Autophagy contributes to lysosomal storage disorders

Degradation in the lysosome/vacuole is not the final step of autophagy. In particular, for starvation-induced autophagy it is necessary to release the breakdown products back into the cytosol. However, some researchers ignore this last step and simply refer to the endpoint of autophagy as degradation, or perhaps even cargo delivery. In many cases this is not a serious issue; however, the analysis of autophagy's role in certain diseases makes clear that this can be a significant error.     

Toward an understanding of autophagosome-lysosome fusion: The unsuspected role of ATG14

Although largely overlooked relative to the process of phagophore formation, the mechanism through which autophagosomes fuse with lysosomes is a critical aspect of macroautophagy that is not fully understood. In particular, this step must be carefully regulated to prevent premature fusion of an incomplete autophagosome (that is, a phagophore) with a lysosome, because such an event would not allow access of the partially sequestered cargo to the lysosome lumen. The identification of the autophagosome-associated SNARE protein STX17 (syntaxin 17) provided some clue in the understanding of this process. STX17 is recruited specifically to mature autophagosomes, and functions in mediating autophagosome-lysosome fusion by forming a complex with the Qbc SNARE SNAP29 and the lysosomal R-SNARE VAMP8. Additionally, STX17 plays a role in the early events of autophagy by interacting with the phosphatidylinositol 3-kinase complex component ATG14. Upon autophagy induction STX17 is strictly required for ATG14 recruitment to the ER-mitochondria contact sites, a critical step for the assembly of the phagophore and therefore for autophagosome formation. In their recent paper, Diao and collaborators now show that the ATG14-STX17-SNAP29 interaction mediates autophagosome-lysosome tethering and fusion events, thus revealing a novel function of ATG14 in the later steps of the autophagy pathway.     

A role for Rac3 GTPase in the regulation of autophagy

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.     

Control of the autophagy maturation step by the MAPK ERK and p38: lessons from environmental carcinogens

Macroautophagy (hereafter referred to as autophagy) is the major degradative pathway of long-lived proteins and organelles that fulfils key functions in cell survival, tissue remodeling and tumor suppression. Consistently, alterations in autophagy have been involved in a growing list of pathologies including toxic injury, infections, neurodegeneration, myopathies and cancers. Although critical, the molecular mechanisms that control autophagy remain largely unknown. We have recently exploited the disruption of autophagy by environmental carcinogens as a powerful model to uncover the underlying signaling pathways. Our work published in Cancer Research revealed that the sustained activation of the MAPK ERK pathway by the carcinogen Lindane or the MEK1(+) oncogene alters autophagy selectively at the maturation step resulting in the accumulation of large defective autolysosomes. Consistent with our findings, a similar defect is observed with other common xenobiotics such as dichlorodiphenyltrichloroethane and biphenol A that specifically activate ERK. Conversely, Pentachlorophenol that activates both ERK and p38, fails to induce autophagic vacuolation. In addition, evidence is provided that abrogation of p38 by SB203580 is sufficient to interfere with the normal autophagic maturation step. Altogether, these findings underscore the critical role played by MAPK ERK and p38 in the tight control of the autophagy process at the maturation step.     

AMPK and mTOR coordinate the regulation of Ulk1 and mammalian autophagy initiation

Ulk1 is a serine/threonine kinase and the mammalian functional homolog of yeast Atg1. It acts at the initiation step of autophagy and forms a complex with mAtg13, FIP200 and Atg101. Assembly of this complex is independent of mTOR signaling, indicating the regulation of autophagy initiation in mammals is different from that in yeast. In a recent study, we reported that Ulk1 can be phosphorylated by mTOR and AMPK kinases. AMPK associates with Ulk1 in nutrient-dependent manner. Rapid dissociation between Ulk1 and AMPK primes cells for fast autophagy induction upon nutrient withdrawal. These studies show that both mTOR and AMPK directly regulate Ulk1 and coordinate the mammalian autophagy initiation.     

自噬在发育及干细胞中的作用

自噬是亚细胞膜结构发生动态变化并经溶酶体介导的细胞内蛋白质和细胞器降解的过程。通过平衡细胞内的合成和分解代谢,自噬可以维持细胞内环境稳态。干细胞是具有自我更新能力和多向分化潜能的细胞,对组织器官再生和维持组织稳态有重要作用。近年的研究表明,自噬在维持干细胞功能方面有非常重要的作用,本文综述了自噬的形成过程和分子机制及其在发育及干细胞中的作用。     

AMPK and mTOR coordinate the regulation of Ulk1 and mammalian autophagy initiation

Ulk1 is a serine/threonine kinase and the mammalian functional homolog of yeast Atg1. It acts at the initiation step of autophagy and forms a complex with mAtg13, FIP200 and Atg101. Assembly of this complex is independent of mTOR signaling, indicating the regulation of autophagy initiation in mammals is different from that in yeast. In a recent study, we reported that Ulk1 can be phosphorylated by mTOR and AMPK kinases. AMPK associates with Ulk1 in nutrient-dependent manner. Rapid dissociation between Ulk1 and AMPK primes cells for fast autophagy induction upon nutrient withdrawal. These studies show that both mTOR and AMPK directly regulate Ulk1 and coordinate the mammalian autophagy initiation.     

TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

Toll‐like receptors (TLRs) are essential immunoreceptors involved in host defence against invading microbes. Recent studies indicate that certain TLRs activate immunological autophagy to eliminate microbes. It remains unknown whether TLRs regulate autophagy to play a role in the heart. This study examined this question. The activation of TLR3 in cultured cardiomyocytes was observed to increase protein levels of autophagic components, including LC3‐II, a specific marker for autophagy induction, and p62/SQSTM1, an autophagy receptor normally degraded in the final step of autophagy. The results of transfection with a tandem mRFP‐GFP‐LC3 adenovirus and use of an autophagic flux inhibitor chloroquine both suggested that TLR3 in cardiomyocytes promotes autophagy induction without affecting autophagic flux. Gene‐knockdown experiments showed that the TRIF‐dependent pathway mediated the autophagic effect of TLR3. In the mouse model of chronic myocardial infarction, persistent autophagy was observed, concomitant with up‐regulated TLR3 expression and increased TLR3‐Trif signalling. Germline knockout (KO) of TLR3 inhibited autophagy, reduced infarct size, attenuated heart failure and improved survival. These protective effects were abolished by in vivo administration of an autophagy inducer rapamycin. Similar to the results obtained in cultured cardiomyocytes, TLR3‐KO did not prevent autophagic flux in mouse heart. Additionally, this study failed to detect the involvement of inflammation in TLR3‐KO‐derived protection, as wild‐type and TLR3‐KO hearts were comparable in inflammatory activity. It is concluded that up‐regulated TLR3 expression and signalling contributes to persistent autophagy following MI, which promotes heart failure and lethality.     

The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae

Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11Delta mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.     

Autophagosome–lysosome fusion in neurons requires INPP5E,a protein associated with Joubert syndrome

Autophagy is a multistep membrane traffic pathway. In contrast to autophagosome formation, the mechanisms underlying autophagosome–lysosome fusion remain largely unknown. Here, we describe a novel autophagy regulator, inositol polyphosphate‐5‐phosphatase E (INPP5E), involved in autophagosome–lysosome fusion process. In neuronal cells, INPP5E knockdown strongly inhibited autophagy by impairing the fusion step. A fraction of INPP5E is localized to lysosomes, and its membrane anchoring and enzymatic activity are necessary for autophagy. INPP5E decreases lysosomal phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P₂), one of the substrates of the phosphatase, that counteracts cortactin‐mediated actin filament stabilization on lysosomes. Lysosomes require actin filaments on their surface for fusing with autophagosomes. INPP5E is one of the genes responsible for Joubert syndrome, a rare brain abnormality, and mutations found in patients with this disease caused defects in autophagy. Taken together, our data reveal a novel role of phosphoinositide on lysosomes and an association between autophagy and neuronal disease.     

综述:细胞自噬的调控机制及其在老年性痴呆发生发展中的作用

细胞自噬是生物体内一种用于清除功能异常的细胞器、错误折叠的蛋白质、被氧化的脂类等有害大分子物质的重要途径．它的机制从低等生物酵母到高等的哺乳动物都高度保守,对维持正常的生命活动至关重要．错误折叠的蛋白质若不能被有效清除,就会造成积聚,致使神经细胞功能丧失乃至死亡,这是神经退行性疾病包括老年性痴呆(Alzheimer's disease, AD)的主要原因．本文回顾了近年来关于细胞自噬及其与老年性痴呆关系的研究进展,主要内容包括以下几点：自噬参与Aβ的产生和清除;γ分泌酶中的Presenilin 1在自噬底物降解中的作用;Tau蛋白调控自噬体转运、融合;老年性痴呆早期自噬对细胞的保护;细胞中感应营养和能量的两个关键蛋白mTOR和AMPK调控自噬及其对老年性痴呆的潜在影响机制．     

Binding Rubicon to cross the Rubicon

Beclin 1 is an antitumor protein, required for mammalian autophagy, but its precise molecular function is poorly understood. Mass spectrometry analysis reveals that two novel proteins, Atg14L and Rubicon, associate with Beclin 1, together with a known Beclin 1-binding protein, UVRAG. The interactions of Atg14L and UVRAG with the Beclin 1-Vps34 (class III PI3-kinase)-Vps15 core complex are mutually exclusive; Rubicon associates with a subpopulation of UVRAG-containing complexes. The Atg14L complex, which positively regulates autophagy at an early step, localizes to the phagophore/isolation membrane, autophagosome and endoplasmic reticulum. In contrast, the Rubicon-UVRAG complex localizes to the late endosome/lysosome and negatively regulates both autophagy at a later step and the endocytic pathway. Thus, the Beclin 1-Vps34-Vps15 complex functions in autophagy and the endocytic pathway, but its function in a given context depends on the identity of its interacting subunits.     

Nix Is Critical to Two Distinct Phases of Mitophagy,Reactive Oxygen Species-mediated Autophagy Induction and Parkin-Ubiquitin-p62-mediated Mitochondrial Priming

Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNA-mediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.     

Recent progress in autophagic lysosome reformation

Autophagic lysosome reformation (ALR) is the terminal step of autophagy and is essential for maintaining lysosome homeostasis during autophagy. During ALR, tubules are extruded from autolysosomes, and small vesicles named proto‐lysosomes, which are composed of lysosomal membrane components, are generated from these tubules. Eventually, proto‐lysosomes mature into functional lysosomes. In this review, we will summarize recent progress in understanding the regulation, mechanisms and physiological functions of ALR.     

Vitamin E as a novel enhancer of macroautophagy in rat hepatocytes and H4-II-E cells

Autophagy is an intracellular bulk degradation process induced by nutrient starvation, and contributes to macromolecular turnover and rejuvenation of cellular organelles. We demonstrated that vitamin E was a novel nutritional enhancer of autophagy in freshly isolated rat hepatocytes and rat hepatoma H4-II-E cells. Supplementation of fresh hepatocytes with vitamin E (up to 100 μM) increased proteolysis significantly in the presence or absence of amino acids in a dose-dependent manner. The cytosolic LC3 ratio, a newly established index of autophagic flux, was significantly increased by vitamin E, strongly suggesting that the possible site of action is the LC3 conversion step, an early step in autophagosome formation. A typical antioxidant, α-lipoic acid, exerted autophagy suppression, while H₂O₂ stimulated autophagy. It is conceivable that autophagy was stimulated by oxidative stress and this stimulation was cancelled by cellular antioxidative effects. However, in our studies, vitamin E could have enhanced autophagy over-stimulation by H₂O₂, rather than suppress it. From these results, using a new cytosolic LC3 ratio, vitamin E increases autophagy by accelerating LC3 conversion through a new signaling pathway, emerging as a novel enhancer of autophagy.     

Atg22 recycles amino acids to link the degradative and recycling functions of autophagy

In response to stress conditions (such as nutrient limitation or accumulation of damaged organelles) and certain pathological situations, eukaryotic cells use autophagy as a survival mechanism. During nutrient stress the main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol. This study elucidates a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies, Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3, and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy.