Mitochondrial Elongation Research Articles

Mitochondrial E3 ubiquitin ligase MARCH5 is required for mouse oocyte meiotic maturation†.

As the most abundant organelles in oocytes, mitochondria play an important role in maintaining oocyte quality. Here, we report that March5, encoding a mitochondrial ubiquitin ligase that promotes mitochondrial elongation, plays a critical role in mouse oocyte meiotic maturation via regulating mitochondrial function. The subcellular localization of MARCH5 was similar to the mitochondrial distribution during mouse oocyte meiotic progression. Knockdown of March5 caused decreased ratios of the first polar body extrusion. March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased reactive oxygen species, decreased ATP content as well as decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore-microtubule detachment. Further study showed that the continuous activation of the spindle assembly checkpoint and the failure of Cyclin B1 degradation caused MI arrest and first polar body (PB1) extrusion failure in March5 knockdown oocytes. Taken together, our results demonstrated that March5 plays an essential role in mouse oocyte meiotic maturation, possibly via regulation of mitochondrial function and/or ubiquitination of microtubule dynamics- or cell cycle-regulating proteins.

FUNDC1 protects against doxorubicin-induced cardiomyocyte PANoptosis through stabilizing mtDNA via interaction with TUFM

Doxorubicin (DOX) is an effective anthracycline chemotherapeutic anticancer drug with its life-threatening cardiotoxicity severely limiting its clinical application. Mitochondrial damage-induced cardiomyocyte death is considered an essential cue for DOX cardiotoxicity. FUN14 domain containing 1 (FUNDC1) is a mitochondrial membrane protein participating in the regulation of mitochondrial integrity in multiple diseases although its role in DOX cardiomyopathy remains elusive. Here, we examined whether PANoptosis, a novel type of programmed cell death closely associated with mitochondrial damage, was involved in DOX-induced heart injury, and FUNDC1-mediated regulation of cardiomyocyte PANoptosis, if any. FUNDC1 was downregulated in heart tissues in patients with dilated cardiomyopathy (DCM) and DOX-challenged mice. FUNDC1 deficiency aggravated DOX-induced cardiac dysfunction, mitochondrial injury, and cardiomyocyte PANoptosis. Further examination revealed that FUNDC1 countered cytoplasmic release of mitochondrial DNA (mtDNA) and activation of PANoptosome through interaction with mitochondrial Tu translation elongation factor (TUFM), a key factor in the translational expression and repair of mitochondrial DNA, via its 96–133 amino acid domain. TUFM intervention reversed FUNDC1-elicited protection against DOX-induced mtDNA cytosolic release and cardiomyocyte PANoptosis. Our findings shed light toward a beneficial role of FUNDC1 in DOX cardiotoxicity and cardiomyocyte PANoptosis, thus offering therapeutic promises in DOX-induced cardiotoxicity.

MRG15 aggravates non-alcoholic steatohepatitis progression by regulating the mitochondrial proteolytic degradation of TUFM.

How hepatic steatosis progresses to non-alcoholic steatohepatitis (NASH) is complicated and remains unclear. The mortality factor 4-like protein 1 (MORF4L1, also called MRG15) was previously identified as a master nuclear chromatin remodeler in the rhythmic regulation of lipid synthesis gene expression in the liver. Whether it also contributes to the progression from liver steatosis to NASH is unclear. We adopted 2 different murine NASH models, liver biopsies from patients with NASH, and primary mouse and human hepatocyte cultures for functional examination of MRG15 in NASH progression. Immunoprecipitation-mass spectrometry was applied to identify protein partners of MRG15, and CRISPR targeting was used for gene depletion in liver cells invivo. The MRG15 level is increased in the livers of humans and mice with NASH. The inflammatory cytokines in NASH livers stabilize MRG15 by increasing its acetylation. Considerable amounts of MRG15 associate with the outer mitochondrial membrane, where it interacts with and deacetylates the mitochondrial Tu translation elongation factor (TUFM). Deacetylated TUFM, especially at the K82 and K91 sites, is subjected to accelerated degradation by the mitochondrial ClpXP protease system. Reduced liver TUFM consequently results in impaired mitophagy, increased oxidative stress and activation of the NLRP3 inflammasome pathway. Blocking MRG15 expression protects the liver from NASH progression by increasing the stability of liver TUFM. Liver samples from patients with NASH also display a clear reduction in TUFM level, which correlates with increased MRG15 expression. Collectively, these findings uncover a mitochondrial MRG15-TUFM regulatory pathway that contributes significantly to progression from simple steatosis to NASH, and which could potentially be targeted to treat NASH. The incidence of non-alcoholic fatty liver diseaseand its progressive form non-alcoholic steatohepatitis (NASH) is increasing, posing a significant global health challenge. Herein,we have uncovered the importance of the MRG15-TUFM pathway in NASH development. This pathway is active in the mitochondria (energy powerhouse of the cell) and could be targeted for the treatment of NASH.

