant, Mic60-low melanoma cells. Citation Format: Jagadish C. Ghosh, Segundo Del Aguila, Haiyin Li, Jessie Villanueva, Dario C. Altieri. Role of the inner mitochondrial membrane protein (Mic60) in MAPKi-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5879.

PsAF5 functions as an essential adapter for PsPHB2-mediated mitophagy under ROS stress in Phytophthora sojae

Host-derived reactive oxygen species (ROS) are an important defense means to protect against pathogens. Although mitochondria are the main intracellular targets of ROS, how pathogens regulate mitochondrial physiology in response to oxidative stress remains elusive. Prohibitin 2 (PHB2) is an inner mitochondrial membrane (IMM) protein, recognized as a mitophagy receptor in animals and fungi. Here, we find that an ANK and FYVE domain-containing protein PsAF5, is an adapter of PsPHB2, interacting with PsATG8 under ROS stress. Unlike animal PHB2 that can recruit ATG8 directly to mitochondria, PsPHB2 in Phytophthora sojae cannot recruit PsATG8 to stressed mitochondria without PsAF5. PsAF5 deletion impairs mitophagy under ROS stress and increases the pathogen’s sensitivity to H2O2, resulting in the attenuation of P. sojae virulence. This discovery of a PsPHB2-PsATG8 adapter (PsAF5) in plant-pathogenic oomycetes reveals that mitophagy induction by IMM proteins is conserved in eukaryotes, but with differences in the details of ATG8 recruitment.

Therapeutic targets for endometriosis: Genome-wide Mendelian randomization and colocalization analyses

BackgroundEndometriosis (EM) greatly affects women's reproductive health, identifying new drug targets for EM is urgently needed. This study utilizes comprehensive genome-wide Mendelian randomization (MR) and colocalization analyses, using genomic data, to identify potential therapeutic approaches for EM. MethodsGenome-wide cis-expression quantitative trait loci (cis-eQTL) data were obtained from GTEx V8, which included 838 participants across 49 tissues or cells, and the eQTLGen consortium, which included 31,684 participants. Genome-wide association analysis (GWAS) data for EM were sourced from the FinnGen study, which consisted of 8,288 cases and 68,969 controls, as well as the UK Biobank study, which included 1,496 cases and 359,698 controls. This study utilized MR analysis to assess the correlation between genes and the risk of EM. Subsequently, colocalization analysis was conducted to investigate potential shared causal variants between the identified genes and EM. ResultsAfter conducting MR and colocalization analyses, we identified a total of 13 genes that showed significant evidence of colocalization. These genes are considered promising therapeutic candidates for treating EM. Among them, inner membrane mitochondrial protein (IMMT), src kinase associated phosphoprotein 1 (SKAP1), lysine methyltransferase 5A (KMT5A), KLF transcription factor 12 (KLF12), GRB10 interacting GYF protein 1 (GIGYF1), Wnt family member 7A (WNT7A), Sad1 and UNC84 domain containing 1 (SUN1), and poly (ADP-ribose) polymerase family member 3 (PARP3) were found to have positive associations with the risk of EM. On the other hand, progestin and adipoQ receptor family member 8 (PAQR8), adaptor related protein complex 3 subunit mu 1 (AP3M1), surfeit 6 (SURF6), TUB bipartite transcription factor (TUB), and DNA polymerase delta interacting protein 2 (POLDIP2) were found to have inverse relationships with the risk of EM. ConclusionsThrough genome-wide MR studies, a comprehensive set of genes associated with EM has been identified. Among them, IMMT, PAQR8, SKAP1, KMT5A, AP3M1, SURF6, KLF12, GIGYF1, TUB, WNT7A, SUN1, POLDIP2, and PARP3 show potential as therapeutic targets for EM treatment. Nonetheless, it is crucial to conduct further rigorous investigations to validate these prospects.

Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5.

AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.

