Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest

Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.     

Targeting autophagy for drug-induced hepatotoxicity

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.     

Targeting autophagy for drug-induced hepatotoxicity

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.     

Endocytosis and Autophagy: Exploitation or Cooperation?

Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here.Autophagy is a lysosome-mediated pathway for the degradation of cytosolic proteins and organelles, which is essential for cell homeostasis, development, and for the prevention of several human diseases and infection (Choi et al. 2013). Importantly, autophagy cannot occur without an active lysosome. However, it is becoming increasingly recognized that the endosomal pathway plays a greater role than just providing the degradative enzymes found in the lysosome. Recent data suggest that in mammalian cells multiple contributions from several stages of the endocytic pathway are essential for efficient autophagy. Here we outline the autophagic pathway and then address the recent data on how different endosomal compartments contribute to autophagy, and the molecular machinery required for the interaction of the endosome and lysosome during the formation, and consumption of the autophagosome. Given the model emerging that the amino-acid-sensitive autophagic pathway originates from the endoplasmic reticulum (ER), several questions arise, including how do recognition and productive interaction occur between an ER-derived membrane and endosomes? How are these interactions mediated, and which are essential for efficient autophagy?     

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages

Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.     

Autophagy: from basic research to its application in food biotechnology

Autophagy is a catabolic process by which the cytoplasm is sequestered into double-membrane vesicles and delivered to the lysosome/vacuole for breaking down and recycling of the low molecular weight degradation products. The isolation in the yeast Saccharomyces cerevisiae of many of the genes involved in autophagy constituted a milestone in understanding the molecular bases of this pathway. The identification of ortholog genes in other eukaryotic models revealed that the mechanism of autophagy is conserved among all eukaryotes. This pathway has been shown to be involved in a growing number of physiological processes and conversely, its deregulation may contribute to the development of several diseases. Recent reports have also shown that autophagy may play an important role in biotechnological processes related with the food industry. In this review we discuss current knowledge of the molecular mechanism of autophagy, including some applied aspects of autophagy in the field of food biotechnology.     

PIP5KIC在自噬体形成过程中的重要作用

自噬（autophagy）是一种在真核生物中十分保守的溶酶体依赖性降解途径,它通过形成双层膜结构包裹胞内堆积的蛋白质和受损细胞器并将其运送到溶酶体中进行降解。在实验中发现,一型磷脂酰肌醇4-磷酸5-激酶C亚型（type I phosphatidylinositol 4-phosphate 5-kinase isoform C,PIP5KIC）会参与到自噬过程中。在哺乳动物细胞中,敲低一型磷脂酰肌醇4-磷酸5-激酶C亚型会造成欧米茄体（omegasome）的形状异常,进而造成自噬水平的降低。同样,在酵母中敲掉其同源物磷脂酰肌醇5-激酶Mss4后也会导致类似的现象。因此,推测一型磷脂酰肌醇4-磷酸5-激酶C亚型在自噬体的生成中起着很重要的作用。     

Inhibition of HIV-1 replication with stable RNAi-mediated knockdown of autophagy factors

Autophagy is a cellular process leading to the degradation of cytoplasmic components such as organelles and intracellular pathogens. It has been shown that HIV-1 relies on several components of the autophagy pathway for its replication, but the virus also blocks late steps of autophagy to prevent its degradation. We generated stable knockdown T cell lines for 12 autophagy factors and analyzed the impact on HIV-1 replication. RNAi-mediated knockdown of 5 autophagy factors resulted in inhibition of HIV-1 replication. Autophagy analysis confirmed a specific defect in the autophagy pathway for 4 of these 5 factors. We also scored the impact on cell viability, but no gross effects were observed. Upon simultaneous knockdown of 2 autophagy factors (Atg16 and Atg5), an additive inhibitory effect was scored on HIV-1 replication. Stable knockdown of several autophagy factors inhibit HIV-1 replication without any apparent cytotoxicity. We therefore propose that targeting of the autophagy pathway can be a novel therapeutic approach against HIV-1.     

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Autophagy is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. It involves the rearrangement of subcellular membranes to sequester cargo for delivery to the lysosome where the sequestered material is degraded and recycled. For many decades, it has been known that autophagy occurs in a wide range of eukaryotic organisms and in multiple different cell types during starvation, cellular and tissue remodeling, and cell death. However, until recently, the functions of autophagy in normal development were largely unknown. The identification of a set of evolutionarily conserved genes that are essential for autophagy has opened up new frontiers for deciphering the role of autophagy in diverse biological processes. In this review, we summarize our current knowledge about the molecular machinery of autophagy and the role of the autophagic machinery in eukaryotic development.     

Autophagy inhibition induces podocyte apoptosis by activating the pro-apoptotic pathway of endoplasmic reticulum stress

Podocyte apoptosis is a major factor inducing podocyte depletion that predicts the progressive course of glomerulosclerosis. However, the molecular mechanisms underlying podocyte apoptosis are still not well understood. Autophagy is a lysosomal degradation system involving the degradation and recycling of obsolete, damaged, or harmful cytoplasmic materials and organelles. Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. However, their cross-talk remains largely obscure until now. Here, we found that podocytes both in vivo and in vitro always exhibited high basal levels of autophagy, whereas autophagy inhibition could induce podocyte apoptosis, suggesting the pro-survival role of autophagy in podocytes. Besides, we found that autophagy inhibition by 3-methyladenine (3-MA) could induce the activation of endoplasmic reticulum stress even without any external stimulations, whereas knockdown of CHOP could effectively improve podocyte apoptosis and down-regulated expression of slit-diaphragm proteins induced by autophagy inhibition. Collectively, this study demonstrated that autophagy might act as a crucial regulatory mechanism of apoptotic cell death by modulating the balance between the pro-survival pathway and the pro-apoptotic pathway of endoplasmic reticulum stress, which might provide a novel mechanistic insight into the interface between autophagy and apoptosis in the progression of podocyte injury.     

