Mitochondria-associated Proteins Research Articles

M133S mutation possibly involve in the ER stress and mitophagy pathway in maintenance hemodialysis patients with occult hepatitis B infection

Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI.

Integrated multi-omics analysis and machine learning developed a prognostic model based on mitochondrial function in a large multicenter cohort for Gastric Cancer

BackgroundGastric cancer (GC) is a common and aggressive type of cancer worldwide. Despite recent advancements in its treatment, the prognosis for patients with GC remains poor. Understanding the mechanisms of cell death in GC, particularly those related to mitochondrial function, is crucial for its development and progression. However, more research is needed to investigate the significance of the interaction between mitochondrial function and GC cell death.MethodsWe employed a robust computational framework to investigate the role of mitochondria-associated proteins in the progression of GC in a cohort of 1,199 GC patients. Ten machine learning algorithms were utilized and combined into 101 unique combinations. Ultimately, we developed a Mitochondrial-related-Score (MitoScore) using the machine learning model that exhibited the best performance. We observed the upregulation of LEMT2 and further explored its function in tumor progression. Mitochondrial functions were assessed by measuring mitochondrial ATP, mitochondrial membrane potential, and levels of lactate, pyruvate, and glucose.ResultsMitoScore showed significant correlations with GC immune and metabolic functions. The higher MitoScore subgroup exhibited enriched metabolic pathways and higher immune activity. Overexpression of LETM2 (leucine zipper and EF-hand containing transmembrane protein 2) significantly enhanced tumor proliferation and metastasis. LETM2 plays a role in promoting GC cell proliferation by activating the mTOR pathway, maintaining mitochondrial homeostasis, and promoting glycolysis.ConclusionThe powerful machine learning framework highlights the significant potential of MitoScore in providing valuable insights and accurate assessments for individuals with GC. This study also enhances our understanding of LETM2 as an oncogene signature in GC. LETM2 may promote tumor progression by maintaining mitochondrial health and activating glycolysis, offering potential targets for diagnosis, treatment, and prognosis of GC.

Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies

The functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome. Biochemical and imaging-based follow-up studies confirm that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. We also validate the CLASP concept in synaptic vesicles, demonstrating its applicability to different sub-cellular compartments. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, and establishes a method for concomitant profiling of sub-organelle and membrane proteomes.

Long-chain acyl-CoA synthetase 4-mediated mitochondrial fatty acid metabolism and dendritic cell antigen presentation.

This study aims to investigate the role of Acyl-CoA synthetase 4 (ACSL4) in mediating mitochondrial fatty acid metabolism and dendritic cell (DC) antigen presentation in the immune response associated with asthma. RNA sequencing was employed to identify key genes associated with mitochondrial function and fatty acid metabolism in DCs. ELISA was employed to assess the levels of fatty acid metabolism in DCs. Mitochondrial morphology was evaluated using laser confocal microscopy, structured illumination microscopy, and transmission electron microscopy. Flow cytometry and immunofluorescence were utilized to detect changes in mitochondrial superoxide generation in DCs, followed by immunofluorescence co-localization analysis of ACSL4 and the mitochondrial marker protein COXIV. Subsequently, pathological changes and immune responses in mouse lung tissue were observed. ELISA was conducted to measure the levels of fatty acid metabolism in lung tissue DCs. qRT-PCR and western blotting were employed to respectively assess the expression levels of mitochondrial-associated genes (ATP5F1A, VDAC1, COXIV, TFAM, iNOS) and proteins (ATP5F1A, VDAC1, COXIV, TOMM20, iNOS) in lung tissue DCs. Flow cytometry was utilized to analyze changes in the expression of surface antigens presented by DCs in lung tissue, specifically the MHCII molecule and the co-stimulatory molecules CD80/86. The sequencing results reveal that ACSL4 is a crucial gene regulating mitochondrial function and fatty acid metabolism in DCs. Inhibiting ACSL4 reduces the levels of fatty acid oxidases in DCs, increases arachidonic acid levels, and decreases A-CoA synthesis. Simultaneously, ACSL4 inhibition leads to an increase in mitochondrial superoxide production (MitoSOX) in DCs, causing mitochondrial rupture, vacuolization, and sparse mitochondrial cristae. In mice, ACSL4 inhibition exacerbates pulmonary pathological changes and immune responses, reducing the fatty acid metabolism levels within lung tissue DCs and the expression of mitochondria-associated genes and proteins. This inhibition induces an increase in the expression of MHCII antigen presentation molecules and co-stimulatory molecules CD80/86 in DCs. The research findings indicate that ACSL4-mediated mitochondrial fatty acid metabolism and dendritic cell antigen presentation play a crucial regulatory role in the immune response of asthma. This discovery holds promise for enhancing our understanding of the mechanisms underlying asthma pathogenesis and potentially identifying novel targets for its prevention and treatment.

LARP4 is an RNA-binding protein that binds nuclear-encoded mitochondrial mRNAs to promote mitochondrial function.

Mitochondria-associated RNA-binding proteins (RBPs) have emerged as key contributors to mitochondrial biogenesis and homeostasis. With few examples known, we set out to identify RBPs that regulate nuclear-encoded mitochondrial mRNAs (NEMmRNAs). Our systematic analysis of RNA targets of 150 RBPs identified RBPs with a preference for binding NEMmRNAs, including LARP4, a La RBP family member. We show that LARP4's targets are particularly enriched in mRNAs that encode respiratory chain complex proteins (RCCPs) and mitochondrial ribosome proteins (MRPs) across multiple human cell lines. Through quantitative proteomics, we demonstrate that depletion of LARP4 leads to a significant reduction in RCCP and MRP protein levels. Furthermore, we show that LARP4 depletion reduces mitochondrial function, and that LARP4 re-expression rescues this phenotype. Our findings shed light on a novel function for LARP4 as an RBP that binds to and positively regulates NEMmRNAs to promote mitochondrial respiratory function.

Engineered biomaterial delivery strategies are used to reduce cardiotoxicity in osteosarcoma.

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Chemotherapy drugs play an integral role in OS treatment. Preoperative neoadjuvant chemotherapy and postoperative conventional adjuvant chemotherapy improve survival in patients with OS. However, the toxic side effects of chemotherapy drugs are unavoidable. Cardiotoxicity is one of the common side effects of chemotherapy drugs that cannot be ignored. Chemotherapy drugs affect the destruction of mitochondrial autophagy and mitochondria-associated proteins to cause a decrease in cardiac ejection fraction and cardiomyocyte necrosis, which in turn causes heart failure and irreversible cardiomyopathy. Biomaterials play an important role in nanomedicine. Biomaterials act as carriers to deliver chemotherapy drugs precisely around tumor cells and continuously release carriers around the tumor. It not only promotes anti-tumor effects but also reduces the cardiotoxicity of chemotherapy drugs. In this paper, we first introduce the mechanism by which chemotherapy drugs commonly used in OS cause cardiotoxicity. Subsequently, we introduce biomaterials for reducing cardiotoxicity in OS chemotherapy. Finally, we prospect biomaterial delivery strategies to reduce cardiotoxicity in OS.

Chronic fluoxetine treatment in socially-isolated rats modulates the prefrontal cortex synaptoproteome