Beneficial effects of bempedoic acid treatment in polycystic kidney disease cells and mice.

ADPKD has few therapeutic options. Tolvaptan slows disease but has side effects limiting its tolerability. Bempedoic acid (BA), an ATP citrate-lyase (ACLY) inhibitor FDA-approved for hypercholesterolemia, catalyzes a key step in fatty acid/sterol synthesis important for cell proliferation. BA is activated by very long-chain acyl-CoA synthetase (FATP2) expressed primarily in kidney and liver. BA also activates AMPK. We hypothesized that BA could be a novel ADPKD therapy by inhibiting cyst growth, proliferation, injury, and metabolic dysregulation via ACLY inhibition and AMPK activation. Pkd1-null kidney cell lines derived from mouse proximal tubule (PT) and collecting duct (IMCD) were grown in 2D or 3D Matrigel cultures and treated ± BA, ± SB-204990 (another ACLY inhibitor) or with Acly shRNA before cyst analysis, immunoblotting or mitochondrial assays using MitoSox and MitoTracker staining. Pkd1 fl/fl ; Pax8-rtTA; Tet-O-Cre C57BL/6J mice were induced with doxycycline injection on postnatal days 10 and 11 (P10-P11) and then treated ± BA (30mg/kg/d) ± tolvaptan (30-100mg/kg/d) by gavage from P12-21. Disease severity was determined by % total-kidney-weight-to-bodyweight (%TKW/BW) and BUN levels at euthanasia (P22). Kidney and liver homogenates were immunoblotted for expression of key biomarkers. ACLY expression and activity were upregulated in Pkd1-null PT and IMCD-derived cells vs. controls. Relative to controls, both BA and SB-204990 inhibited cystic growth in Pkd1-null kidney cells, as did Acly knockdown. BA inhibited mitochondrial superoxide production and promoted mitochondrial elongation, suggesting improved mitochondrial function. In ADPKD mice, BA reduced %TKW/BW and BUN to a similar extent as tolvaptan vs. untreated controls. Addition of BA to tolvaptan caused a further reduction in %TKW/BW and BUN vs. tolvaptan alone. BA generally reduced ACLY and stimulated AMPK activity in kidneys and livers vs. controls. BA also inhibited mTOR and ERK signaling and reduced kidney injury markers. In liver, BA treatment, both alone and together with tolvaptan, increased mitochondrial biogenesis while inhibiting apoptosis. We conclude that BA and ACLY inhibition inhibited cyst growth in vitro, and BA decreased ADPKD severity in vivo. Combining BA with tolvaptan further improved various ADPKD disease parameters. Repurposing BA may be a promising new ADPKD therapy, having beneficial effects alone and along with tolvaptan.

Gene Editing and Multi-Omics Reveal Novel Metabolic Vulnerabilities in t(4;14)-Associated Multiple Myeloma