IMMT promotes hepatocellular carcinoma formation via PI3K/AKT/mTOR pathway

Abstract Objectives Hepatocellular carcinoma (HCC) remains a global challenge. Finding new specific targets has significant clinical value for the treatment of hepatocellular carcinoma. In this study, we discovered a new biomarker targeting HCC. At present, the role of Inner Membrane Mitochondrial Protein (IMMT) in various malignant tumors is receiving increasing attention, but its molecular mechanism of action in the malignant process of HCC has not been fully elucidated. This study aims to investigate the key regulatory mechanisms of IMMT in the formation of HCC. Methods Using TCGA data and clinical HCC samples, we first studied the expression of IMMT in HCC tissues and its correlation with malignant prognosis of patients. The regulatory effect of IMMT on HCC was studied by lentivirus infection. In vitro, the effects of IMMT on the proliferation, migration and apoptosis of cells were investigated by CCK8, colony formation assay, transwell and flow cytometry. Consistently, in vivo experiments, the regulation of tumor growth by IMMT was studied by constructing subcutaneous transplanted tumor and liver carcinoma in situ. In terms of mechanism, we predicted and verified the downstream genes of IMMT with the help of string database. Results We found that IMMT was significantly up-regulated in HCC tissues and was significantly positively correlated with poor prognosis of patients. Functionally, we demonstrated that IMMT knockdown significantly inhibited HCC cell proliferation, migration and promoted cell apoptosis in vitro. Similarly, the knockdown of IMMT also significantly weakened the progression of tumors in vivo. In terms of mechanism, we demonstrate for the first time that IMMT can regulate the progression of HCC by influencing the activation of PI3K-AKT-mTOR pathway. Conclusions Collectively, our research findings elucidate the hitherto unexplored important role of the IMMT/PI3K/AKT/mTOR axis in the formation of HCC, and provide a new biomarker for clinical diagnosis and treatment of HCC.

Unraveling the mechanisms of cardiolipin function: The role of oxidative polymerization of unsaturated acyl chains

Cardiolipin is a unique phospholipid of the inner mitochondrial membrane (IMM) as well as in bacteria. It performs several vital functions such as resisting osmotic rupture and stabilizing the supramolecular structure of large membrane proteins, like ATP synthases and respirasomes. The process of cardiolipin biosynthesis results in the production of immature cardiolipin. A subsequent step is required for its maturation when its acyl groups are replaced with unsaturated acyl chains, primarily linoleic acid. Linoleic acid is the major fatty acid of cardiolipin across all organs and tissues, except for the brain. Linoleic acid is not synthesized by mammalian cells. It has the unique ability to undergo oxidative polymerization at a moderately accelerated rate compared to other unsaturated fatty acids. This property can enable cardiolipin to form covalently bonded net-like structures essential for maintaining the complex geometry of the IMM and gluing the quaternary structure of large IMM protein complexes. Unlike triglycerides, phospholipids possess only two covalently linked acyl chains, which constrain their capacity to develop robust and complicated structures through oxidative polymerization of unsaturated acyl chains. Cardiolipin, on the other hand, has four fatty acids at its disposal to form covalently bonded polymer structures. Despite its significance, the oxidative polymerization of cardiolipin has been overlooked due to the negative perception surrounding biological oxidation and methodological difficulties. Here, we discuss an intriguing hypothesis that oxidative polymerization of cardiolipin is essential for the structure and function of cardiolipin in the IMM in physiological conditions. In addition, we highlight current challenges associated with the identification and characterization of oxidative polymerization of cardiolipin in vivo. Altogether, the study provides a better understanding of the structural and functional role of cardiolipin in mitochondria.

UCP2 45bp Ins/Del Gene Polymorphism in Association with Overweight and Obesity: A Meta-Analysis

Uncoupling Protein (UCP) is a mitochondrial inner-membrane protein that is involved in energy homeostasis. In human tissues, UCP2 is widely expressed, acts as an uncoupling agent for oxidative phosphorylation, and is involved in the regulation of metabolism and ATP production.

Supervised Learning and Multi-Omics Integration Reveals Clinical Significance of Inner Membrane Mitochondrial Protein (IMMT) in Prognostic Prediction, Tumor Immune Microenvironment and Precision Medicine for Kidney Renal Clear Cell Carcinoma.