Exposure to chronic social isolation (CSIS) and synapse dysfunction have been implicated in the etiology of major depressive disorder (MDD). Fluoxetine (Flx) has been widely used to treat MDD, but its mechanisms of action remain elusive. We employed comparative synaptoproteomics to investigate the changes in the levels of proteins and molecular signaling pathways in prefrontal cortical samples of adult male Wistar rats exposed to CSIS, a rat model of depression, and CSIS rats treated with chronic Flx and controls, using liquid chromatography coupled to tandem mass spectrometry. Flx-treated control rats showed a decreased level of proteins involved in vesicle-mediated transport, and a predominantly increased level of exocytosis-associated proteins. CSIS significantly reduced the level of proteins involved in the ATP metabolic process, clathrin-dependent endocytosis, and proteolysis. Flx treatment in CSIS rats stimulated synaptic vesicle trafficking by increasing the regulation of exo/endocytosis-associated proteins, proteins involved in synaptic plasticity including neurogenesis, Cox5a, mitochondria-associated proteins involved in oxidative phosphorylation, and ion transport proteins (Slc8a2, Atp1b2). Flx treatment resulted in an increased synaptic vesicle dynamic, plasticity and mitochondrial functionality, and a suppression of CSIS-induced impairment of these processes. Biological significanceIdentifying biomarkers of MDD and treatment response is the goal of many studies. Contemporary studies have shown that many molecular alterations associated with the pathophysiology of MDD reside within the synapse. As part of this research, a growing importance is the use of proteomics, as monitoring the changes in protein levels enables the identification of (possible) biochemical pathways and processes of importance for the development of depressive-like behavior and the efficacy of antidepressant treatments. We profiled proteomic changes representative of the development of CSIS-induced depressive-like behavior and the antidepressant effects of Flx. Our study has identified synaptosomal proteins and altered molecular pathways that may be potential markers of prefrontal cortical synaptic dysfunction associated with depressive-like behavior, and further clarified the mechanisms of depressive-like behavior and mode of action of Flx. Our findings indicate potential PFC synaptic targets for antidepressant treatment.

Mitochondria-associated gene expression perturbation predicts clinical outcomes and shows potential for targeted therapy in neuroblastoma.

Mitochondria have long been considered a potential target in cancer therapy because malignant cells are known for their altered energy production. However, there is a lack of comprehensive research on the involvement of mitochondria-associated proteins (MAPs) in neuroblastoma (NB), and their potential as therapeutic targets is yet to be fully explored. MAP genes were defined based on the protein-coding genes with mitochondrial localization. The mRNA expression patterns and dynamics of MAP genes associated with NB were investigated by integrating publicly available transcriptional profiles at the cellular and tissue levels. Multivariate Cox regression analysis was conducted to reveal the association of MAP genes with the overall survival (OS) and clinical subgroups of NB patients. The single-cell RNA-seq dataset and gene dependency screening datasets were analyzed to reveal the therapeutic potential of targeting MAP genes. We compiled a total of 1,712 MAP genes. We found the global and cell type-specific mRNA expression changes of the MAP genes associated with NB status and survival. Our analyses revealed a group of MAP gene signatures independent of MYCN-amplification status associated with NB outcome. We provided computational evidence with selected MAP genes showing good performance in predicting long-term prognosis. By analyzing gene dependency of the MAP genes in NB cell lines and ex vivo human primary T cells, we demonstrated the therapeutic potential of targeting several MAP genes in NB tumors. Collectively, our study provides evidence for the MAP genes as extended candidates in NB tumor stratification and staging, prognostic prediction, and targeted drug development.

Silibinin Protects against H2O2-Induced Oxidative Damage in SH-SY5Y Cells by Improving Mitochondrial Function.

Oxidative stress plays a critical role in the pathogenesis of various neurodegenerative diseases. Increasing evidence suggests the association of mitochondrial abnormalities with oxidative stress-related neural damage. Silibinin, a natural flavonol compound isolated from Silybum marianum, exhibits multiple biological activities. The present study investigated the effects of silibinin on H2O2-induced oxidative stress in human neuroblastoma SH-SY5Y cells. Exposure to H2O2 (750 µM) reduced the viability of SH-SY5Y cells, which was coupled with increased reactive oxygen species (ROS), abnormal cell morphology, and mitochondrial dysfunction. Remarkably, silibinin (1, 5, and 10 µM) treatment attenuated the H2O2-induced cell death. Moreover, silibinin reduced ROS production and the levels of malondialdehyde (MDA), increased the levels of superoxide dismutase (SOD) and glutathione (GSH), and increased mitochondrial membrane potential. Moreover, silibinin normalized the expression of nuclear factor 2-related factor 2 (Nrf2)-related and mitochondria-associated proteins. Taken together, our findings demonstrated that silibinin could attenuate H2O2-induced oxidative stress by regulating Nrf2 signaling and improving mitochondrial function in SH-SY5Y cells. The protective effect against oxidative stress suggests silibinin as a potential candidate for preventing neurodegeneration.

Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins.

Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER–mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.

The Effects Of Exercise Preconditioning On Serum -free Mtdna And Mitochondrial Autoimmunity In Mice

PURPOSE: To explore the effects of exercise preconditioning on serum free mtDNA and mitochondria-associated immune proteins in skeletal muscles under acute centrifugation injury. METHODS: Eight-week-old C57BL male mice were randomly divided into three groups according to their body weight. Each group consisted of 6 mice (NC, RC, RE). Exercise pre-adaptation program: take a non-slope 20 m/min treadmill training for 40 min/d, training for 6 weeks. Acute Sports Injury Program: In the 6th week of exercise training in RE group, 3 days of 10 m/min and 5 minutes of adaptive training without gradients on the RC group were started in the RC group; all mice in the RC and RE groups took 16 m/min,-16 ° Downhill running, training 90 min. After 16 hours of injury training, the mice were sacrificed, and one side of the gastrocnemius muscle was fixed with paraformaldehyde and awaited for HE staining. The tissue samples were detected by PCR, RT-PCR, Western Blotting, and ELISA. RESULTS:1) Serum free mtDNA content in RC group was significantly higher than NC and RE group (P < 0.05) 2) IL6 levels in mouse serum were significantly lower in NC group than in RC and RE group (P < 0.05, P < 0.01); The group was significantly higher than the NC group and RE group (P < 0.05, P < 0.01); 3) The results of STING mRNA detection showed that NC group was higher than RE (P < 0.05) There was no significant difference between RC group and RC group, but RC group was significantly higher than that of RE group (P < 0.01). The results of mRNA expression tests of other molecules in STING pathway showed that only IRF3 expression was significantly lower in RE group than in NC group. (P < 0.05);4) The expression of PINK1 in skeletal muscle was highest in the RC group and significantly higher than that in the RE group (P < 0.01). Parkin was the highest in NC group in skeletal muscle, (P < 0.01). CONCLUSIONS: (1) Increase in serum free mtDNA content caused by acute centrifugation injury exercise is not a cause of inflammatory reaction to skeletal muscle injury, because acute centrifugation injury inhibits the transcription of mitochondria-associated immune proteins in skeletal muscle, and cGAS-STING-TBK1-IRF3 - The IFN-β signaling pathway is not activated. (2) Acute centrifugal injury exercise and exercise preconditioning did not affect PINK1/Parkin-mediated mitochondrial autophagy.

Prevention of HIV-1 TAT Protein-Induced Peripheral Neuropathy and Mitochondrial Disruption by the Antimuscarinic Pirenzepine.

HIV-associated distal sensory polyneuropathy (HIV-DSP) affects about one third of people with HIV and is characterized by distal degeneration of axons. The pathogenesis of HIV-DSP is not known and there is currently no FDA-approved treatment. HIV trans-activator of transcription (TAT) is associated with mitochondrial dysfunction and neurotoxicity in the brain and may play a role in the pathogenesis of HIV-DSP. In the present study, we measured indices of peripheral neuropathy in the doxycycline (DOX)-inducible HIV-TAT (iTAT) transgenic mouse and investigated the therapeutic efficacy of a selective muscarinic subtype-1 receptor (M1R) antagonist, pirenzepine (PZ). PZ was selected as we have previously shown that it prevents and/or reverses indices of peripheral neuropathy in multiple disease models. DOX alone induced weight loss, tactile allodynia and paw thermal hypoalgesia in normal C57Bl/6J mice. Conduction velocity of large motor fibers, density of small sensory nerve fibers in the cornea and expression of mitochondria-associated proteins in sciatic nerve were unaffected by DOX in normal mice, whereas these parameters were disrupted when DOX was given to iTAT mice to induce TAT expression. Daily injection of PZ (10 mg/kg s.c.) prevented all of the disorders associated with TAT expression. These studies demonstrate that TAT expression disrupts mitochondria and induces indices of sensory and motor peripheral neuropathy and that M1R antagonism may be a viable treatment for HIV-DSP. However, some indices of neuropathy in the DOX-inducible TAT transgenic mouse model can be ascribed to DOX treatment rather than TAT expression and data obtained from animal models in which gene expression is modified by DOX should be accompanied by appropriate controls and treated with due caution.