Background: Chromosomal translocation t(4;14) is a recurrent genetic lesion in multiple myeloma (MM) which drives aberrant overexpression of the histone methyltransferase NSD2 and is associated with a more guarded prognosis. Despite its oncogenic outcomes, NSD2 overexpression in MM cells could simultaneously confer cellular vulnerabilities that can be therapeutically exploited. Aim: To define potential therapeutic susceptibilities driven by NSD2 overexpression in t(4;14) MM. Methods: We performed genome-wide knockout screening in isogenic NSD2 high and low KMS11 cells using the Brunello CRISPR library, followed by validation of key target genes in several different t(4;14) MM cell lines. Results: These screens revealed increased reliance of NSD2-overexpressing cells on multiple cellular processes including mitochondrial respiration, transcriptional elongation and proteasomal degradation. One of the top preferential dependencies in NSD2-high MM cells is the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). AK2 catalyzes the reversible interconversion of ADP to ATP and AMP. Cancer Dependency Map (depmap) analysis indicated that AK2 loss is detrimental to most MM cell lines but it is not a dependency in most solid tumor cells. Reliance on AK2 was modest in t(4;14) MM cells engineered to express low levels of NSD2 but the detrimental effect of AK2 depletion by shRNA or CRISPR was exacerbated upon enforced overexpression of NSD2. Gene expression studies showed that AK2 suppression leads to induction of genes involved in oxidative stress response. Metabolomic profiling of NSD2 high and low isogenic MM cells demonstrated striking reduction of NAD+ and elevation of nicotinamide (NAM) levels in NSD2 overexpressing cells, which correlates with a 10-fold induction of expression of the NAD+ consuming enzyme CD38 by NSD2. NAD+ and its metabolite NADP+ are essential co-factors of multiple antioxidant enzymes and its depletion can exacerbate oxidative damage. Therefore, NAD+ depletion likely accounts for the increased susceptibility of NSD2 high MM cells to AK2 loss. In vitro cell proliferation/viability assays demonstrated increased sensitivity of NSD2 high MM cells to PI exposure suggesting that NSD2 overexpression drives a higher load of unfolded proteins, correlating with loss of NAD+ and redox system activity. AK2 suppression in MM cells also impairs activation of the adaptive unfolded protein response through the IRE1a-XBP1 axis leading to decreased protein folding capacity. The combination of high unfolded protein load and poor ATP shuttling to the endoplasmic reticulum (ER) secondary to AK2 loss may represent an additional mechanism of selective lethality. Furthermore, AK2 deficient MM cells displayed increased sensitivity to PIs. Transcriptomic analyses in NSD2 high and low MM cells revealed that NSD2 overexpression, besides driving an oncogenic transcriptional program, alters the expression of several genes involved in nucleotide metabolism. Perturbed nucleotide metabolism was further documented by untargeted metabolite profiling which showed a notable depletion of several pyrimidine metabolites when NSD2 is overexpressed. Intriguingly, induced reduction of pyrimidine synthesis by treatment with DHODH inhibitors appeared to be more detrimental in NSD2 overexpressing MM cells. Conclusions: NSD2 overexpression in MM cells alters cellular metabolism and confers higher susceptibility to oxidative stress and unfolded protein loads. By inducing the expression of NAD+ consuming enzymes, NSD2 overexpression results in NAD+ depletion which likely underlies the described vulnerabilities. Loss of the mitochondrial adenine nucleotide regulator AK2 is more detrimental to NSD2 overexpressing MM cells as it diminishes efficient ATP utilization in the ER perturbing the ability of MM cells to handle high loads of unfolded immunoglobulins and mitigate oxidative stress. Furthermore, integrated analyses of the different omics datasets highlighted other NSD2-driven liabilities, including mitochondrial respiration, transcriptional elongation and nucleotide metabolism, that can be targeted to improve prognosis of MM.

ACC1 Inhibition Enhances Treg Gvhd Treatment Efficacy through Reciprocal Fatty-Acid Synthesis and Oxidative Phosphorylation Regulation

Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not eliminate grades II-IV graft-vs-host disease (GVHD). We hypothesized that increasing fatty acid oxidation (FAO) would augment Treg potency, and ultimately enhance Treg-mediated GVHD reduction. Acetyl-CoA carboxylase-1 (ACC1) negatively regulates FAO by augmenting fatty acid synthesis (FAS), which results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 genetic deletion or inhibition increases T-cell FAO and skews naive CD4 T-cell differentiation from Th17 to Treg. However, the role of ACC1 in thymic Treg function and metabolic fitness is unknown. Here we show that a 2-hour treatment of murine Treg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, enhances Treg suppressive function. As compared to control Tregs, ND630 treatment also increased expression of suppressive molecules Lag3, TIGIT, and IL-10. Treatment of Tregs with either of two other ACC1 inhibitors (TOFA, sorafin A) resulted in similar augmentations in Treg function, disfavoring the likelihood of off-target effects. Additionally, ND630 treatment increased Treg oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. Similar to murine Treg, ND630 treatment of in vitro expanded human Treg enhanced their suppressive function and OCR/OXPHOS. IACS, an OXPHOS inhibitor, abrogated the positive ND630 effect on in vitro Treg suppressive function and OXPHOS. Given the augmentation in OXPHOS with ND630 treatment, we further evaluated ND630 effects on Treg mitochondria. Compared to controls, ND630 treatment increased mitochondrial mass and membrane potential, essential for energy storage during OXPHOS, as well as mitochondrial polarization, which is required for full Treg functionality. Microscopy analysis revealed enhanced mitochondrial fusion and elongation after ND630 or TOFA treatment (FigA). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition on mitochondrial fitness. Since ACC1-driven FAS ultimately inhibits FAO, we next evaluated contributors to FAO. Expression of CPT1a, a critical enzyme for mitochondrial FAO, fatty acid binding protein 5, a key shuttle of fatty acids (FA), and exogenous FA uptake were all enhanced after ND630 treatment. Using an unbiased metabolomic screen, a metabolic signature was observed that was consistent with enhanced FAO. Together, these data suggest that ND630 increases mitochondrial fitness, and augments both FA uptake and FAO. To identify additional ND603-induced intracellular effects in Treg, activated murine Treg were subjected to RNAseq. Differential gene expression analysis indicated that 82 genes differed between DMSO and ND630 treated Treg. These included increased expression of genes associated with OXPHOS, as well as mTOR, consistent with high OXPHOS and suppressor cell function. Flow cytometry analysis of phosphorylated proteins showed reduced pFOXO3a and pAKT, both mTORC2 targets, but increased mTORC1 downstream phospho-proteins. An imbalance of mTORC1/2 is consistent to our previous studies reflective of higher Treg function and metabolic fitness (McDonald-Hyman C, JCI 2018). To exclude potential bystander effects of ND630 on Treg function, we utilized murine Treg with genetic deletion of ACC1 (ACC1KO). Supporting our studies with chemical ACC1 inhibitors, ACC1KO Treg were functionally and metabolically superior to wild-type Treg. Furthermore, as our goal is to use ACC1 inhibited or deleted Treg in treatment of GVHD, we tested ACC1KO Treg in treating established bronchiolitis obliterans (BOS) in a murine model of chronic GVHD. ACC1KO Treg were more potent than wild-type Treg in reversing BOS pathology (FigB), a finding consistent in each of 3 replicate transplants. In summary, ACC1 inhibition or deletion amplifies murine (and human) Treg potency resulting in superior metabolic fitness, and greater efficacy in treating established BOS in murine chronic GVHD. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing ACC1 inhibited Treg infusion to improve GVHD therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology.

Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane-localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.

Effects of short-term waterfall forest aerosol air exposure on rat lung proteomics

Chronic exposure to airborne microparticles has been shown to increase the incidence of several chronic diseases. Previous studies have found that waterfall forest aerosols contribute to a diminished immune stress response in patients with asthma. However, the specific effects of short-term waterfall forest aerosol exposure on lung proteins have not been fully elucidated. This study used liquid chromatography-tandem mass spectrometry (LC-MS) to analyze changes in protein expression in the lungs of rats exposed to short-term waterfall forest aerosol environments. Specific protein markers were identified using bioconductivity analysis screening and validated using immunohistochemistry. Waterfall forest aerosol environment exposure on day 5 downregulated the expression of the classical inflammatory pathway nuclear factor κB (NF-κB) signaling pathway. As the waterfall forest aerosol environment increased due to the duration of exposure, it was involved in oxidative phosphorylation and then hormone signaling in lung cells from the very beginning. In contrast, at day 15 of exposure, there is an effect on the regulation of the immune-related high-affinity IgE receptor pathway. In addition, iron-sulfur Rieske protein (Uqcrfs1), mitochondrial Tu translation elongation factor (Tufm) and ribosomal protein L4 (Rpl4) were identified as possible bioindicators for the evaluation of air quality. These results provide a comprehensive proteomic analysis that supports the positive contribution of a good air quality environment to lung health.

Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism.

