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Racing performance traits are the main indicators for evaluating the performance and value of sport horses. The aim of this study was to identify the key genes for racing performance traits in Yili horses by performing a genome-wide association study (GWAS). Breeding values for racing performance traits were calculated for Yili horses (n = 827) using an animal model. Genome-wide association analysis of racing performance traits in horses (n = 236) was carried out using the Blink, and FarmCPU models in GAPIT software, and genes within the significant regions were functionally annotated. The results of GWAS showed that a total of 24 significant SNP markers (P < 6.05 × 10− 9) and 22 suggestive SNP markers (P < 1.21 × 10− 7) were identified. Among them, the Blink associated 16 significant SNP loci and FarmCPU associated 12 significant SNP loci. A total of 127 candidate genes (50 significant) were annotated. Among these, CNTN6 (motor coordination), NIPA1 (neuronal development), and DCC (dopamine pathway maturation) may be the main candidate genes affecting speed traits. SHANK2 (neuronal synaptic regulation), ISCA1 (mitochondrial protein assembly), and KCNIP4 (neuronal excitability) may be the main candidate genes affecting ranking score traits. A common locus (ECA1: 22698579) was significantly associated with racing performance traits, and the function of the genes at this locus needs to be studied in depth. These findings will provide new insights into the detection and selection of genetic variants for racing performance and will help to accelerate the genetic improvement of Yili horses. [ABSTRACT FROM AUTHOR]

Presenilin-associated protein (PSAP) is a mitochondrial proapoptotic protein as established in cell biology studies. It remains unknown whether it involves in neurodegenerative diseases. Here, we explored PASP expression in adult and aged human brains and its alteration relative to Alzheimer-disease (AD)-type neuropathology. In pathology-free brains, light PASP immunoreactivity (IR) occurred among largely principal neurons in the cerebrum and subcortical structures. In the brains with AD pathology, enhanced PSAP IR occurred in neuronal and neuritic profiles with a tangle-like appearance, with PSAP and pTau protein levels elevated in neocortical lysates relative to control. Neuronal/neuritic profiles with enhanced PSAP IR partially colocalized with pTau, but invariably with Amylo-Glo labelled tangles. The neuronal somata with enhanced PASP IR also showed diminished IR for casein kinase 1 delta (Ck1δ), a marker of granulovacuolar degeneration; and diminished IR for sortilin, which is normally expressed in membrane and intracellular protein sorting/trafficking organelles. In old 3xTg-AD mice with β-amyloid and pTau pathologies developed in the brain, PSAP IR in the cerebral sections exhibited no difference relative to wildtype mice. These findings indicate that PSAP upregulation is involved in the course of tangle formation especially in the human brain during aging and in AD pathogenesis. [ABSTRACT FROM AUTHOR]

The most common mutation in southern Chinese individuals with late-onset multiple acyl-coenzyme A dehydrogenase deficiency (MADD; a fatty acid metabolism disorder) is c.250G > A (p.Ala84Thr) in the electron transfer flavoprotein dehydrogenase gene (ETFDH). Various phenotypes, including episodic weakness or rhabdomyolysis, exercise intolerance, and peripheral neuropathy, have been reported in both muscular and neuronal contexts. Our cellular models of MADD exhibit neurite growth defects and excessive apoptosis. Given that axonal degeneration and neuronal apoptosis may be regulated by B-cell lymphoma (BCL)-2 family proteins and mitochondrial outer membrane permeabilization through the activation of proapoptotic molecules, we measured the expression levels of proapoptotic BCL-2 family proteins (e.g., BCL-2-associated X protein and p53-upregulated modulator of apoptosis), cytochrome c, caspase-3, and caspase-9 in NSC-34 cells carrying the most common ETFDH mutation. The levels of these proteins were higher in the mutant cells than in the wide-type cells. Subsequent treatment of the mutant cells with coenzyme Q10 downregulated activated protein expression and mitigated neurite growth defects. These results suggest that the activation of the BCL-2/mitochondrial outer membrane permeabilization/apoptosis pathway promotes apoptosis in cellular models of MADD and that coenzyme Q10 can reverse this effect. Our findings aid the development of novel therapeutic strategies for reducing axonal degeneration and neuronal apoptosis in MADD. [ABSTRACT FROM AUTHOR]

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with β-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis. [ABSTRACT FROM AUTHOR]

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria. [ABSTRACT FROM AUTHOR]

Selective degradation of dysfunctional or excess mitochondria is a fundamental process crucial for cell homeostasis in almost all eukaryotes. This process relies on autophagy, an intracellular self-eating system conserved from yeast to humans and is thus called mitophagy. Detailed mechanisms of mitophagy remain to be fully understood. Here we show that mitochondrial degradation in budding yeast, which requires the pro-mitophagic protein Atg32, is strongly reduced in cells lacking Egd1, a beta subunit of the nascent polypeptide-associated complex acting in cytosolic ribosome attachment and protein targeting to mitochondria. By contrast, loss of the sole alpha subunit Egd2 or the beta subunit paralogue Btt1 led to only a partial or slight reduction in mitophagy. We also found that phosphorylation of Atg32, a crucial step for priming mitophagy, is decreased in the absence of Egd1. Forced Atg32 hyperphosphorylation almost completely restored mitophagy in egd1-null cells. Together, we propose that Egd1 acts in Atg32 phosphorylation to facilitate mitophagy. [ABSTRACT FROM AUTHOR]

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels. [ABSTRACT FROM AUTHOR]

Cellular senescence is a therapy endpoint in melanoma, and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and mitochondrial energy metabolism supports resistance to therapy in melanoma. In a previous report we showed that senescence, induced by the DNA methylating agent temozolomide, increased the level of fusion proteins mitofusin 1 and 2 in melanoma, and silencing Mfn1 or Mfn2 expression reduced interleukin-6 secretion by senescent cells. Here we expanded these observations evaluating the secretome of senescent melanoma cells using shotgun proteomics, and explored the impact of silencing Mfn1 on the SASP. A significant increase in proteins reported to reduce the immune response towards the tumor was found in the media of senescent cells. The secretion of several of these immunomodulatory proteins was affected by Mfn1 silencing, among them was galectin-9. In agreement, tumors lacking mitofusin 1 responded better to treatment with the methylating agent dacarbazine, tumor size was reduced and a higher immune cell infiltration was detected in the tumor. Our results highlight mitochondrial dynamic proteins as potential pharmacological targets to modulate the SASP in the context of melanoma treatment. [ABSTRACT FROM AUTHOR]

The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria. [ABSTRACT FROM AUTHOR]

Artesunate is a derivative of artemisinin, an active compound isolated from Artemisia annua which has been used in Traditional Chinese Medicine and to treat malaria worldwide. Artemisinin derivatives have exhibited anti-cancer activity against both solid tumors and leukemia. The direct target(s) of artesunate are controversial; although, heme-bound proteins in the mitochondria have been implicated. We utilized computational modeling to calculate the predicted binding score of artesunate with heme-bound mitochondrial proteins and identified cytochrome c as potential artesunate target. UV–visible spectroscopy showed changes in the absorbance spectrum, and thus protein structure, when cytochrome c was incubated with artesunate. Artesunate induces apoptosis, disrupts mitochondrial membrane potential, and is antagonized by methazolamide in pediatric AML cells indicating a probable mechanism of action involving cytochrome c. We utilized a multi-disciplinary approach to show that artesunate can interact with and is dependent on cytochrome c release to induce cell death in pediatric AML cell lines. [ABSTRACT FROM AUTHOR]

Mitochondria morphology and function, and their quality control by mitophagy, are essential for heart function. We investigated whether these are influenced by time of the day (TOD), sex, and fed or fasting status, using transmission electron microscopy (EM), mitochondrial electron transport chain (ETC) activity, and mito-QC reporter mice. We observed peak mitochondrial number at ZT8 in the fed state, which was dependent on the intrinsic cardiac circadian clock, as hearts from cardiomyocyte-specific BMAL1 knockout (CBK) mice exhibit different TOD responses. In contrast to mitochondrial number, mitochondrial ETC activities do not fluctuate across TOD, but decrease immediately and significantly in response to fasting. Concurrent with the loss of ETC activities, ETC proteins were decreased with fasting, simultaneous with significant increases of mitophagy, mitochondrial antioxidant protein SOD2, and the fission protein DRP1. Fasting-induced mitophagy was lost in CBK mice, indicating a direct role of BMAL1 in regulating mitophagy. This is the first of its kind report to demonstrate the interactions between sex, fasting, and TOD on cardiac mitochondrial structure, function and mitophagy. These studies provide a foundation for future investigations of mitochondrial functional perturbation in aging and heart diseases. [ABSTRACT FROM AUTHOR]

Lack of efficient insulin secretion from the pancreas can lead to impaired glucose tolerance (IGT), prediabetes, and diabetes. We have previously identified two IGT-associated single nucleotide polymorphisms (SNPs) rs62212118 and rs13052524 located at two overlapping genes: MRPS6 and SLC5A3. In this study, we show that MRPS6 but not SLC5A3 regulates glucose-stimulated insulin secretion (GSIS) in primary human β-cell and a mouse pancreatic insulinoma β-cell line. Data mining and biochemical studies reveal that MRPS6 is positively regulated by the mitochondrial unfolded protein response (UPRmt), but feedback inhibits UPRmt. Disruption of such feedback by MRPS6 knockdown causes UPRmt hyperactivation in high glucose conditions, hence elevated ROS levels, increased apoptosis, and impaired GSIS. Conversely, MRPS6 overexpression reduces UPRmt, mitigates high glucose-induced ROS levels and apoptosis, and enhances GSIS in an ATF5-dependent manner. Consistently, UPRmt up-regulation or down-regulation by modulating ATF5 expression is sufficient to decrease or increase GSIS. The negative role of UPRmt in GSIS is further supported by analysis of public transcriptomic data from murine islets. In all, our studies identify MRPS6 and UPRmt as novel modulators of GSIS and apoptosis in β-cells, contributing to our understanding of the molecular and cellular mechanisms of IGT, prediabetes, and diabetes. [ABSTRACT FROM AUTHOR]

A Kinase Interacting Protein 1 (AKIP1) is a signalling adaptor that promotes mitochondrial respiration and attenuates mitochondrial oxidative stress in cultured cardiomyocytes. We sought to determine whether AKIP1 influences mitochondrial function and the mitochondrial adaptation in response to exercise in vivo. We assessed mitochondrial respiratory capacity, as well as electron microscopy and mitochondrial targeted-proteomics in hearts from mice with cardiomyocyte-specific overexpression of AKIP1 (AKIP1-TG) and their wild type (WT) littermates. These parameters were also assessed after four weeks of voluntary wheel running. In contrast to our previous in vitro study, respiratory capacity measured as state 3 respiration on palmitoyl carnitine was significantly lower in AKIP1-TG compared to WT mice, whereas state 3 respiration on pyruvate remained unaltered. Similar findings were observed for maximal respiration, after addition of FCCP. Mitochondrial DNA damage and oxidative stress markers were not elevated in AKIP1-TG mice and gross mitochondrial morphology was similar. Mitochondrial targeted-proteomics did reveal reductions in mitochondrial proteins involved in energy metabolism. Exercise performance was comparable between genotypes, whereas exercise-induced cardiac hypertrophy was significantly increased in AKIP1-TG mice. After exercise, mitochondrial state 3 respiration on pyruvate substrates was significantly lower in AKIP1-TG compared with WT mice, while respiration on palmitoyl carnitine was not further decreased. This was associated with increased mitochondrial fission on electron microscopy, and the activation of pathways associated with mitochondrial fission and mitophagy. This study suggests that AKIP1 regulates the mitochondrial proteome involved in energy metabolism and promotes mitochondrial turnover after exercise. Future studies are required to unravel the mechanistic underpinnings and whether the mitochondrial changes are required for the AKIP1-induced physiological cardiac growth. [ABSTRACT FROM AUTHOR]

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type. [ABSTRACT FROM AUTHOR]

Our study utilizes a longitudinal isotopic metabolic labeling approach in vivo in combination with organelle fraction proteomics to address the role of parkin in mitochondrial protein turnover in mice. The use of metabolic labeling provides a method to quantitatively determine the global changes in protein half-lives whilst simultaneously assessing protein expression. Studying two diverse mitochondrial populations, we demonstrated the median half-life of brain striatal synaptic mitochondrial proteins is significantly greater than that of hepatic mitochondrial proteins (25.7 vs. 3.5 days). Furthermore, loss of parkin resulted in an overall, albeit modest, increase in both mitochondrial protein abundance and half-life. Pathway and functional analysis of our proteomics data identified both known and novel pathways affected by loss of parkin that are consistent with its role in both mitochondrial quality control and neurodegeneration. Our study therefore adds to a growing body of evidence suggesting dependence on parkin is low for basal mitophagy in vivo and provides a foundation for the investigation of novel parkin targets. [ABSTRACT FROM AUTHOR]

Maintaining mitochondrial function is critical to an improved healthspan and lifespan. Introducing mild stress by inhibiting mitochondrial translation invokes the mitochondrial unfolded protein response (UPRmt) and increases lifespan in several animal models. Notably, lower mitochondrial ribosomal protein (MRP) expression also correlates with increased lifespan in a reference population of mice. In this study, we tested whether partially reducing the gene expression of a critical MRP, Mrpl54, reduced mitochondrial DNA-encoded protein content, induced the UPRmt, and affected lifespan or metabolic health using germline heterozygous Mrpl54 mice. Despite reduced Mrpl54 expression in multiple organs and a reduction in mitochondrial-encoded protein expression in myoblasts, we identified few significant differences between male or female Mrpl54+/− and wild type mice in initial body composition, respiratory parameters, energy intake and expenditure, or ambulatory motion. We also observed no differences in glucose or insulin tolerance, treadmill endurance, cold tolerance, heart rate, or blood pressure. There were no differences in median life expectancy or maximum lifespan. Overall, we demonstrate that genetic manipulation of Mrpl54 expression reduces mitochondrial-encoded protein content but is not sufficient to improve healthspan in otherwise healthy and unstressed mice. [ABSTRACT FROM AUTHOR]

Organellar genomes of liverworts are considered as one of the most stable among plants, with rare events of gene loss and structural rearrangements. However, not all lineages of liverworts are equally explored in the field of organellar genomics, and subclass Pellidae is one of the less known. Hybrid assembly, using both short- and long-read technologies enabled the assembly of repeat-rich mitogenomes of Pellia and Apopellia revealing extraordinary reduction of length in the latter which impacts only intergenic spacers. The mitogenomes of Apopellia were revealed to be the smallest among all known liverworts—109 k bp, despite retaining all introns. The study also showed the loss of one tRNA gene in Apopellia mitogenome, although it had no impact on the codon usage pattern of mitochondrial protein coding genes. Moreover, it was revealed that Apopellia and Pellia differ in codon usage by plastome CDSs, despite identical tRNA gene content. Molecular identification of species is especially important where traditional taxonomic methods fail, especially within Pellidae where cryptic speciation is well recognized. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. Application of super-barcodes, based on complete mitochondrial or plastid genomes sequences enable identification of all cryptic lineages within Apopellia and Pellia genera, however in some particular cases, mitogenomes were more efficient in species delimitation than plastomes. [ABSTRACT FROM AUTHOR]

Lysine Nɛ-acylations, such as acetylation or succinylation, are post-translational modifications that regulate protein function. In mitochondria, lysine acylation is predominantly non-enzymatic, and only a specific subset of the proteome is acylated. Coenzyme A (CoA) can act as an acyl group carrier via a thioester bond, but what controls the acylation of mitochondrial lysines remains poorly understood. Using published datasets, here we found that proteins with a CoA-binding site are more likely to be acetylated, succinylated, and glutarylated. Using computational modeling, we show that lysine residues near the CoA-binding pocket are highly acylated compared to those farther away. We hypothesized that acyl-CoA binding enhances acylation of nearby lysine residues. To test this hypothesis, we co-incubated enoyl-CoA hydratase short chain 1 (ECHS1), a CoA-binding mitochondrial protein, with succinyl-CoA and CoA. Using mass spectrometry, we found that succinyl-CoA induced widespread lysine succinylation and that CoA competitively inhibited ECHS1 succinylation. CoA-induced inhibition at a particular lysine site correlated inversely with the distance between that lysine and the CoA-binding pocket. Our study indicated that CoA acts as a competitive inhibitor of ECHS1 succinylation by binding to the CoA-binding pocket. Together, this suggests that proximal acylation at CoA-binding sites is a primary mechanism for lysine acylation in the mitochondria. [ABSTRACT FROM AUTHOR]

Constitutive expression of the chemokine Mcp1 in mouse cardiomyocytes creates a model of inflammatory cardiomyopathy, with death from heart failure at age 7-8 months. A critical pathogenic role has previously been proposed for induced oxidative stress, involving NADPH oxidase activation. To test this idea, we exposed the mice to elevated oxygen levels. Against expectation, this prevented, rather than accelerated, the ultrastructural and functional signs of heart failure. This result suggests that the immune signaling initiated by Mcp1 leads instead to the inhibition of cellular oxygen usage, for which mitochondrial respiration is an obvious target. To address this hypothesis, we combined the Mcp1 model with xenotopic expression of the alternative oxidase (AOX), which provides a sink for electrons blocked from passage to oxygen via respiratory complexes III and IV. Ubiquitous AOX expression provided only a minor delay to cardiac functional deterioration and did not prevent the induction of markers of cardiac and metabolic remodeling considered a hallmark of the model. Moreover, cardiomyocyte-specific AOX expression resulted in exacerbation of Mcp1-induced heart failure, and failed to rescue a second cardiomyopathy model directly involving loss of cIV. Our findings imply that mitochondrial involvement in the pathology of inflammatory cardiomyopathy is multifaceted and complex.

Both the identity and the amount of a carbon source present in laboratory or industrial cultivation media have major impacts on the growth and physiology of a microbial species. In the case of the yeast Saccharomyces cerevisiae, sucrose is arguably the most important sugar used in industrial biotechnology, whereas glucose is the most common carbon and energy source used in research, with many well-known and described regulatory effects, e.g. glucose repression. Here we compared the label-free proteomes of exponentially growing S. cerevisiae cells in a defined medium containing either sucrose or glucose as the sole carbon source. For this purpose, bioreactor cultivations were employed, and three different strains were investigated, namely: CEN.PK113-7D (a common laboratory strain), UFMG-CM-Y259 (a wild isolate), and JP1 (an industrial bioethanol strain). These strains present different physiologies during growth on sucrose; some of them reach higher specific growth rates on this carbon source, when compared to growth on glucose, whereas others display the opposite behavior. It was not possible to identify proteins that commonly presented either higher or lower levels during growth on sucrose, when compared to growth on glucose, considering the three strains investigated here, except for one protein, named Mnp1—a mitochondrial ribosomal protein of the large subunit, which had higher levels on sucrose than on glucose, for all three strains. Interestingly, following a Gene Ontology overrepresentation and KEGG pathway enrichment analyses, an inverse pattern of enriched biological functions and pathways was observed for the strains CEN.PK113-7D and UFMG-CM-Y259, which is in line with the fact that whereas the CEN.PK113-7D strain grows faster on glucose than on sucrose, the opposite is observed for the UFMG-CM-Y259 strain. [ABSTRACT FROM AUTHOR]

Imeglimin is a recently launched antidiabetic drug structurally related to metformin. To provide insight into the pharmacological properties of imeglimin, we investigated its effects on hepatocytes and compared them with those of metformin. The effects of imeglimin on mitochondrial function in HepG2 cells or mouse primary hepatocytes were examined with an extracellular flux analyzer and on gene expression in HepG2 cells by comprehensive RNA-sequencing analysis. The effects of the drug on AMPK activity in HepG2 cells, mouse primary hepatocytes, and mouse liver were also examined. Treatment of HepG2 cells or mouse primary hepatocytes with imeglimin reduced the oxygen consumption rate coupled to ATP production. Imeglimin activated AMPK in these cells whereas the potency was smaller than metformin. Bolus administration of imeglimin in mice also activated AMPK in the liver. Whereas the effects of imeglimin and metformin on gene expression in HepG2 cells were similar overall, the expression of genes encoding proteins of mitochondrial respiratory complex III and complex I was upregulated by imeglimin but not by metformin. Our results suggest that imeglimin and metformin exert similar pharmacological effects on mitochondrial respiration, AMPK activity, and gene expression in cultured hepatocytes, whereas the two drugs differ in their effects on the expression of certain genes related to mitochondrial function. [ABSTRACT FROM AUTHOR]

Autophagy of damaged mitochondria, called mitophagy, is an important organelle quality control process involved in the pathogenesis of inflammation, cancer, aging, and age-associated diseases. Many of these disorders are associated with altered expression of the inner mitochondrial membrane (IMM) protein Prohibitin 1. The mechanisms whereby dysfunction occurring internally at the IMM and matrix activate events at the outer mitochondrial membrane (OMM) to induce mitophagy are not fully elucidated. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a specific role of Prohibitin-induced mitophagy in maintaining intestinal homeostasis. We demonstrate that Prohibitin 1 induces mitophagy in response to increased mitochondrial reactive oxygen species (ROS) through binding to mitophagy receptor Nix/Bnip3L and independently of Parkin. Prohibitin 1 is required for ROS-induced Nix localization to mitochondria and maintaining homeostasis of epithelial cells highly susceptible to mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1. [ABSTRACT FROM AUTHOR]

Mitochondrial reactive oxygen species (ROS) are essential regulators of cellular signaling, metabolism and epigenetics underlying the pathophysiology of numerous diseases. Despite the critical function of redox regulation in mitochondria, currently there are limited methods available to monitor protein oxidation in this key subcellular organelle. Here, we describe compounds for imaging sulfenylated proteins in mitochondria: DCP-NEt2-Coumarin (DCP-NEt2C) and rhodamine-based DCP-Rho1. Side-by-side comparison studies are presented on the reactivity of DCP-NEt2C and DCP-Rho1 with a model protein sulfenic acid (AhpC-SOH) and mitochondrial localization to identify optimized experimental conditions for labeling and visualization of protein sulfenylation that would be independent of mitochondria membrane potential and would not impact mitochondrial function. These probes are applied to image mitochondrial protein sulfenylation under conditions of serum starvation and in a cell culture model of lung cancer exposed to ionizing radiation and silver nanoparticles, agents serving dual functions as environmental stressors and cancer therapeutics.

DNA damaging agents are a mainstay of standard chemotherapy for ovarian cancer. Unfortunately, resistance to such DNA damaging agents frequently develops, often due to increased activity of DNA repair pathways. Sideroflexin 4 (SFXN4) is a little-studied inner mitochondrial membrane protein. Here we demonstrate that SFXN4 plays a role in synthesis of iron sulfur clusters (Fe-S) in ovarian cancer cells and ovarian cancer tumor-initiating cells, and that knockdown of SFXN4 inhibits Fe-S biogenesis in ovarian cancer cells. We demonstrate that this has two important consequences that may be useful in anti-cancer therapy. First, inhibition of Fe-S biogenesis triggers the accumulation of excess iron, leading to oxidative stress. Second, because enzymes critical to multiple DNA repair pathways require Fe-S clusters for their function, DNA repair enzymes and DNA repair itself are inhibited by reduction of SFXN4. Through this dual mechanism, SFXN4 inhibition heightens ovarian cancer cell sensitivity to DNA-damaging drugs and DNA repair inhibitors used in ovarian cancer therapy, such as cisplatin and PARP inhibitors. Sensitization is achieved even in drug resistant ovarian cancer cells. Further, knockout of SFXN4 decreases DNA repair and profoundly inhibits tumor growth in a mouse model of ovarian cancer metastasis. Collectively, these results suggest that SFXN4 may represent a new target in ovarian cancer therapy. [ABSTRACT FROM AUTHOR]

Human mitochondrial chaperonin mHsp60 is broadly associated with various human health conditions and the V72I mutation in mHsp60 causes a form of hereditary spastic paraplegia, a neurodegenerative disease. The main function of mHsp60 is to assist folding of mitochondrial proteins in an ATP-dependent manner. In this study, we unexpectedly found that mutant mHsp60V72I was more stable structurally and more active in the ATPase activity than the wildtype. Analysis of our recently solved cryo-EM structure of mHsp60 revealed allosteric roles of V72I in structural stability and ATPase activity, which were supported by studies including those using the V72A mutation. Despite with the increases in structural stability and ATPase activity, mHsp60V72I was less efficient in folding malate dehydrogenase, a putative mHsp60 substrate protein in mitochondria and also commonly used in chaperonin studies. In addition, although mHsp60V72I along with its cochaperonin mHsp10 was able to substitute the E. coli chaperonin system in supporting cell growth under normal temperature of 37 °C, it was unable under heat shock temperature of 42 °C. Our results support the importance of structural dynamics and an optimal ATP turnover that mHsp60 has evolved for its function and physiology. We propose that unproductive energy utilization, or hyperactive ATPase activity and compromised folding function, not mutually exclusive, are responsible for the V72I pathology in neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Epigenetic silencing in Friedreich ataxia (FRDA), induced by an expanded GAA triplet-repeat in intron 1 of the FXN gene, results in deficiency of the mitochondrial protein, frataxin. A lesser known extramitochondrial isoform of frataxin detected in erythrocytes, frataxin-E, is encoded via an alternate transcript (FXN-E) originating in intron 1 that lacks a mitochondrial targeting sequence. We show that FXN-E is deficient in FRDA, including in patient-derived cell lines, iPS-derived proprioceptive neurons, and tissues from a humanized mouse model. In a series of FRDA patients, deficiency of frataxin-E protein correlated with the length of the expanded GAA triplet-repeat, and with repeat-induced DNA hypermethylation that occurs in close proximity to the intronic origin of FXN-E. CRISPR-induced epimodification to mimic DNA hypermethylation seen in FRDA reproduced FXN-E transcriptional deficiency. Deficiency of frataxin E is a consequence of FRDA-specific epigenetic silencing, and therapeutic strategies may need to address this deficiency. [ABSTRACT FROM AUTHOR]

Mitochondria, the powerhouse and the vital signaling hub of the cell, participate in a variety of biological processes, such as apoptosis, redox responses, cell senescence, autophagy, and iron homeostasis. Mitochondria form a mostly tubular network, made up of an outer and a cristeae-forming inner membrane. The network undergoes dynamic fusion and fission that change its morphological structure according to the functional needs. Approximately 1500 mitochondrial proteins encoded by nuclear genome plus over 10 proteins encoded by mitochondrial DNA are folded and assembled in the mitochondria under a high-fidelity control system. These proteins are involved in oxidative phosphorylation, metabolism, network and cristae dynamics, mitophagy, import machinery, ion channels, and mitochondrial DNA maintenance. This Collection gathers original research that advances our understanding of the monitoring techniques and pathophysiological significance of mitochondrial dynamics in health and disease. [ABSTRACT FROM AUTHOR]

Post-translational modifications, such as lysine acetylation, regulate the activity of diverse proteins across many cellular compartments. Protein deacetylation in mitochondria is catalyzed by the enzymatic activity of the NAD+-dependent deacetylase sirtuin 3 (SIRT3), however it remains unclear whether corresponding mitochondrial acetyltransferases exist. We used a bioinformatics approach to search for mitochondrial proteins with an acetyltransferase catalytic domain, and identified a novel splice variant of ELP3 (mt-ELP3) of the elongator complex, which localizes to the mitochondrial matrix in mammalian cells. Unexpectedly, mt-ELP3 does not mediate mitochondrial protein acetylation but instead induces a post-transcriptional modification of mitochondrial-transfer RNAs (mt-tRNAs). Overexpression of mt-ELP3 leads to the protection of mt-tRNAs against the tRNA-specific RNase angiogenin, increases mitochondrial translation, and furthermore increases expression of OXPHOS complexes. This study thus identifies mt-ELP3 as a non-canonical mt-tRNA modifying enzyme. [ABSTRACT FROM AUTHOR]

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein—DWEORG1—from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a complex, chronic, relapsing and heterogeneous disease induced by environmental, genomic, microbial and immunological factors. MCJ is a mitochondrial protein that regulates the metabolic status of macrophages and their response to translocated bacteria. Previously, an acute murine model of DSS-induced colitis showed increased disease severity due to MCJ deficiency. Unexpectedly, we now show that MCJ-deficient mice have augmented tumor necrosis factor α converting enzyme (TACE) activity in the context of chronic inflammation. This adaptative change likely affects the balance between soluble and transmembrane TNF and supports the association of the soluble form and a milder phenotype. Interestingly, the general shifts in microbial composition previously observed during acute inflammation were absent in the chronic model of inflammation in MCJ-deficient mice. However, the lack of the mitochondrial protein resulted in increased alpha diversity and the reduction in critical microbial members associated with inflammation, such as Ruminococcus gnavus, which could be associated with TACE activity. These results provide evidence of the dynamic metabolic adaptation of the colon tissue to chronic inflammatory changes mediated by the control of mitochondrial function. [ABSTRACT FROM AUTHOR]

In the last decade, there has been an increased appreciation for mitochondria as central hubs in diverse processes, such as cellular energy, immunity, and signal transduction. As such, we have become aware that mitochondrial dysfunction underlies many diseases, including primary (mutations in genes encoding mitochondrial proteins) and secondary mitochondrial diseases (mutations in non-mitochondrial genes critical for mitochondrial biology), as well as complex diseases with mitochondrial dysfunction (chronic or degenerative diseases). Evidence suggests that mitochondrial dysfunction may often precede other pathological signs in these disorders, further modulated by genetics, environment, and lifestyle. [ABSTRACT FROM AUTHOR]

Fancd2 is a component of the Fanconi anemia (FA) DNA repair pathway, which is frequently found defective in human cancers. The full repertoire of Fancd2 functions in normal development and tumorigenesis remains to be determined. Here we developed a Flag- and hemagglutinin-tagged Fancd2 knock-in mouse strain that allowed a high throughput mass spectrometry approach to search for Fancd2-binding proteins in different mouse organs. In addition to DNA repair partners, we observed that many Fancd2-interacting proteins are mitochondrion-specific. Fancd2 localizes in the mitochondrion and associates with the nucleoid complex components Atad3 and Tufm. The Atad3-Tufm complex is disrupted in Fancd2-/- mice and those deficient for the FA core component Fanca. Fancd2 mitochondrial localization requires Atad3. Collectively, these findings provide evidence for Fancd2 as a crucial regulator of mitochondrion biosynthesis, and of a molecular link between FA and mitochondrial homeostasis.

Extant symbioses illustrate endosymbiosis is a driving force for evolution and diversification. In the ciliate Paramecium bursaria, the endosymbiotic alga Chlorella variabilis in perialgal vacuole localizes beneath the host cell cortex by adhesion between the perialgal vacuole membrane and host mitochondria. We investigated whether host mitochondria are also affected by algal endosymbiosis. Transmission electron microscopy of host cells showed fewer mitochondria beneath the algae-bearing host cell cortex than that of alga-free cells. To compare the density and distribution of host mitochondria with or without symbiotic algae, we developed a monoclonal antibody against Paramecium mitochondria. Immunofluorescence microscopy with the monoclonal antibody showed that the mitochondrial density of the algae-bearing P. bursaria was significantly lower than that of the alga-free cells. The total cell protein concentration of alga-free P. bursaria cells was approximately 1.8-fold higher than that of algae-bearing cells, and the protein content of mitochondria was significantly higher in alga-free cells than that in the algae-bearing cells. These results corresponded with those obtained by transmission electron and immunofluorescence microscopies. This paper shows that endosymbiotic algae affect reduced mitochondrial number in the host P. bursaria significantly. [ABSTRACT FROM AUTHOR]

Heart failure is a multifactorial disease that affects an estimated 38 million people worldwide. Current pharmacotherapy of heart failure with reduced ejection fraction (HFrEF) includes combination therapy with angiotensin-converting enzyme inhibitors (ACEi) and β-adrenergic receptor blockers (β-AR blockers), a therapy also used as treatment for non-cardiac conditions. Our knowledge of the molecular changes accompanying treatment with ACEi and β-AR blockers is limited. Here, we applied proteomics and phosphoproteomics approaches to profile the global changes in protein abundance and phosphorylation state in cardiac left ventricles consequent to combination therapy of β-AR blocker and ACE inhibitor in HFrEF and control hearts. The phosphorylation changes induced by treatment were profoundly different for failing than for non-failing hearts. HFrEF was characterized by profound downregulation of mitochondrial proteins coupled with derangement of β-adrenergic and pyruvate dehydrogenase signaling. Upon treatment, phosphorylation changes consequent to HFrEF were reversed. In control hearts, treatment mainly led to downregulation of canonical PKA signaling. The observation of divergent signaling outcomes depending on disease state underscores the importance of evaluating drug effects within the context of the specific conditions present in the recipient heart. [ABSTRACT FROM AUTHOR]

Morphine is one of the most potent opioid analgesic used for pain treatment. Morphine action in the central nervous system requires crossing the blood–brain barrier. Due to the controversial relationship between morphine and oxidative stress, the potential pro- or antioxidant effects of morphine in the blood–brain barrier is important to be understood, as oxidative stress could cause its disruption and predispose to neurodegenerative diseases. However, investigation is scarce in human brain endothelial cells. Therefore, the present study evaluated the impact of morphine exposure at three different concentrations (1, 10 and 100 µM) for 24 h and 48 h on primary human brain microvascular endothelial cells. A quantitative data-independent acquisition mass spectrometry strategy was used to analyze proteome modulations. Almost 3000 proteins were quantified of which 217 were reported to be significantly regulated in at least one condition versus untreated control. Pathway enrichment analysis unveiled dysregulation of the Nrf2 pathway involved in oxidative stress response. Seahorse assay underlined mitochondria dysfunctions, which were supported by significant expression modulations of relevant mitochondrial proteins. In conclusion, our study revealed the dysregulation of the Nrf2 pathway and mitochondria dysfunctions after morphine exposure, highlighting a potential redox imbalance in human brain endothelial cells. [ABSTRACT FROM AUTHOR]

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification on serine and threonine residues of cytosolic, nuclear and mitochondrial proteins. O-GlcNAcylation level is regulated by OGT (O-GlcNAc transferase), which adds GlcNAc on proteins, and OGA (O-GlcNAcase), which removes it. Abnormal level of protein O-GlcNAcylation has been observed in numerous cancer cell types, including cervical cancer cells. In the present study, we have evaluated the effect of increasing protein O-GlcNAcylation on cervical cancer-derived CaSki cells. We observed that pharmacological enhancement of protein O-GlcNAcylation by Thiamet G (an inhibitor of OGA) and glucosamine (which provides UDP-GlcNAc substrate to OGT) increases CaSki cells proliferation, migration and survival. Moreover, we showed that increased O-GlcNAcylation promotes IGF-1 receptor (IGF1R) autophosphorylation, possibly through inhibition of protein tyrosine-phosphatase 1B activity. This was associated with increased IGF-1-induced phosphatidyl-Inositol 3-phosphate production at the plasma membrane and increased Akt activation in CaSki cells. Finally, we showed that protein O-GlcNAcylation and Akt phosphorylation levels were higher in human cervical cancer samples compared to healthy cervix tissues, and a highly positive correlation was observed between O-GlcNAcylation level and Akt phosphorylation in theses tissues. Together, our results indicate that increased O-GlcNAcylation, by activating IGF1R/ Phosphatidyl inositol 3-Kinase (PI-3K)/Akt signaling, may participate in cervical cancer cell growth and proliferation. [ABSTRACT FROM AUTHOR]

Despite limited reports on glutamine methylation, methylated glutamine is found to be highly conserved in a "GGQ" motif in both prokaryotes and eukaryotes. In bacteria, glutamine methylation of peptide chain release factors 1/2 (RF1/2) by the enzyme PrmC is essential for translational termination and transcript recycling. Two PrmC homologs, HEMK1 and HEMK2, are found in mammals. In contrast to those of HEMK2, the biochemical properties and biological significance of HEMK1 remain largely unknown. In this study, we demonstrated that HEMK1 is an active methyltransferase for the glutamine residue of the GGQ motif of all four putative mitochondrial release factors (mtRFs)—MTRF1, MTRF1L, MRPL58, and MTRFR. In HEMK1-deficient HeLa cells, GGQ motif glutamine methylation was absent in all the mtRFs. We examined cell growth and mitochondrial properties, but disruption of the HEMK1 gene had no considerable impact on the overall cell growth, mtDNA copy number, mitochondrial membrane potential, and mitochondrial protein synthesis under regular culture condition with glucose as a carbon source. Furthermore, cell growth potential of HEMK1 KO cells was still maintained in the respiratory condition with galactose medium. Our results suggest that HEMK1 mediates the GGQ methylation of all four mtRFs in human cells; however, this specific modification seems mostly dispensable in cell growth and mitochondrial protein homeostasis at least for HeLa cells under fermentative culture condition. [ABSTRACT FROM AUTHOR]

In the mitochondria-mediated vicious cycle of Alzheimer's disease (AD), intracellular amyloid β (Aβ) induces mitochondrial dysfunction and reactive oxygen species, which further accelerate Aβ accumulation. This vicious cycle is thought to play a pivotal role in the development of AD, although the molecular mechanism remains unclear. Here, we examined the effects of human mitochondrial transcriptional factor A (hTFAM) on the pathology of a mouse model of AD (3xTg-AD), because TFAM is known to protect mitochondria from oxidative stress through maintenance of mitochondrial DNA (mtDNA). Expression of hTFAM significantly improved cognitive function, reducing accumulation of both 8-oxoguanine, an oxidized form of guanine, in mtDNA and intracellular Aβ in 3xTg-AD mice and increasing expression of transthyretin, known to inhibit Aβ aggregation. Next, we found that AD model neurons derived from human induced pluripotent stem cells carrying a mutant PSEN1(P117L) gene, exhibited mitochondrial dysfunction, accumulation of 8-oxoguanine and single-strand breaks in mtDNA, and impaired neuritogenesis with a decreased expression of transthyretin, which is known to be downregulated by oxidative stress. Extracellular treatment with recombinant hTFAM effectively suppressed these deleterious outcomes. Moreover, the treatment increased expression of transthyretin, accompanied by reduction of intracellular Aβ. These results provide new insights into potential novel therapeutic targets.

Abuse of the potent psychostimulant cocaine is widely established to have cardiovascular consequences. The cardiotoxicity of cocaine is mainly associated with oxidative stress and mitochondrial dysfunction. Mitochondrial dynamics and biogenesis, as well as the mitochondrial unfolded protein response (UPRmt), guarantee cardiac mitochondrial homeostasis. Collectively, these mechanisms act to protect against stress, injury, and the detrimental effects of chemicals on mitochondria. In this study, we examined the effects of cocaine on cardiac mitochondrial dynamics, biogenesis, and UPRmt in vivo. Rats administered cocaine via the tail vein at a dose of 20 mg/kg/day for 7 days showed no structural changes in the myocardium, but electron microscopy revealed a significant increase in the number of cardiac mitochondria. Correspondingly, the expressions of the mitochondrial fission gene and mitochondrial biogenesis were increased after cocaine administration. Significant increase in the expression and nuclear translocation of activating transcription factor 5, the major active regulator of UPRmt, were observed after cocaine administration. Accordingly, our findings show that before any structural changes are observable in the myocardium, cocaine alters mitochondrial dynamics, elevates mitochondrial biogenesis, and induces the activation of UPRmt. These alterations might reflect cardiac mitochondrial compensation to protect against the cardiotoxicity of cocaine. [ABSTRACT FROM AUTHOR]

The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation. [ABSTRACT FROM AUTHOR]

The effect of uridine on the myocardial ischemic and reperfusion injury was investigated. A possible mechanism of its cardioprotective action was established. Two rat models were used: (1) acute myocardial ischemia induced by occlusion of the left coronary artery for 60 min; and (2) myocardial ischemia/reperfusion with 30-min ischemia and 120-min reperfusion. In both models, treatment with uridine (30 mg/kg) prevented a decrease in cell energy supply and in the activity of the antioxidant system, as well as an increase in the level of lipid hydroperoxides and diene conjugates. This led to a reduction of the necrosis zone in the myocardium and disturbances in the heart rhythm. The blocker of the mitochondrial ATP-dependent potassium (mitoKATP) channel 5-hydroxydecanoate limited the positive effects of uridine. The data indicate that the cardioprotective action of uridine may be related to the activation of the mitoKATP channel. Intravenously injected uridine was more rapidly eliminated from the blood in hypoxia than in normoxia, and the level of the mitoKATP channel activator UDP in the myocardium after uridine administration increased. The results suggest that the use of uridine can be a potentially effective approach to the management of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Several studies reported that mitochondrial stress induces cytosolic proteostasis in yeast and C. elegans. Notably, inhibition of mitochondrial translation with doxcycyline decreases the toxicity of β-amyloid aggregates, in a C. elegans. However, how mitochondrial stress activates cytosolic proteostasis remains unclear. Further whether doxycycline has this effect in mammals and in disease relevant tissues also remains unclear. We show here that doxycycline treatment in mice drastically reduces the accumulation of proteins destined for degradation by the proteasome in a CNS region-specific manner. This effect is associated with the activation of the ERα axis of the mitochondrial unfolded protein response (UPRmt), in both males and females. However, sexually dimorphic mechanisms of proteasome activation were observed. Doxycycline also activates the proteasome in fission yeast, where ERα is not expressed. Rather, the ancient ERα-coactivator Mms19 regulates this response in yeast. Our results suggest that the UPRmt initiates a conserved mitochondria-to-cytosol stress signal, resulting in proteasome activation, and that this signal has adapted during evolution, in a sex and tissue specific-manner. Therefore, while our results support the use of doxycycline in the prevention of proteopathic diseases, they also indicate that sex is an important variable to consider in the design of future clinical trials using doxycycline. [ABSTRACT FROM AUTHOR]

Extraction of biosurfactants from plants is advantageous than from microbes. The properties and robustness of biosurfactant derived from the mesocarp of Balanites aegyptiaca have been reported. However, the dark brown property of biosurfactant and lack of knowledge of its biocompatibility limits its scope. In the present work, the decolorization protocol for this biosurfactant was optimized using hydrogen peroxide. The hemolytic potential and biocompatibility based on cell toxicity and proliferation were also investigated. This study is the first report on the decolorization and toxicity assay of this biosurfactant. For decolorization of biosurfactant, 34 full factorial design was used, and the data were subjected to ANOVA. Results indicate that 1.5% of hydrogen peroxide can decolorize the biosurfactant most efficiently at 40 °C in 70 min at pH 7. Mitochondrial reductase (MTT) and reactive oxygen species (ROS) assays on M5S mouse skin fibroblast cells revealed that decolorized biosurfactant up to 50 µg/mL for 6 h had no significant toxic effect. Hemolysis assay showed ~ 2.5% hemolysis of human RBCs, indicating the nontoxic effect of this biosurfactant. The present work established a decolorization protocol making the biosurfactant chromatically acceptable. Biocompatibility assays confirm its safer use as observed by experiments on M5S skin fibroblast cells under in vitro conditions. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a common neurodegenerative disease with poor prognosis. New options for drug discovery targets are needed. We developed an imaging based arrayed CRISPR method to interrogate the human genome for modulation of in vitro correlates of AD features, and used this to assess 1525 human genes related to tau aggregation, autophagy and mitochondria. This work revealed (I) a network of tau aggregation modulators including the NF-κB pathway and inflammatory signaling, (II) a correlation between mitochondrial morphology, respiratory function and transcriptomics, (III) machine learning predicted novel roles of genes and pathways in autophagic processes and (IV) individual gene function inferences and interactions among biological processes via multi-feature clustering. These studies provide a platform to interrogate underexplored aspects of AD biology and offer several specific hypotheses for future drug discovery efforts. [ABSTRACT FROM AUTHOR]

In this study, we analyzed the complete mitochondrial genome (mitogenome) of Speiredonia retorta, which is a pest and a member of the Lepidoptera order. In total, the S. retorta mitogenome was found to contain 15,652 base pairs encoding 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, as well as an adenine (A) + thymine (T)-rich region. These findings were consistent with the mitogenome composition of other lepidopterans, as we identified all 13 PCGs beginning at ATN codons. We also found that 11 PCGs terminated with canonical stop codons, whereas cox2 and nad4 exhibited incomplete termination codons. By analyzing the mitogenome of S. retorta using Bayesian inference (BI) and maximum likelihood (ML) models, we were able to further confirm that this species is a member of the Erebidae family. [ABSTRACT FROM AUTHOR]

Oxidative stress is characterized by an excessive concentration of reactive oxygen species (ROS) resulting from a disturbance in the balance between ROS production and their removal by antioxidant systems (SOD, CAT, GPx). Prolonged and intense oxidative stress can cause various forms of damage to cells, which markers are total antioxidant capacity (TAC), reactive oxygen species modulator (ROMO1), and malondialdehyde (MDA). It has been demonstrated that magnetic fields can positively affect human health, for example, by reducing oxidative stress. Determination of the effect of a rotating magnetic field (RMF) on the activity/concentration of selected oxidative stress markers. A group of 30 healthy volunteers (15 women and 15 men) (mean age 24.8 ± 5.1) in the study classified into the following groups: internal control group (CG);1 h 25 Hz (samples placed in the field for one hour at 25 Hz); 3 h 25 Hz (samples placed in the field for 3 h at 25 Hz), the 1 h 50 Hz group ( placed in RMF for an hour at 50 Hz), and a group of 3 h 50 Hz (samples placed in the field for 3 h at 50 Hz). Serum samples were collected in K 2 EDTA tubes.. The magnetic induction value obtained for RMF is 37.06 mT and 42.64 mT.Activity/concentration of selected oxidative stress markers was analyzed by ELISA. The influence of an RMF on the activity/concentration of SOD, MDA, TAC, and ROMO1 was demonstrated (p < 0.001; p = 0.0013; p < 0.001; p = 0.003). The RFM can reduce oxidative stress, as evidenced by higher SOD and CAT activities in the CG than in samples placed in the RFM. Prolonged exposure to the RFM at 50 Hz increased the TAC level, indicating an intensification of oxidative stress in these samples. The optimal conditions for staying in the RFM (reducing oxidative stress) are 1 h 50 Hz for SOD and MDA; 3 h 25 Hz for CAT and TAC. In the case of ROMO1, it is stated that 1 h 25 Hz are the optimal conditions for no increased production of ROS., (© 2024. The Author(s).)

Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels. Isolated mitochondria are a powerful model for separating global cellular changes from intrinsic mitochondrial alterations. However, common laboratory practices for isolating mitochondria (e.g., differential centrifugation) routinely results in organelle preparations with variable mitochondrial purity. To overcome this issue, we developed a mass spectrometry-based method that quantitatively evaluates sample-specific percent mitochondrial enrichment. Sample-specific mitochondrial enrichment was then used to correct various biochemical readouts of mitochondrial function to a 'fixed' amount of mitochondrial protein, thus allowing for intrinsic mitochondrial bioenergetics, relative to the underlying proteome, to be assessed across multiple mouse tissues (e.g., heart, brown adipose, kidney, liver). Our results support the use of mitochondrial-targeted nLC-MS/MS as a method to quantitate mitochondrial enrichment on a per-sample basis, allowing for unbiased comparison of functional parameters between populations of mitochondria isolated from metabolically distinct tissues. This method can easily be applied across multiple experimental settings in which intrinsic shifts in the mitochondrial network are suspected of driving a given physiological or pathophysiological outcome. [ABSTRACT FROM AUTHOR]

Mitochondrial lysine acetylation regulates several metabolic pathways in cardiac cells. The current study investigated whether GCN5L1-mediated lysine acetylation regulates cardiac mitochondrial metabolic proteins in response to a high fat diet (HFD). GCN5L1 cardiac-specific knockout (cKO) mice showed significantly reduced mitochondrial protein acetylation following a HFD relative to wildtype (WT) mice. GCN5L1 cKO mice did not display any decrease in ex vivo cardiac workload in response to a HFD. In contrast, ex vivo cardiac function in HFD-fed WT mice dropped ~ 50% relative to low fat diet (LFD) fed controls. The acetylation status of electron transport chain Complex I protein NDUFB8 was significantly increased in WT mice fed a HFD, but remained unchanged in GCN5L1 cKO mice relative to LFD controls. Finally, we observed that inhibitory acetylation of superoxide dismutase 2 (SOD2) at K122 was increased in WT (but not cKO mice) on a HFD. This correlated with significantly increased cardiac lipid peroxidation in HFD-fed WT mice relative to GCN5L1 cKO animals under the same conditions. We conclude that increased GCN5L1 expression in response to a HFD promotes increased lysine acetylation, and that this may play a role in the development of reactive oxygen species (ROS) damage caused by nutrient excess. [ABSTRACT FROM AUTHOR]