Mitochondrial dysfunction as a driver of NLRP3 inflammasome activation and its modulation through mitophagy for potential therapeutics.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is responsible for the sensation of various pathogenic and non-pathogenic damage signals and has a vital role in neuroinflammation and neural diseases. Various stimuli, such as microbial infection, misfolded protein aggregates, and aberrant deposition of proteins can induce NLRP3 inflammasome in neural cells. Once triggered, the NLRP3 inflammasome leads to the activation of caspase-1, which in turn activates inflammatory cytokines, such as interleukin-1β and interleukin -18, and induces pyroptotic cell death. Mitochondria are critically involved in diverse cellular processes and are involved in regulating cellular redox status, calcium levels, inflammasome activation, and cell death. Mitochondrial dysfunction and subsequent accumulation of mitochondrial reactive oxygen species, mitochondrial deoxyribonucleic acid, and other mitochondria-associated proteins and lipids play vital roles in the instigation of the NLRP3 inflammasome. In addition, the processes of mitochondrial dynamics, such as fission and fusion, are essential in the maintenance of mitochondrial integrity and their imbalance also promotes NLRP3 inflammasome activation. In this connection, mitophagy-mediated maintenance of mitochondrial homeostasis restricts NLRP3 inflammasome hyperactivation and its consequences in various neurological disorders. Hence, mitophagy can be exploited as a potential strategy to target damaged mitochondria induced NLRP3 inflammasome activation and its lethal consequences. Therefore, the identification of novel mitophagy modulators has promising therapeutic potential for NLRP3 inflammasome-associated neuronal diseases.

Toxoplasma gondii association with host mitochondria requires key mitochondrial protein import machinery

Host mitochondrial association (HMA) is a well-known phenomenon during Toxoplasma gondii infection of the host cell. The T. gondii locus mitochondrial association factor 1 (MAF1) is required for HMA and MAF1 encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified ∼1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1b-targeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the T. gondii vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell.

Effects of Exercise and a High-Fat, High-Sucrose Restriction Diet on Metabolic Indicators, Nr4a3, and Mitochondria-Associated Protein Expression in the Gastrocnemius Muscles of Mice with Diet-Induced Obesity.

BackgroundExercise and high fat, high sucrose restriction diets are well known treatments for obesity. The aim of this study was to measure the effects of those lifestyle interventions on molecular transducers of exercise, such as Nr4a3, mitochondria-associated proteins, and muscle function.MethodsWe conducted 8 weeks of treadmill exercise and sucrose or fat restriction diets in obese mice. The mice were divided into eight groups: the normal diet (CON) group, normal diet with exercise (CONEX) group, high fat, high sucrose diet (HFHS) group, HFHS with exercise (HFHSEX) group, sucrose restriction (SR) group, SR with exercise (SREX) group, high fat, high sucrose restriction (ND) group, and ND with exercise (NDEX) group.ResultsThe 8 weeks of exercise reduced body weight, improved lipid profiles (total cholesterol, triglycerides), and increased hanging time. The combination of exercise and a fat and sucrose restriction diet improved glucose tolerance and increased grip strength. The 8 weeks of intervention did not significantly affect the Nr4a3 protein level. The sucrose and fat restriction diet increased the phosphorylated protein kinase B (pAkt)/Akt ratio, and its level was lower in the HFHS group. Exercise increased the protein expression level of PGC-1α in obese conditions. Moreover, SR led reduced the phosphorylated AMP-activated protein kinase (pAMPK)/AMPK ratio and PGC-1α to the control level.ConclusionThe 8 weeks of exercise or a sucrose and fat restriction diet improved metabolic indicators and muscle function. SR reduced pAMPK/AMPK and PGC-1α to the control level. Nr4a3 protein expression was not significantly changed by either exercise or a fat and sucrose restriction diet.

Tissue-specific roles of GCN2 in aging and autosomal dominant retinitis pigmentosa

The organisms have the capacity to sense and adapt to their surroundings for their life in a dynamic environment. In response to amino acid starvation, cells activate a rectifying physiological program, termed the integrated stress response (ISR), to restore cellular homeostasis. General controlled non-repressed (GCN2) kinase is a master regulator of the ISR and modulates protein synthesis in response to amino acid starvation. We previously established the GCN2/ATF4/4E-BP pathway in development and aging. Here, we investigated the tissue-specific roles of GCN2 upon dietary restriction of amino acid in a Drosophila model. The knockdown of GCN2 in the gut and fat body, an energy sensing organ in Drosophila, abolished the beneficial effect of GCN2 in lifespan extension upon dietary restriction of amino acids. Proteome analysis in an autosomal dominant retinitis pigmentosa (ADRP) model showed that dietary restriction of amino acids regulates the synthesis of proteins in several pathways, including mitochondrial translation, mitochondrial gene expression, and regulation of biological quality, and that gcn2-mutant flies have reduced levels of these mitochondria-associated proteins, which may contribute to retinal degeneration in ADRP. These results indicate that the tissue-specific regulation of GCN2 contributes to normal physiology and ADRP progression.

Conditional Deletion of PGC-1α Results in Energetic and Functional Defects in NK Cells.

SummaryDuring an immune response, natural killer (NK) cells activate specific metabolic pathways to meet the increased energetic and biosynthetic demands associated with effector functions. Here, we found in vivo activation of NK cells during Listeria monocytogenes infection-augmented transcription of genes encoding mitochondria-associated proteins in a manner dependent on the transcriptional coactivator PGC-1α. Using an Ncr1Cre-based conditional knockout mouse, we found that PGC-1α was crucial for optimal NK cell effector functions and bioenergetics, as the deletion of PGC-1α was associated with decreased cytotoxic potential and cytokine production along with altered ADP/ATP ratios. Lack of PGC-1α also significantly impaired the ability of NK cells to control B16F10 tumor growth in vivo, and subsequent gene expression analysis showed that PGC-1α mediates transcription required to maintain mitochondrial activity within the tumor microenvironment. Together, these data suggest that PGC-1α-dependent transcription of specific target genes is required for optimal NK cell function during the response to infection or tumor growth.

Mechanism of miRNA-26a on the Proliferation of Pancreatic Tumor Cells by Regulating the Expression of Cycline2

Background: qRT-PCR was used to measure the expression of miRNA-26a in pancreatic epithelial cells (HPDE) and human pancreatic cancer cell lines PANC-1 and MIA PaCa-2. Methods: PANC-1 and MIA PaCa-2 cell lines were infected with lentiviruses to construct PANC-miR-26a and MIA-miR-26a, and RT-PCR was used to detect the infection efficiency. The cell proliferation ability of PANC-miR-26a and MIA-miR-26a were examined by CCK-8 assay, and apoptosis was detected by flow cytometry. Western blotting was used to detect the expressions of CyclinE2 protein and mitochondria-associated apoptotic proteins. Results: miR-26a was expressed in human normal pancreatic epithelial cells (HPDE), and not detected in PANC-1 and MIA PaCa-2; miR-26a was highly expressed in the cell lines PANC-miR-26a and MIA-miR-26a infected by the virus particles. The absorbance values of PANC-miR-26a and MIA-miR-26a were lower than those of NC1 and PANC-1 in control group. The apoptosis rates of PANC-miR-26a and MIA-miR-26a were substantially higher than those of the control group. The overexpression of miR-26a inhibited the expression of the target protein CyclinE2 in PANC-miR-26a and MIA-miR-26a. The expression of the anti-apoptotic protein Bcl-2 was decreased in PANC-miR-26a and MIA-miR-26a, while the expression of the pro-apoptotic protein Bax was increased. Compared with HPDE, miR-26a was down-regulated in PANC-1 and MIA PaCa-2. After overexpression of miR-26a, the proliferation of PANC-1 and MIA PaCa-2 cell lines was weakened. Conclusion: Molecular mechanism is the negative regulation of CyclinE2 by miR-26a as well as the expressions of downstream mitochondrial apoptosis proteins Bcl-2 and Bax.