Phosphoglycerate dehydrogenase (PHGDH) is a key serine biosynthesis enzyme whose aberrant expression promotes various types of tumors. Recently, PHGDH has been found to have some non-canonical functions beyond serine biosynthesis, but its specific mechanisms in tumorigenesis remain unclear. Here, we show that PHGDH localizes to the inner mitochondrial membrane and promotes the translation of mitochondrial DNA (mtDNA)-encoded proteins in liver cancer cells. Mechanistically, we demonstrate that mitochondrial PHGDH directly interacts with adenine nucleotide translocase 2 (ANT2) and then recruits mitochondrial elongation factor G2 (mtEFG2) to promote mitochondrial ribosome recycling efficiency, thereby promoting mtDNA-encoded protein expression and subsequent mitochondrial respiration. Moreover, we show that treatment with a mitochondrial translation inhibitor or depletion of mtEFG2 diminishes PHGDH-mediated tumor growth. Collectively, our findings uncover a previously unappreciated function of PHGDH in tumorigenesis acting via promotion of mitochondrial translation and bioenergetics.

I.01Congenital myasthenic syndromes

Neuromuscular junction disorders are a heterogeneous group of acquired (Myasthenia Gravis, MG) and inherited (Congenital Myasthenic Syndromes, CMS) disorders associated with distinctive clinical, electrophysiological, laboratory and ultrastructural abnormalities. The genetic defects in CMS either impair neuromuscular transmission directly or result in secondary impairments, which eventually compromise the safety margin of neuromuscular transmission. More recently, we have identified two genes (<i>DOK7, GFPT1</i>) that cause fatigable weakness of muscles in a limb-girdle distribution, but rarely affecting facial or eye muscles. Next-generation sequencing and deep phenotyping, in combination with international data sharing, reveals new genetic causes of CMS, but also unusual, overlapping clinical phenotypes, which blur the boundaries with primary myopathies and motor neuropathies. Mitochondrial dysfunction has been implicated with both myopathies and neuromuscular transmission defects. TEFM is a nuclear gene that codes for a mitochondrial transcription elongation factor – the protein responsible for prolonging activity of RNA polymerase to ensure distal transcripts of the mitochondrial genome are transcribed. We identified the first human <i>TEFM</i> variants associated with mitochondrial respiratory chain deficiency in seven patients with varying clinical presentations. While symptoms included classical features of a mitochondrial disease, some patients also presented with a treatable neuromuscular transmission defect. We will cover the significant progress made in understanding the molecular pathogenesis of CMS, which is important for both patients and clinicians in terms of reaching a definite diagnosis and selecting the most appropriate treatment.

Valproic acid regulates MIEF1 through MST2-HIPPO to suppress breast cancer growth

AimsTo determine the effects of valproic acid (VPA) on anti-proliferative effects and mitochondrial function in breast cancer cells and the underlying mechanisms involved in the effects, with a focus on signal transduction. Main methodsThe inhibitory effect of valproic acid on breast cancer in vivo and in vitro was evaluated by cellular and animal experiments. Mitochondria-related proteins as well as hippo pathway were monitored by western blotting. The effects of VPA on mitochondrial membrane potential, reactive oxygen species, and apoptosis were confirmed by flow cytometry. In addition, the involvement of hippo pathway in the regulation of mitochondrial function by VPA was verified by XMU-MP-1 (MST2 inhibitor). Key findingsIn this study, we highlight that VPA significantly attenuates mitochondrial function, leading to inhibited cell proliferation and reduced colony formation in MCF-7 and MDA-MB-231 breast cancer cells. Mechanistically, VPA-induced suppression of mitochondrial aerobic respiration was mediated by decreased expression of mitochondrial elongation factor 1 through activation of the hippo pathway, resulting in impaired breast cancer growth. In summary, we uncover a novel mechanism of VPA in regulating mitochondrial aerobic respiration, which is essential for developing an effective approach in breast cancer therapy. SignificanceMitochondrial aerobic respiration and its products are the main sources of energy for tumors; therefore, studying the role of mitochondrial function in tumor cells is significant. VPA has been used as a therapeutic agent for cancer. However, the detail mechanism underlying the effects of VPA on mitochondrial function in breast cancer remains unclear.

CRISPR-based knockout screening identifies the loss of MIEF2 to enhance oxaliplatin resistance in colorectal cancer through inhibiting the mitochondrial apoptosis pathway.