Kidney renal clear cell carcinoma (KIRC) accounts for approximately 75% of all renal cancers. The prognosis for patients with metastatic KIRC is poor, with less than 10% surviving five years after diagnosis. Inner membrane mitochondrial protein (IMMT) plays a crucial role in shaping the inner mitochondrial membrane (IMM), regulation of metabolism and innate immunity. However, the clinical relevance of IMMT in KIRC is not yet fully understood, and its role in shaping the tumor immune microenvironment (TIME) remains unclear. This study aimed to investigate the clinical significance of IMMT in KIRC using a combination of supervised learning and multi-omics integration. The supervised learning principle was applied to analyze a TCGA dataset, which was downloaded and split into training and test datasets. The training dataset was used to train the prediction model, while the test and the entire TCGA dataset were used to evaluate its performance. Based on the risk score, the cutoff between the low and high IMMT group was set at median value. A Kaplan-Meier curve, receiver operating characteristic (ROC) curve, principal component analysis (PCA) and Spearman's correlation were conducted to evaluate the prediction ability of the model. Gene Set Enrichment Analysis (GSEA) was used to investigate the critical biological pathways. Immunogenicity, immunological landscape and single-cell analysis were performed to examine the TIME. Databases including Gene Expression Omnibus (GEO), Human Protein Atlas (HPA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) were employed for inter-database verification. Pharmacogenetic prediction was analyzed via single-guide RNA (sgRNA)-based drug sensitivity screening using Q-omics v.1.30. Low expressions of IMMT in tumor predicted dismal prognosis in KIRC patients and correlated with KIRC progression. GSEA revealed that low expressions of IMMT were implicated in mitochondrial inhibition and angiogenetic activation. In addition, low IMMT expressions had associations with reduced immunogenicity and an immunosuppressive TIME. Inter-database verification corroborated the correlation between low IMMT expressions, KIRC tumors and the immunosuppressive TIME. Pharmacogenetic prediction identified lestaurtinib as a potent drug for KIRC in the context of low IMMT expressions. This study highlights the potential of IMMT as a novel biomarker, prognostic predictor and pharmacogenetic predictor to inform the development of more personalized and effective cancer treatments. Additionally, it provides important insights into the role of IMMT in the mechanism underlying mitochondrial activity and angiogenesis development in KIRC, which suggests IMMT as a promising target for the development of new therapies.

UCP2 45bp Ins/Del gene polymorphism in association with overweight and obesity: A meta-analysis

Uncoupling protein (UCP) is a mitochondrial inner-membrane protein that is involved in energy homeostasis. In human tissues

Multi-omics and experimental analysis unveil theragnostic value and immunological roles of inner membrane mitochondrial protein (IMMT) in breast cancer

BackgroundThe inner membrane mitochondrial protein (IMMT) is a central unit of the mitochondrial contact site and cristae organizing system (MICOS). While researchers continue to demonstrate the physiological function of IMMT in regulating mitochondrial dynamics and preserving mitochondrial structural integrity, the roles of IMMT in clinicopathology, the tumor immune microenvironment (TIME), and precision oncology in breast cancer (BC) remain unclear.MethodsMulti-omics analysis was used here to evaluate the diagnostic and prognostic value of IMMT. Web applications aimed at analyzing the whole tumor tissue, single cells, and spatial transcriptomics were used to examine the relationship of IMMT with TIME. Gene set enrichment analysis (GSEA) was employed to determine the primary biological impact of IMMT. Experimental verification using siRNA knockdown and clinical specimens of BC patients confirmed the mechanisms behind IMMT on BC cells and the clinical significance, respectively. Potent drugs were identified by accessing the data repositories of CRISPR-based drug screenings.ResultsHigh IMMT expression served as an independent diagnostic biomarker, correlated with advanced clinical status, and indicated a poor relapse-free survival (RFS) rate for patients with BC. Although, the contents of Th1, Th2, MSC, macrophages, basophil, CD4 + T cell and B cell, and TMB levels counteracted the prognostic significance. Single-cell level and whole-tissue level analyses revealed that high IMMT was associated with an immunosuppressive TIME. GSEA identified IMMT perturbation as involved in cell cycle progression and mitochondrial antioxidant defenses. Experimental knockdown of IMMT impeded the migration and viability of BC cells, arrested the cell cycle, disturbed mitochondrial function, and increased the ROS level and lipid peroxidation. The clinical values of IMMT were amenable to ethnic Chinese BC patients, and can be extrapolated to some other cancer types. Furthermore, we discovered that pyridostatin acted as a potent drug candidate in BC cells harboring an elevated IMMT expression.ConclusionThis study combined a multi-omics survey with experimental verification to reveal the novel clinical significance of IMMT in BC, demonstrating its role in TIME, cancer cell growth and mitochondrial fitness, and identified pyridostatin as a promising drug candidate for the development of precision medicine.

The leucine zipper EF-hand containing transmembrane protein-1 EF-handis an atypical tripartite calcium, temperature, and pH sensor.

Mitochondrial calcium (mtCa2+) is integral to ATP production; however, excessive Ca2+ leads to cell death. Both these processes are crucial to cellular function, and thus mtCa2+ homeostasis is tightly regulated. Leucine zipper EF-hand containing transmembrane protein-1 (LETM1) is an essential inner-mitochondrial membrane protein that regulates mtCa2+ through exchange of Ca2+ for protons (H+). LETM1 haploinsufficiency is associated with Wolf-Hirschhorn syndrome, characterized by microcephaly, growth retardation, and epileptic seizures, indicating a fundamental function for LETM1 in humans. LETM1 contains a conserved Ca2+ binding EF-hand motif (EF1). Deletion of EF1 abrogates LETM1 mediated mtCa2+ flux, but the precise mechanism by which EF1 regulates exchanger function remains unknown. To begin understanding the role of EF1 in LETM1 function, we biophysical and structurally characterized isolated EF1. Our data show that EF1 exhibits augmented α-helical secondary structure, solvent exposed hydrophobicity and oligomerization in the presence of Ca2+ due to a weak but specific, Ca2+-binding affinity. The low affinity is, in part, due to a terminal Asp within the binding loop, where mutation to Glu increases the affinity ∼4-fold. EF1 is structurally sensitive to pH changes within the physiological range for mitochondria, and remarkably, unfolds at high and low temperatures. Using solution NMR spectroscopy, we reveal that EF1 adopts an atypical EF-hand arrangement by pairing with an upstream loop-helix motif. Collectively, our data suggest that EF1 functions as a divergent multipartite environmental sensor within the EF-hand protein family by adopting an inimitable three-helix EF-hand domain arrangement in LETM1.

PRKN regulates inner mitochondrial membrane PHB2 during mitophagy

PINK1 (PTEN induced kinase 1) and PRKN-mediated mitophagy is an important mitochondrial quality control pathway which selectively degrades damaged mitochondria and is tightly associated with neurodegenerative diseases, including Parkinson disease and amyotrophic lateral sclerosis. The current model of PINK1-PRKN-mediated mitophagy is that PRKN ubiquitinates multiple outer mitochondrial membrane (OMM) proteins, and then the ubiquitin chains on the OMM interact with autophagy receptors which bind Atg8-family protein labeled phagophores. However, little work has been focused on the PRKN-mediated ubiquitination of inner mitochondrial membrane (IMM) proteins during mitophagy. Our recent work revealed that PRKN binds and ubiquitinates PHB2 (prohibitin 2), an essential IMM protein which was previously recognized as a mitophagy receptor, after the OMM is ruptured by proteasomal degradation. Using biochemical and microscopy approaches, we found that mutations of PRKN-targeted ubiquitination sites on PHB2 decrease the recognition of damaged mitochondria by the phagophore and the clearance of damaged mitochondria. In conclusion, our findings revealed a critical role for PRKN-PHB2 interaction in mitochondrial quality control by regulating IMM-associated recognition of mitochondria by the autophagy machinery.

The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy

The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.

Mitochondrial fitness and cancer risk.

Changes in metabolism are a hallmark of cancer, but molecular signatures of altered bioenergetics to aid in clinical decision-making do not currently exist. We recently identified a group of human tumors with constitutively reduced expression of the mitochondrial structural protein, Mic60, also called mitofilin or inner membrane mitochondrial protein (IMMT). These Mic60-low tumors exhibit severe loss of mitochondrial fitness, paradoxically accompanied by increased metastatic propensity and upregulation of a unique transcriptome of Interferon (IFN) signaling and Senescence-Associated Secretory Phenotype (SASP). Here, we show that an optimized, 11-gene signature of Mic60-low tumors is differentially expressed in multiple malignancies, compared to normal tissues, and correlates with poor patient outcome. When analyzed in three independent patient cohorts of pancreatic ductal adenocarcinoma (PDAC), the Mic60-low gene signature was associated with aggressive disease variants, local inflammation, FOLFIRINOX failure and shortened survival, independently of age, gender, or stage. Therefore, the 11-gene Mic60-low signature may provide an easily accessible molecular tool to stratify patient risk in PDAC and potentially other malignancies.

Mitochondrial retrograde signaling through UCP1-mediated inhibition of the plant oxygen-sensing pathway

SummaryMitochondrial retrograde signaling is an important component of intracellular stress signaling in eukaryotes. UNCOUPLING PROTEIN (UCP)1 is an abundant plant inner-mitochondrial membrane protein with multiple functions including uncoupled respiration and amino-acid transport1,2 that influences broad abiotic stress responses. Although the mechanism(s) through which this retrograde function acts is unknown, overexpression of UCP1 activates expression of hypoxia (low oxygen)-associated nuclear genes.3,4 Here we show in Arabidopsis thaliana that UCP1 influences nuclear gene expression and physiological response by inhibiting the cytoplasmic PLANT CYSTEINE OXIDASE (PCO) branch of the PROTEOLYSIS (PRT)6 N-degron pathway, a major mechanism of oxygen and nitric oxide (NO) sensing.5 Overexpression of UCP1 (UCP1ox) resulted in the stabilization of an artificial PCO N-degron pathway substrate, and stability of this reporter protein was influenced by pharmacological interventions that control UCP1 activity. Hypoxia and salt-tolerant phenotypes observed in UCP1ox lines resembled those observed for the PRT6 N-recognin E3 ligase mutant prt6-1. Genetic analysis showed that UCP1 regulation of hypoxia responses required the activity of PCO N-degron pathway ETHYLENE RESPONSE FACTOR (ERF)VII substrates. Transcript expression analysis indicated that UCP1 regulation of hypoxia-related gene expression is a normal component of seedling development. Our results show that mitochondrial retrograde signaling represses the PCO N-degron pathway, enhancing substrate function, thus facilitating downstream stress responses. This work reveals a novel mechanism through which mitochondrial retrograde signaling influences nuclear response to hypoxia by inhibition of an ancient cytoplasmic pathway of eukaryotic oxygen sensing.

Identification of Novel Mitochondrial Pyruvate Carrier Inhibitors by Homology Modeling and Pharmacophore-Based Virtual Screening.

The mitochondrial pyruvate carrier (MPC) is an inner-mitochondrial membrane protein complex that has emerged as a drug target for treating a variety of human conditions. A heterodimer of two proteins, MPC1 and MPC2, comprises the functional MPC complex in higher organisms; however, the structure of this complex, including the critical residues that mediate binding of pyruvate and inhibitors, remain to be determined. Using homology modeling, we identified a putative substrate-binding cavity in the MPC dimer. Three amino acid residues (Phe66 (MPC1) and Asn100 and Lys49 (MPC2)) were validated by mutagenesis experiments to be important for substrate and inhibitor binding. Using this information, we developed a pharmacophore model and then performed a virtual screen of a chemical library. We identified five new non-indole MPC inhibitors, four with IC50 values in the nanomolar range that were up to 7-fold more potent than the canonical inhibitor UK-5099. These novel compounds possess drug-like properties and complied with Lipinski’s Rule of Five. They are predicted to have good aqueous solubility, oral bioavailability, and metabolic stability. Collectively, these studies provide important information about the structure-function relationships of the MPC complex and for future drug discovery efforts targeting the MPC.

Loss of mpv17 affected early embryonic development via mitochondria dysfunction in zebrafish

MVP17 encodes a mitochondrial inner-membrane protein, and mutation of human MVP17 can cause mitochondria DNA depletion syndrome (MDDS). However, the underlying function of mpv17 is still elusive. Here, we developed a new mutant with mpv17 knockout by using the CRISPR/Cas9 system. The mpv17−/− zebrafish showed developmental defects in muscles, liver, and energy supply. The mpv17−/− larvae hardly survived beyond a month, and they showed abnormal growth during the development stage. Abnormal swimming ability was also found in the mpv17−/− zebrafish. The transmission electron microscope (TEM) observation indicated that the mpv17−/− zebrafish underwent severe mitochondria dysfunction and the disorder of mitochondrial cristae. As an energy producer, the defects of mitochondria significantly reduced ATP content in mpv17−/− zebrafish, compared to wild-type zebrafish. We hypothesized that the disorder of mitochondria cristae was contributed to the dysfunction of muscle and liver in the mpv17−/− zebrafish. Moreover, the content of major energy depot triglycerides (TAG) was decreased dramatically. Interestingly, after rescued with normal exogenous mitochondria by microinjection, the genes involved in the TAG metabolism pathway were recovered to a normal level. Taken together, this is the first report of developmental defects in muscles, liver, and energy supply via mitochondria dysfunction, and reveals the functional mechanism of mpv17 in zebrafish.

Dual function of Bim is switched by ANT2 (SLC25A5) to balance apoptotic and effective signals in CD8+ T cells

Abstract The immune responses to anti-PD-1 therapy in cancer patients are not completely defined due to lack of reliable biomarkers that identifies responsive and functional T cells. Bim, a molecule of the Bcl-2 family, was recently identified as a downstream signaling molecule of PD-1 pathway in CD8+ T cells and has both pro-apoptotic and effective roles in CD8 T cells. Our clinical data shown that high level of Bim in CD8+ T cells (before checkpoint blockade) was predictive of response in metastatic melanoma whereas in colorectal cancer, low level of Bim was associated with treatment response. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2, an inner membrane protein of mitochondria) as a protein interacting with Bim. Accordingly, we found CD8+ T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratio, and high ANT2/Bim ratio fall in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival of ANT2, our results indicate that ANT2 may function to balance the pro-apoptotic and effective role of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interaction in CD8+ T cells may provide a novel marker to identify resilient CD8+ T cells responsive to anti-PD-1 therapy, and predict clinical response to the therapy.

Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination.

Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell ("mosaic distribution"). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high-cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.