The product of the UTH1 gene,required for Bax-induced cell death in yeast,is involved in the response to rapamycin

A yeast mutant was isolated that was resistant to Bax-induced cell death. It supports a mutation leading to decreased amounts of the protein Uth1p. A strain in which the UTH1 gene is disrupted also exhibits resistance to Bax expression. The absence of Uth1p does not change the mitochondrial localization of Bax, its insertion in the mitochondrial outer membrane or its cytochrome c-releasing activity. On the other hand, the absence of Uth1p does prevent the appearance of other hallmarks related to Bax expression in yeast, such as oxidation of mitochondrial lipid, production of reactive oxygen species and maintenance of plasma membrane properties after ethanol stress. The absence of Uth1p was also found to induce resistance to rapamycin, a specific inducer of autophagy. This resistance only appears when cells are grown under respiratory conditions, but not under fermentative conditions, suggesting that Uth1p acts in an autophagic pathway involving mitochondria, in accordance with its main localization in the outer mitochondrial membrane. Taken together, these data show that Bax is able to activate a death pathway related to autophagy in yeast, which also exhibits typical hallmarks of apoptosis, revealing a possible dual function of Bax in both types of death. This hypothesis is discussed in the light of observations suggesting a co-regulation of apoptosis and autophagy in mammalian cells.     

Uth1p is involved in the autophagic degradation of mitochondria

The absence of the outer mitochondrial membrane protein Uth1p was found to induce resistance to rapamycin treatment and starvation, two conditions that induce the autophagic process. Biochemical studies showed the onset of a fully active autophagic activity both in wild-type and Deltauth1 strains. On the other hand, the disorganization of the mitochondrial network induced by rapamycin treatment or 15 h of nitrogen starvation was followed in cells expressing mitochondria-targeted green fluorescent protein; a rapid colocalization of green fluorescent protein fluorescence with vacuole-selective FM4-64 labeling was observed in the wild-type but not in the Deltauth1 strain. Degradation of mitochondrial proteins, followed by Western blot analysis, did not occur in mutant strains carrying null mutations of the vacuolar protease Pep4p, the autophagy-specific protein Atg5p, and Uth1p. These data show that, although the autophagic machinery was fully functional in the absence of Uth1p, this protein is involved in the autophagic degradation of mitochondria.     

线粒体自噬

细胞自噬(autophagy)是细胞依赖溶酶体对蛋白和细胞器进行降解的一条重要途径.目前,将通过细胞自噬降解线粒体的途径称为线粒体自噬(mitophagy).最近几年的证据表明,线粒体自噬是一个特异性的选择过程,并受到各种因子的精密调节,是细胞清除体内损伤线粒体和维持自身稳态的一种重要调节机制.自噬相关分子,如“核心”Atg 复合物,酵母线粒体外膜分子Atg32、Atg33、Uth1和Aup1,哺乳细胞线粒体外膜蛋白PINK1、NIX和胞质的Parkin等,在线粒体自噬中起关键的作用. 线粒体自噬异常与神经退行性疾病如帕金森氏病（Parkinson’s disease,PD）的发生密切相关. 本文就线粒体自噬的研究进展做简要的介绍.     

The neuA/flmD gene cluster of Helicobacter pylori is involved in flagellar biosynthesis and flagellin glycosylation

The Saccharomyces cerevisiae SUN family gene products, namely Sim1p, Uth1p, Nca3p and Sun4p, show a high degree of homology among themselves and are closely related to beta-glucosidase of Candida wickerhamii; however, these proteins do not bear such an activity. Dithiothreitol-treatment of intact cells induces the release of Uth1p, Sun4p and Sim1p from the cell wall. These highly glycosylated proteins are thus non-covalently bound to the cell wall. Two of them, Uth1p and Sun4p, have also been found in mitochondria. Sub-localization experiments show that Uth1p is inserted in the outer mitochondrial membrane and that Sun4p is preferentially a matrix protein. The physiological significance of this double localization is discussed in relation to the roles of these proteins in different cellular processes, namely mitochondrial biogenesis and cell septation.     