The first-line anticancer agent oxaliplatin (OXL) is the preferred drug for treating colorectal cancer (CRC); however, the development of drug resistance is common in patients treated with OXL, which considerably reduces the efficacy of OXL-based regimens. By performing genome-wide CRISPR/Cas9 library knockdown screening, we found that mitochondrial elongation factor 2 (MIEF2) was among the top candidate genes. The OXL-resistant cell lines and organoids developed in the present study showed stable but low expression of MIEF2. Reduced MIEF2 expression may enhance CRC resistance to OXL by reducing mitochondrial stability and inhibiting apoptosis by decreasing cytochrome C release. In conclusion, among the different biomarkers of OXL resistance in CRC, MIEF2 may serve as a specific biomarker of OXL responsiveness and a potential target for the development of therapies to improve chemotherapeutic effectiveness.

Abstract P2033: Neddylation Regulates Cardiac Mitochondria Homeostasis

Neddylation is a reversible post-translational modification that covalently attaches ubiquitin-like protein NEDD8 to target proteins via NEDD8-specific E1-E2-E3 enzymes. Through regulating the function of various cellular proteins, neddylation impacts diverse cellular processes and disease states. We have previously identified an indispensable role for neddylation in cardiac chamber maturation through sustaining YAP signaling in the developing heart. However, the importance of neddylation in post-mitotic hearts remains unclear. Here we report neddylation as a novel post-translation mechanism regulating mitochondrial homeostasis in adult heart. Inducible deletion of NAE1, a regulatory subunit of NEDD8 E1 enzyme, in adult mouse heart resulted in dilated cardiomyopathy, heart failure, and eventually premature lethality. The severe cardiac phenotype was preceded by accumulation of mitophagic vesicles and mitochondrial elongation, accompanied by pronounced upregulation of mitophagic markers and alterations in mitochondrial dynamics proteins. Moreover, we found that neddylation is required for mitochondrial ubiquitination and turnover. Consequently, pharmacological and genetic inhibition of neddylation accumulated damaged mitochondria, impaired mitochondrial membrane potential, inhibited mitochondrial respiration, and increased cardiomyocyte cell death in both neonatal and adult cardiomyocytes. Mechanistically, neddylation of Cullins is essential for the assembly of a functional multi-subunit Cullin-RING ubiquitin ligase, which appears to mediate mitochondrial ubiquitination following mitochondrial damage. Taken together, our findings suggest that neddylation serves as a novel mechanism surveilling mitochondrial quality and is essential for the function of normal adult heart.

The Bacterial ClpXP-ClpB Family Is Enriched with RNA-Binding Protein Complexes.

In the matrix of bacteria/mitochondria/chloroplasts, Lon acts as the degradation machine for soluble proteins. In stress periods, however, proteostasis and survival depend on the strongly conserved Clp/Hsp100 family. Currently, the targets of ATP-powered unfoldases/disaggregases ClpB and ClpX and of peptidase ClpP heptameric rings are still unclear. Trapping experiments and proteome profiling in multiple organisms triggered confusion, so we analyzed the consistency of ClpP-trap targets in bacteria. We also provide meta-analyses of protein interactions in humans, to elucidate where Clp family members are enriched. Furthermore, meta-analyses of mouse complexomics are provided. Genotype–phenotype correlations confirmed our concept. Trapping, proteome, and complexome data retrieved consistent coaccumulation of CLPXP with GFM1 and TUFM orthologs. CLPX shows broad interaction selectivity encompassing mitochondrial translation elongation, RNA granules, and nucleoids. CLPB preferentially attaches to mitochondrial RNA granules and translation initiation components; CLPP is enriched with them all and associates with release/recycling factors. Mutations in CLPP cause Perrault syndrome, with phenotypes similar to defects in mtDNA/mtRNA. Thus, we propose that CLPB and CLPXP are crucial to counteract misfolded insoluble protein assemblies that contain nucleotides. This insight is relevant to improve ClpP-modulating drugs that block bacterial growth and for the treatment of human infertility, deafness, and neurodegeneration.

Predicting the Prognostic Value of POLI Expression in Different Cancers via a Machine Learning Approach.

Translesion synthesis (TLS) is a cell signaling pathway that facilitates the tolerance of replication stress. Increased TLS activity, the particularly elevated expression of TLS polymerases, has been linked to resistance to cancer chemotherapeutics and significantly altered patient outcomes. Building upon current knowledge, we found that the expression of one of these TLS polymerases (POLI) is associated with significant differences in cervical and pancreatic cancer survival. These data led us to hypothesize that POLI expression is associated with cancer survival more broadly. However, when cancers were grouped cancer type, POLI expression did not have a significant prognostic value. We presented a binary cancer random forest classifier using 396 genes that influence the prognostic characteristics of POLI in cervical and pancreatic cancer selected via graphical least absolute shrinkage and selection operator. The classifier was then used to cluster patients with bladder, breast, colorectal, head and neck, liver, lung, ovary, melanoma, stomach, and uterus cancer when high POLI expression was associated with worsened survival (Group I) or with improved survival (Group II). This approach allowed us to identify cancers where POLI expression is a significant prognostic factor for survival (p = 0.028 in Group I and p = 0.0059 in Group II). Multiple independent validation approaches, including the gene ontology enrichment analysis and visualization tool and network visualization support the classification scheme. The functions of the selected genes involving mitochondrial translational elongation, Wnt signaling pathway, and tumor necrosis factor-mediated signaling pathway support their association with TLS and replication stress. Our multidisciplinary approach provides a novel way of identifying tumors where increased TLS polymerase expression is associated with significant differences in cancer survival.

Mitochondrial Elongation and OPA1 Play Crucial Roles during the Stemness Acquisition Process in Pancreatic Ductal Adenocarcinoma.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is a highly lethal neoplasia and the currently used treatments are not effective in a wide range of patients. Presently, the evidence points out that cancer stem cells (CSCs) are key players during tumor development, metastasis, chemoresistance, and tumor relapse. The study of the metabolism of CSCs, specifically the mitochondrial alterations, could pave the way to the discovery of new therapeutical targets. In this study, we show that during progressive de-differentiation, pancreatic CSCs undergo changes in mitochondrial mass, dynamics, and function. Interestingly, the silencing of OPA1, a protein involved in mitochondrial fusion, significantly inhibits the formation of CSCs. These results reveal new insight into mitochondria and stemness acquisition that could be useful for the design of novel potential therapies in PDAC.Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.

Identification of the shared gene signatures and pathways between polycystic ovary syndrome and endometrial cancer: An omics data based combined approach.

ObjectivePolycystic ovary syndrome (PCOS) is a common endocrine disorder with high incidence. Recently it has been implicated as a significant risk factor for endometrial cancer (EC). Our study aims to detect shared gene signatures and biological mechanism between PCOS and EC by bioinformatics analysis.MethodsBioinformatics analysis based on GEO database consisted of data integration, network construction and functional enrichment analysis was applied. In addition, the pharmacological methodology and molecular docking was also performed.ResultsTotally 10 hub common genes, MRPL16, MRPL22, MRPS11, RPL26L1, ESR1, JUN, UBE2I, MRPL17, RPL37A, GTF2H3, were considered as shared gene signatures for EC and PCOS. The GO and KEGG pathway analysis of these hub genes showed that “mitochondrial translational elongation”, “ribosomal subunit”, “structural constituent of ribosome” and “ribosome” were highly correlated. Besides, associated transcription factors (TFs) and miRNAs network were constructed. We identified candidate drug molecules including fenofibrate, cinnarizine, propanil, fenthion, clindamycin, chloramphenicol, demeclocycline, hydrochloride, azacitidine, chrysene and artenimol according to these hub genes. Molecular docking analysis verified a good binding interaction of fenofibrate against available targets (JUN, ESR1, UBE2I).ConclusionGene signatures and regulatory biological pathways were identified through bioinformatics analysis. Moreover, the molecular mechanisms of these signatures were explored and potential drug molecules associated with PCOS and EC were screened out.

L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through nitric oxide-mediated Drp1 inhibition in mouse C2C12 myoblasts.

Severe heat exposure causes mitochondrial fragmentation and dysfunction, which contribute to the pathogenesis of heat-related illness. L-citrulline is a naturally occurring amino acid and has been suggested to influence heat shock responses. This study aimed to test whether L-citrulline supplementation would preserve mitochondrial integrity and attenuate heat-induced skeletal muscle injury, and elucidate the underlying mechanisms. At 37°C, L-citrulline (2 mM) increased mitochondrial elongation in mouse C2C12 myoblasts, a process associated with a reduction in mitochondrial fission protein Drp1 levels. Mechanistic studies revealed that L-citrulline increased cellular nitric oxide (NO) levels, but not S-nitrosylation of Drp1. L-citrulline caused a decrease in phosphorylation of Drp1 at Ser 616 and an increase in phosphorylation of Drp1 at Ser 637, which resulted in a reduced mitochondrial localization of Drp1. L-NAME, a non-selective NO synthase inhibitor, abolished the increase in L-citrulline-induced NO levels and inhibited Drp1 phosphorylation changes and mitochondrial elongation, which indicates involvement of a NO-dependent pathway. Under 43°C heat stress conditions, L-citrulline prevented translocation of Drp1 to mitochondria, mitochondrial fragmentation and decreased membrane potential. Finally, L-citrulline pretreatment inhibited heat-induced reactive oxygen species (ROS) overproduction, caspase 3/7 activation, apoptotic cell death, and improved cell viability. NO inhibitor L-NAME abolished all the above protective effects of L-citrulline under heat stress. Our results suggest that L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through NO-mediated Drp1 inhibition in C2C12 myoblasts. L-citrulline may be an effective treatment for heat-related illnesses and other mitochondrial diseases.

Expression and prognosis analysis of mitochondrial ribosomal protein family in breast cancer

Breast cancer (BC) is characterized by high morbidity. Mitochondrial ribosomal protein (MRP) family participates in mitochondrial energy metabolism, underlying BC progression. This study aims to analyze the expression and prognosis effect of the MRP genes in BC patients. GEPIA2, UALCAN, cBioPortal, and MethSurv were used to demonstrate the differential expression, genomic alteration profiles, and DNA methylation of the MRP gene family in BC. Functional enrichment analysis and protein–protein interaction network construction were performed to understand the biological function. Based on 1056 TCGA samples with the transcriptional level of MRPs, Kaplan–Meier curves, Cox, and LASSO regression were applied to explore their prognostic effects. 12 MRPs were upregulated in BC, which were associated with gene amplification and DNA methylation. MRP genetic alteration occurred in 42% of BC patients, and amplification was the most frequent variation. Functioning in its entirety, the MRP family was involved in mitochondrial translational termination, elongation, translation, and poly(A) RNA binding. High expression of MRPL1, MRPL13, MRPS6, MRPS18C, and MRPS35, as well as low levels of MRPL16, and MRPL40 significantly indicated poor prognosis in BC patients. Thus, a novel MRP-based prognostic nomogram was established and verified with favorable discrimination and calibration. We not only provided a thorough expression and prognosis analysis of the MRP family in BC patients but also constructed an MRP-based prognostic nomogram. It was suggested that MRPs acted as biomarkers in individualized risk prediction and may serve as potential therapeutic targets in BC patients.

Piperine Derivatives Enhance Fusion and Axonal Transport of Mitochondria by Activating Mitofusins

Piperine (1-piperoylpiperidine) is the major pungent component of black pepper (Piper nigrum) and exhibits a spectrum of pharmacological activities. The molecular bases for many of piperine’s biological effects are incompletely defined. We noted that the chemical structure of piperine generally conforms to a pharmacophore model for small bioactive molecules that activate mitofusin (MFN)-mediated mitochondrial fusion. Piperine, but not its isomer chavicine, stimulated mitochondrial fusion in MFN-deficient cells with EC50 of ~8 nM. We synthesized piperine analogs having structural features predicted to optimize mitofusin activation and defined structure-activity relationships (SAR) in live-cell mitochondrial elongation assays. When optimal spacing was maintained between amide and aromatic groups the derivatives were potent mitofusin activators. Compared to the prototype phenylhexanamide mitofusin activator, 2, novel molecules containing the piperidine structure of piperine exhibited markedly enhanced passive membrane permeability with no loss of fusogenic potency. Lead compounds 5 and 8 enhanced mitochondrial motility in cultured murine Charcot-Marie-Tooth disease type 2A (CMT2A) neurons, but only 8 improved mitochondrial transport in sciatic nerve axons of CMT2A mice. Piperine analogs represent a new chemical class of mitofusin activators with potential pharmaceutical advantages.