Protective effects of cardiac resynchronization therapy in a canine model with experimental heart failure by improving mitochondrial function: a mitochondrial proteomics study.

Cardiac resynchronization therapy (CRT) is well acknowledged as an effective treatment for dyssynchronous heart failure. However, the molecular mechanism is unclear to date. Mitochondrial dysfunction and impaired energetic metabolism are two important mechanisms that lead to heart failure. Therefore, we aim to screen the changes of mitochondria-associated proteins and signaling pathways involved in heart failure and CRT treatment. A total of 24 beagle dogs were randomly assigned into control (CON), heart failure (HF), or CRT group. Myocardial mitochondria from the free wall of left ventricle was extracted for isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with two-dimensional liquid chromatography tandem mass spectrometry analysis (2DLC-MS/MS). A total of 2190 proteins were identified, among which 234 proteins were differentially expressed in HF compared with CON group, 151 proteins were differentially expressed in CRT compared with HF group. A total of 192 of the 234 differentially expressed proteins in HF group were changed oppositely by CRT treatment, and 128 of the 151 CRT-induced differentially expressed proteins showed opposite trend of expression to HF/CON. Gene Ontology analysis of the 128 proteins revealed that 16 were localized in mitochondria, 17 were associated with calcium signaling, and 7 could be secreted extracellularly for cell-to-cell signaling. Calpain-1 (CAPN1), which is localized to mitochondria and related to calcium signaling, was upregulated in HF and downregulated after CRT treatment. CRT treatment also improved mitochondrial morphology and function and reduced collagen areas of both interstitial and perivascular fibrosis. CRT treatment significantly improved cardiac function, reduced myocardial fibrosis, and enhanced mitochondrial function in the failing heart through CAPN1 downregulation.

Protective effects of nicotinamide mononucleotide against oxidative stress-induced PC12 cell death via mitochondrial enhancement

Oxidative stress is involved in most neurodegenerative diseases. Although it has been reported that the NAD+ precursor nicotinamide mononucleotide (NMN) improves symptoms in some oxidative stress-associated disease models, the detailed mechanisms have yet to be elucidated. In this study, we hypothesized that NMN attenuates oxidative stress-induced cell damage via sirtuin activation and increased numbers of mitochondria. To investigate whether NMN can protect neuronal cells from oxidative stress via the enhancement of mitochondrial function, we used PC12 cells treated with 6-hydroxydopamine (6-OHDA) or hydrogen peroxide (H2O2) as oxidative stress models. With regard to cytoprotective effects, pretreatment with NMN significantly reduced cell death induced by these stressors. These protective effects were attenuated by the sirtuin inhibitor, sirtinol, and knock-down of the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α), which activates mitochondrial biogenesis. To clarify if NMN increased mitochondria, intracellular mitochondria were quantified using cytometric analysis. NMN clearly increased the numbers of intracellular mitochondria, and this increase was attenuated by sirtinol. Moreover, we quantified the mitochondria-associated proteins Sirt3 and superoxide dismutase 2 (SOD2) and found that treatment with NMN increased these proteins. We conclude that NMN protects neuronal cells from oxidative stress via sirtuin activation followed by mitochondrial enhancement.