Liver autophagy: physiology and pathology

Autophagy has long been thought of as a bulk degradation system in which cytoplasmic components are sequestered by double-membrane structures called autophagosomes, and the contents are then degraded after autophagosomes fuse with lysosomes. Genetic experiments in yeast identified a set of Autophagy-related (ATG) genes that are essential for autophagy. We have since elucidated many of the molecular underpinnings of autophagy and the physiologic roles of these processes in various systems. This review summarizes the physiologic roles of autophagy with a particular focus on liver autophagy based on analyses of knockout mice lacking Atg genes.     

Autophagy: for better or for worse

Autophagy is a lysosomal degradation pathway that degrades damaged or superfluous cell components into basic biomolecules, which are then recycled back into the cytosol. In this respect, autophagy drives a flow of biomolecules in a continuous degradation-regeneration cycle. Autophagy is generally considered a pro-survival mechanism protecting cells under stress or poor nutrient conditions. Current research clearly shows that autophagy fulfills numerous functions in vital biological processes. It is implicated in development, differentiation, innate and adaptive immunity, ageing and cell death. In addition, accumulating evidence demonstrates interesting links between autophagy and several human diseases and tumor development. Therefore, autophagy seems to be an important player in the life and death of cells and organisms. Despite the mounting knowledge about autophagy, the mechanisms through which the autophagic machinery regulates these diverse processes are not entirely understood. In this review, we give a comprehensive overview of the autophagic signaling pathway, its role in general cellular processes and its connection to cell death. In addition, we present a brief overview of the possible contribution of defective autophagic signaling to disease.     

Molecular interplay of autophagy and endocytosis in human health and diseases

Autophagy, an evolutionarily conserved process for maintaining the physio‐metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras‐like GTPases (Rabs), soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal‐associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane‐bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy–endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.     

自噬的相关分子机制

细胞自噬是真核生物中高度保守的一类生物学途径,它通过降解细胞浆内不同组分,维持细胞自身平衡并帮助细胞在应激情况下生存。自噬在生物体生长发育、免疫防御、肿瘤抑制及神经退行性疾病中都有重大的意义。哺乳动物细胞中,自噬过程主要由自噬相关蛋白（Atg）所形成的一系列复合物所调控,这些蛋白质分别在自噬的启动、自噬泡的形成、延伸及成熟和降解过程中发挥重要的作用。在此,本文针对一些重要的自噬相关蛋白质对近年来自噬分子机制的研究进展做一总结。     

植物细胞自噬基因的功能与转录调控机制

自噬(autophagy)是真核生物长期进化形成的一种高度保守的细胞内物质降解和周转途径,通过形成双层膜结构的自噬体将包裹其中的待降解大分子物质,如受损伤的蛋白质、蛋白质复合物和细胞器,运送至液泡或溶酶体进行降解并产生可循环利用的降解产物。细胞自噬在植物生长发育和环境应答等过程中发挥重要作用。在拟南芥(Arabidop...     

Dynamin Regulates Autophagy by Modulating Lysosomal Function

Autophagy is a central lysosomal degradation pathway required for maintaining cellular homeostasis and its dysfunction is associated with numerous human diseases. To identify players in autophagy, we tested w1200 chemically induced mutations on the X chromosome in Drosophila fat body clones and discovered that shibire(shi) plays an essential role in starvation-induced autophagy. shi encodes a dynamin protein required for fission of clathrin-coated vesicles from the plasma membrane during endocytosis. We showed that Shi is dispensable for autophagy initiation and autophagosomeelysosome fusion, but required for lysosomal/autolysosomal acidification. We also showed that other endocytic core machinery components like clathrin and AP2 play similar but not identical roles in regulating autophagy and lysosomal function as dynamin. Previous studies suggested that dynamin directly regulates autophagosome formation and autophagic lysosome reformation(ALR) through its excision activity. Here, we provide evidence that dynamin also regulates autophagy indirectly by regulating lysosomal function.     

植物细胞自噬研究进展

细胞自噬是一类依赖于溶酶体和液泡的蛋白质降解途径。在动物细胞中, 靶物质通过自噬体包裹被运送到溶酶体中,由特定的水解酶降解; 而植物和酵母细胞中该过程在液泡内进行。近年来, 在模式植物拟南芥(Arabidopsis thaliana)中鉴定到多个关键ATG基因, 它们对植物细胞自噬体的形成及自噬调控起到关键作用。该文全面综述了植物细胞自噬的调控及其在植物逆境胁迫中的生理功能。     

Viruses and the autophagy machinery

Autophagy is a major intracellular pathway for degradation and recycling of long-lived proteins and cytoplasmic organelles that plays an essential role in maintenance of homeostasis in response to starvation and other cellular stresses. Autophagy is also important for a variety of other processes including restriction of intracellular pathogen replication. Our understanding of the fascinating relationship between viruses and the autophagy machinery is still in its infancy but it is clear that autophagy is a newly recognized facet of innate and adaptive immunity against viral infection. Although the autophagy pathway is emerging as a component of host defense, certain viruses have developed strategies to counteract these antiviral mechanisms, and others appear to have co-opted the autophagy machinery as proviral host factors favoring viral replication. The complex interplay between autophagy and viral infection will be discussed in this review.