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells

Autophagy is originally named as a process of protein recycling. It begins with sequestering cytoplasmic organelles in a membrane vacuole called autophagosome. Autophagosomes then fuse with lysosomes, where the materials inside are degraded and recycled. To date, however, little is known about the role of autophagy in cancer therapy. In this study, we present that temozolomide (TMZ), a new alkylating agent, inhibited the viability of malignant glioma cells in a dose-dependent manner and induced G2/M arrest. At a clinically achievable dose (100 microM), TMZ induced autophagy, but not apoptosis in malignant glioma cells. After the treatment with TMZ, microtubule-associated protein light-chain 3 (LC3), a mammalian homologue of Apg8p/Aut7p essential for amino-acid starvation-induced autophagy in yeast, was recruited on autophagosome membranes. When autophagy was prevented at an early stage by 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, not only the characteristic pattern of LC3 localization, but also the antitumor effect of TMZ was suppressed. On the other hand, bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, that prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes, sensitized tumor cells to TMZ by inducing apoptosis through activation of caspase-3 with mitochondrial and lysosomal membrane permeabilization, while LC3 localization pattern stayed the same. These results indicate that TMZ induces autophagy in malignant glioma cells. Application of an autophagy inhibitor that works after the association of LC3 with autophagosome membrane, such as bafilomycin A1, is expected to enhance the cytotoxicity of TMZ for malignant gliomas.     

Albumin prevents reactive oxygen species-induced mitochondrial damage, autophagy, and apoptosis during serum starvation

Aberrant levels of reactive oxygen species (ROS) rapidly generated from NADPH oxidase (NOX) activation can be cytotoxic due to activating pro-apoptotic signals. However, ROS also induce pro-survival autophagy through the engulfment of damaged mitochondria. This study is aimed at investigating the cytoprotective role of albumin against NOX/ROS-induced autophagy and apoptosis under serum starvation. Serum starvation induced apoptosis following a myeloid cell leukemia sequence 1 (Mcl-1)/Bax imbalance, loss of the mitochondrial transmembrane potential, and caspase activation accompanied by pro-survival autophagy following canonical inhibition of mammalian target of rapamycin complex 1 (mTORC1). Aberrant ROS generation, initially occurring through NOX, facilitated mitochondrial damage, autophagy, and apoptosis. Autophagy additionally regulated the accumulation of ROS-generating mitochondria. NOX/ROS permitted p38 mitogen-activated protein kinase (p38 MAPK)-regulated mitochondrial apoptosis, accompanied by non-canonical induction of autophagy. In addition, activation of glycogen synthase kinase (GSK)-3β by NOX/ROS-inactivated Akt facilitated a decrease in Mcl-1, followed by mitochondrial apoptosis as well as autophagy. Restoring albumin conferred an anti-oxidative effect against serum starvation-deregulated NOX, p38 MAPK, and Akt/GSK-3β/Mcl-1/caspase-3 signaling. Albumin also prevented autophagy by sustaining mTORC1. These results indicate an anti-oxidative role for albumin via preventing NOX/ROS-mediated mitochondrial signaling to stimulate apoptosis as well as autophagy. Autophagy, initially induced by canonical inhibition of mTORC1 and enhanced by non-canonical mitochondrial damage, acts physically as a pro-survival mechanism.     

p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53^K382R failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.     

Mitochondrial autophagy by Bnip3 involves Drp1-mediated mitochondrial fission and recruitment of Parkin in cardiac myocytes

The Bcl2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) is an atypical BH3-only protein that is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of mitochondrial autophagy, and in this study we have investigated the mechanisms by which Bnip3 induces autophagy in cardiac myocytes. We found that Bnip3 induced mitochondrial translocation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission in adult myocytes. Drp1-mediated mitochondrial fission correlated with increased autophagy, and inhibition of Drp1 reduced Bnip3-mediated autophagy. Overexpression of Drp1K38E, a dominant negative of Drp1, or mitofusin 1 prevented mitochondrial fission and autophagy by Bnip3. Also, inhibition of mitochondrial fission or autophagy resulted in increased death of myocytes overexpressing Bnip3. Moreover, Bnip3 promoted translocation of the E3 ubiquitin ligase Parkin to mitochondria, which was prevented in the presence of a Drp1 inhibitor. Interestingly, induction of autophagy by Bnip3 was reduced in Parkin-deficient myocytes. Thus our data suggest that induction of autophagy in response to Bnip3 is a protective response activated by the cell that involves Drp1-mediated mitochondrial fission and recruitment of Parkin.     

Drp1-dependent mitochondrial fission regulates p62-mediated autophagy in LPS-induced activated microglial cells

Microglial activation is known to be an important event during innate immunity, but microglial inflammation is also thought to play a role in the etiology of neurodegenerative diseases. Recently, it was reported that autophagy could influence inflammation and activation of microglia. However, little is known about the regulation of autophagy during microglial activation. In this study, we demonstrated that mitochondrial fission-induced ROS can promote autophagy in microglia. Following LPS-induced autophagy, GFP-LC3 puncta were increased, and this was suppressed by inhibiting mitochondrial fission and mitochondrial ROS. Interestingly, inhibition of mitochondrial fission and mitochondrial ROS also resulted in decreased p62 expression, but Beclin1 and LC3B were unaffected. Taken together, these results indicate that ROS induction due to increased LPS-stimulated mitochondrial fission triggers p62 mediated autophagy in microglial cells. Our findings provide the first important clues towards understanding the correlation between mitochondrial ROS and autophagy.

Abbreviations: Drp1; Dynamin related protein 1, LPS; Lipopolysaccharide, ROS; Reactive Oxygen Species, GFP; Green Fluorescent Protein, CNS; Central Nervous System, AD; Alzheimer’s Disease, PD; Parkinson’s Disease, ALIS; Aggresome-like induced structures, iNOS; inducible nitric oxide synthase, Cox-2; Cyclooxygenase-2, MAPK; Mitogen-activated protein kinase; SODs; Superoxide dismutase, GPXs; Glutathione Peroxidase, Prxs; Peroxiredoxins     

Chemerin‐induced mitochondrial dysfunction in skeletal muscle

Chemerin is a novel adipocyte‐derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin‐mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real‐time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3‐I to LC3‐II and higher expression levels of Beclin1 and autophagy‐related protein‐5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine‐like receptor 1 or the use of mitochondria‐targeting antioxidant Mito‐TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin‐stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin‐induced mitochondrial autophagy likely through an AKT‐FoxO3α‐dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.     

HMGB1 Promotes Mitochondrial Dysfunction–Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation

Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP–induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP–induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.     

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.     

Autophagy in the fission yeast Schizosaccharomyces pombe

Autophagy is a non-selective degradation process in eukaryotic cells. The genome sequence of the fission yeast Schizosaccharomyces pombe has revealed that many of the genes required for autophagy are common between the fission yeast and budding yeast, suggesting that the basic machinery of autophagy is conserved between these species. Autophagy in fission yeast is specifically induced by nitrogen starvation based on monitoring a GFP-Atg8p marker. Upon nitrogen starvation, fission yeast cells exit the vegetative cell cycle and initiate sexual differentiation to produce spores. Most of the nitrogen used for de novo protein synthesis during sporulation derives from the autophagic protein degradation system. This review focuses on the recent advances in the role of autophagy in fission yeast.     

Neurochemical effects of the R form of α-lipoic acid and its neuroprotective mechanism in cellular models of Parkinson’s disease

ObjectiveParkinson’s disease (PD) is a common neurodegenerative disease. This study aimed to investigate the effects of the R form of α-lipoic acid (RLA) in cellular models of PD induced by 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).MethodsCell viability and apoptosis were detected using CCK8 and Annexin V-FITC assays, respectively. Intracellular reactive oxygen species (ROS) were detected by fluorescence staining. ELISA assays were performed to detect the levels of dopamine and α-synuclein. To evaluate the effects of RLA on mitochondrial function, cytotoxicity, ATP levels, and mitochondrial gene expression were assayed. Additionally, the expression levels of autophagy-related proteins, including Parkin, PINK1, p62, ATG12, and LC3, were analyzed by western blot, and cell autophagy was visualized by immunofluorescence.ResultsRLA increased cell viability and decreased apoptosis, intracellular ROS, and cytotoxicity, and induced cell autophagy in PD models induced by 6-OHDA and MPTP. RLA also reversed the decreased dopamine and increased α-synuclein expression induced by 6-OHDA and MPTP. The mitochondrial regulatory protein PGC-1α was significantly up-regulated by RLA. The expression levels of autophagy-related proteins, including Parkin, PINK1, p62, and ATG12, were down-regulated after RLA treatment, while LC3 expression was up-regulated.ConclusionsRLA has a protective effect against cellular damage induced by 6-OHDA and MPTP. The neuroprotective mechanism of RLA may be associated with improvement of mitochondrial function and autophagy. Therefore, RLA may serve as a promising potential adjuvant for PD treatment.     

Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis

Arh1p is an essential mitochondrial protein of yeast with reductase activity. Here we show that this protein is involved in iron metabolism. A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter. Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer. Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron. Iron trafficking within the cell was also disturbed. Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control. Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered. Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron. Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins. Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins.