Control of GABARAP‐mediated autophagy by the Golgi complex,centrosome and centriolar satellites

Within minutes of induction of autophagy by amino‐acid starvation in mammalian cells, multiple autophagosomes form throughout the cell cytoplasm. During their formation, the autophagosomes sequester cytoplasmic material and deliver it to lysosomes for degradation. How these organelles can be so rapidly formed and how their formation is acutely regulated are major questions in the autophagy field. Protein and lipid trafficking from diverse cell compartments contribute membrane to, or regulate the formation of the autophagosome. In addition, recruitment of Atg8 (in yeast), and the ATG8‐family members (in mammalian cells) to autophagosomes is required for efficient autophagy. Recently, it was discovered that the centrosome and centriolar satellites regulate autophagosome formation by delivery of an ATG8‐family member, GABARAP, to the forming autophagosome membrane, the phagophore. We propose that GABARAP regulates phagophore expansion by activating the ULK complex, the amino‐acid controlled initiator complex. This finding reveals a previously unknown link between the centrosome, centriolar satellites and autophagy.     

死亡相关蛋白激酶（DAPK）是负调控还是正调控自噬?

自噬(autophagy)是1个严谨调控的代谢途径. 哺乳动物细胞通过自噬能够降解和循环利用大分子和某些细胞器.自噬作为一种适应的机制,保护有机体对抗各种病理病变,包括感染、癌症、退行性病变、衰老和心脏疾病等.在移除蛋白聚集体、以及受损或过剩细胞器时,自噬发挥着很关键的作用,从而能够维持细胞能量平衡并适应环境压力.当自噬不足或过剩时,自噬也可作为一种促死亡的机制.在不同的情况下,自噬活化的程度通过自噬信号通路网络而精确地调节.死亡相关蛋白激酶(DAPK)是1个刺激调节蛋白激酶,它由几个功能结构域组成.这些结构域能让它参与广泛的信号通路中,包括凋亡、自噬和膜泡.DAPK在调节自噬中发挥着关键的作用.本文综述了在不同的情况下DAPK负调控或是正调控自噬的路径.     

TMEM41B is a novel regulator of autophagy and lipid mobilization

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well‐characterized autophagy‐related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2‐ and DFCP1‐positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β‐oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER‐localized regulator of autophagosome biogenesis and lipid mobilization.     

Eating the endoplasmic reticulum: quality control by autophagy

Autophagy is connected to a surprising range of cellular processes, including the stress response, developmental remodeling, organelle homeostasis and disease pathophysiology. The inducible, predominant form of autophagy, macroautophagy, involves dynamic membrane rearrangements, culminating in the formation of a double-membrane cytosolic vesicle, an autophagosome, which sequesters cytoplasm and organelles. The signal transduction mechanisms that regulate autophagy are poorly understood and have focused on extracellular nutrient sensing. Similarly, little is known about the contribution of the endomembrane organelles to autophagy-related processes. Recent studies have provided interesting links between these topics, revealing that the secretory pathway provides membrane for autophagosome formation, and that autophagy has an important role in organelle homeostasis.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment

Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the host's autophagy machinery is activated during HBV infection to enhance HBV replication.     

DAP-kinase and autophagy

DAP-kinase (DAPK) is a Ca²⁺-calmodulin regulated kinase with various, diverse cellular activities, including regulation of apoptosis and caspase-independent death programs, cytoskeletal dynamics, and immune functions. Recently, DAPK has also been shown to be a critical regulator of autophagy, a catabolic process whereby the cell consumes cytoplasmic contents and organelles within specialized vesicles, called autophagosomes. Here we present the latest findings demonstrating how DAPK modulates autophagy. DAPK positively contributes to the induction stage of autophagosome nucleation by modulating the Vps34 class III phosphatidyl inositol 3-kinase complex by two independent mechanisms. The first involves a kinase cascade in which DAPK phosphorylates protein kinase D, which then phosphorylates and activates Vps34. In the second mechanism, DAPK directly phosphorylates Beclin 1, a necessary component of the Vps34 complex, thereby releasing it from its inhibitor, Bcl-2. In addition to these established pathways, we will discuss additional connections between DAPK and autophagy and potential mechanisms that still remain to be fully validated. These include myosin-dependent trafficking of Atg9-containing vesicles to the sites of autophagosome formation, membrane fusion events that contribute to expansion of the autophagosome membrane and maturation through the endocytic pathway, and trafficking to the lysosome on microtubules. Finally, we discuss how DAPK's participation in the autophagic process may be related to its function as a tumor suppressor protein, and its role in neurodegenerative diseases.     

Regulation of macroautophagy by mTOR and Beclin 1 complexes

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.     

UNC51‐like kinase 1, autophagic regulator and cancer therapeutic target

Autophagy, the cell process of self‐digestion, plays a pivotal role in maintaining energy homoeostasis and protein synthesis. When required, it causes degradation of long‐lived proteins and damaged organelles, indicating that it may play a dual role in cancer, by both protecting against and promoting cell death. The autophagy‐related gene (Atg) family, with more than 35 members, regulates multiple stages of the process. Serine/threonine protein kinase Atg1 in yeast, for example, can interact with other ATG gene products, functioning in autophagosome formation. One mammalian homologue of Atg1, UNC‐51‐like kinase 1 (ULK1) and its related complex ULK1–mAtg13–FIP200 can mediate autophagy under nutrient‐deprived conditions, by protein–protein interactions and post‐translational modifications. Although specific mechanisms of how ULK1 and its complex transduces upstream signals to the downstream central autophagy pathways is not fully understood, past studies have indicated that ULK1 can both suppress and promote tumour growth under different conditions. Here, we summarize some properties of ULK1 which can regulate autophagy in cancer, which may shed new light on future cancer therapy strategies, utilizing ULK1 as a potential new target.     

TBC1D14 sets the TRAPP for ATG9

Amino acid withdrawal induces the formation of autophagosomes, which results in dozens of these large double-membrane vesicles appearing in the starved cell within 10–15 min, and the initiation of autophagy. This vesicle-mediated response clearly requires an adequate supply of membrane and a tight molecular regulation creating a substantial challenge for the cell in terms of vesicle trafficking pathways. Several membrane sources, which contribute to autophagosome initiation and formation, have been identified including the ER, Golgi, plasma membrane, mitochondria and recycling endosomes. How contributions from these organelles are regulated is an intensive area of study. Members of several families of membrane traffic regulators, including small GTPases, such as RAB proteins, and their regulators, SNARE proteins and BAR domain-containing proteins, have recently been shown to support autophagosome formation.     

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.     

LC3B is indispensable for selective autophagy of p62 but not basal autophagy

Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.     

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.     

Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy

Autophagy is a catabolic process essential for cell homeostasis, at the core of which is the formation of double-membrane organelles called autophagosomes. Atg9 is the only known transmembrane protein required for autophagy and is proposed to deliver membrane to the preautophagosome structures and autophagosomes. We show here that mammalian Atg9 (mAtg9) is required for the formation of DFCP1-positive autophagosome precursors called phagophores. mAtg9 is recruited to phagophores independent of early autophagy proteins, such as ULK1 and WIPI2, but does not become a stable component of the autophagosome membrane. In fact, mAtg9-positive structures interact dynamically with phagophores and autophagosomes without being incorporated into them. The membrane compartment enriched in mAtg9 displays a unique sedimentation profile, which is unaltered upon starvation-induced autophagy. Correlative light electron microscopy reveals that mAtg9 is present on tubular-vesicular membranes emanating from vacuolar structures. We show that mAtg9 resides in a unique endosomal-like compartment and on endosomes, including recycling endosomes, where it interacts with the transferrin receptor. We propose that mAtg9 trafficking through multiple organelles, including recycling endosomes, is essential for the initiation and progression of autophagy; however, rather than acting as a structural component of the autophagosome, it is required for the expansion of the autophagosome precursor.     

Atg27 is required for autophagy-dependent cycling of Atg9

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.     

The role of ncRNA in the co-regulation of autophagy and exosome pathways during cancer progression

Since its discovery a few decades ago, autophagy has been recognized as a crucial signaling pathway, linked to the recycling of cellular components in nutrient stress. Autophagy is a two-way sword, playing a dual role in tumorigenesis. In this catabolic process, dysfunctional organelles, biomolecules, and misfolded proteins are sequestered in the autophagosome and sent to the lysosome for degradation. Alongside, there are cellular messengers called exosomes, which are released from cells and are known to communicate and regulate metabolism in recipient cells. Multivesicular bodies (MVB) act as the intricate link between autophagy and exosome pathways. The continuous crosstalk between the two pathways is coordinated and regulated by multiple players among which ncRNA is the emerging candidates. The exosomes carry varied cargo of which non-coding RNA exerts an immediate regulatory effect on recipient cells. ncRNA is known to exhibit dual behavior in both promoting and inhibiting tumor growth. There is increasing evidence for the involvement of ncRNAs' in the regulation of different hallmarks of cancer. Different ncRNAs are involved in the co-regulation of autophagy and exosome pathways and therefore represent a superior therapeutic approach to target cancer chemoresistance. Here, we will discuss the ncRNA involved in regulating autophagy, and exosomes pathways and its relevance in cancer therapeutics.     

Remodeling of ER‐exit sites initiates a membrane supply pathway for autophagosome biogenesis

Autophagosomes are double‐membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER–Golgi intermediate compartment (ERGIC) generates ERGIC‐derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super‐resolution microscopy to show that starvation induces the enlargement of ER‐exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation‐induced enlargement of ERES independent of the other subunits of this complex and associates via its C‐terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation‐induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation.     

Autophagy: a lysosomal degradation pathway with a central role in health and disease

Autophagy delivers cytoplasmic material and organelles to lysosomes for degradation. The formation of autophagosomes is controlled by a specific set of autophagy genes called atg genes. The magnitude of autophagosome formation is tightly regulated by intracellular and extracellular amino acid concentrations and ATP levels via signaling pathways that include the nutrient sensing kinase TOR. Autophagy functions as a stress response that is upregulated by starvation, oxidative stress, or other harmful conditions. Remarkably, autophagy has been shown to possess important housekeeping and quality control functions that contribute to health and longevity. Autophagy plays a role in innate and adaptive immunity, programmed cell death, as well as prevention of cancer, neurodegeneration and aging. In addition, impaired autophagic degradation contributes to the pathogenesis of several human diseases including lysosomal storage disorders and muscle diseases.     

HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy

Autophagy is primarily considered a non‐selective degradation process induced by starvation. Nutrient‐independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin‐binding deacetylase, histone deacetylase‐6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin‐dependent, actin‐remodelling machinery, which in turn assembles an F‐actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build‐up, and neurodegeneration. Remarkably, HDAC6 and F‐actin assembly are completely dispensable for starvation‐induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.