The regulation of autophagy in eukaryotic cells: do all roads pass through Atg1?

The induction of autophagy appears to be tightly controlled in all eukaryotic cells. This highly conserved, degradative process is induced by a variety of signals, including nutrient deprivation, and is generally thought to be incompatible with rapid cell growth. Recent work in the budding yeast, Saccharomyces cerevisiae, has suggested that the Atg1 protein kinase is at the center of this control. Atg1, and its associated proteins, appear to be directly targeted by multiple signaling pathways important for the control of both autophagy and cell growth. These pathways involve the small GTP-binding Ras proteins, the Tor protein kinases and the AMP-activated protein kinase, Snf1, respectively. A key question that remains is whether this regulatory paradigm has been evolutionarily conserved. In other words, is Atg1 the primary target of those signaling pathways responsible for coordinating growth with environmental influences in other eukaryotes? Here, we suggest that Atg1 is very likely to fulfill this role but that a truly definitive answer will require that we develop a better understanding of this protein kinase and its targets in all eukaryotes.     

Atg11的功能研究进展

Atg11利用自身众多螺旋结构域作为支架蛋白,主要介导选择性自噬过程中自噬体的形成.选择性自噬可特异性清除损坏的生物大分子和细胞器,在真核生物的胞内物质周转及细胞器质量控制中起重要作用.本文首先介绍了Atg11的结构特点,其次重点介绍了Atg11在3种选择性自噬(细胞质到液泡靶向(Cvt)途径、过氧化物酶体自噬和线粒体自噬)中的作用,最后概括了Atg11的其他功能.本文系统总结了近几年关于Atg11的研究进展,以期为自噬体形成机制研究及Atg11在自噬体形成过程中的功能研究提供参考.     

Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12–5-16L2 complex

A large protein complex consisting of Atg5, Atg12 and Atg16L1 has recently been shown to be essential for the elongation of isolation membranes (also called phagophores) during mammalian autophagy. However, the precise function and regulation of the Atg12–5-16L1 complex has largely remained unknown. In this study we identified a novel isoform of mammalian Atg16L, termed Atg16L2, that consists of the same domain structures as Atg16L1. Biochemical analysis revealed that Atg16L2 interacts with Atg5 and self-oligomerizes to form an ~800-kDa complex, the same as Atg16L1 does. A subcellular distribution analysis indicated that, despite forming the Atg12–5-16L2 complex, Atg16L2 is not recruited to phagophores and is mostly present in the cytosol. The results also showed that Atg16L2 is unable to compensate for the function of Atg16L1 in autophagosome formation, and knockdown of endogenous Atg16L2 did not affect autophagosome formation, indicating that Atg16L2 does not possess the ability to mediate canonical autophagy. Moreover, a chimeric analysis between Atg16L1 and Atg16L2 revealed that their difference in function in regard to autophagy is entirely attributable to the difference between their middle regions that contain a coiled-coil domain. Based on the above findings, we propose that formation of the Atg12–5-16L complex is necessary but insufficient to mediate mammalian autophagy and that an additional function of the middle region (especially around amino acid residues 229–242) of Atg16L1 (e.g., interaction with an unidentified binding partner on phagophores) is required for autophagosome formation.     

Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12–5-16L2 complex

A large protein complex consisting of Atg5, Atg12 and Atg16L1 has recently been shown to be essential for the elongation of isolation membranes (also called phagophores) during mammalian autophagy. However, the precise function and regulation of the Atg12–5-16L1 complex has largely remained unknown. In this study we identified a novel isoform of mammalian Atg16L, termed Atg16L2, that consists of the same domain structures as Atg16L1. Biochemical analysis revealed that Atg16L2 interacts with Atg5 and self-oligomerizes to form an ~800-kDa complex, the same as Atg16L1 does. A subcellular distribution analysis indicated that, despite forming the Atg12–5-16L2 complex, Atg16L2 is not recruited to phagophores and is mostly present in the cytosol. The results also showed that Atg16L2 is unable to compensate for the function of Atg16L1 in autophagosome formation, and knockdown of endogenous Atg16L2 did not affect autophagosome formation, indicating that Atg16L2 does not possess the ability to mediate canonical autophagy. Moreover, a chimeric analysis between Atg16L1 and Atg16L2 revealed that their difference in function in regard to autophagy is entirely attributable to the difference between their middle regions that contain a coiled-coil domain. Based on the above findings, we propose that formation of the Atg12–5-16L complex is necessary but insufficient to mediate mammalian autophagy and that an additional function of the middle region (especially around amino acid residues 229–242) of Atg16L1 (e.g., interaction with an unidentified binding partner on phagophores) is required for autophagosome formation.     

自噬新蛋白Atg101的相关研究进展

自噬是真核细胞特有的生命现象,它控制着细胞内蛋白质和细胞器的降解,并在机体的生长发育和维持能量平衡中起重要的作用。目前在酵母中已被鉴定的自噬相关基因有40余种。Atg101是一种全新的自噬相关蛋白,近年来,其分子结构及功能逐步被阐明,其在疾病发展中的作用也引起了广泛关注。该综述总结了近年来Atg101在分子生物学和病理生理领域的相关研究进展。     

Selective Transport of α-Mannosidase by Autophagic Pathways: STRUCTURAL BASIS FOR CARGO RECOGNITION BY Atg19 AND Atg34*

In the yeast Saccharomyces cerevisiae, a precursor form of aminopeptidase I (prApe1) and α-mannosidase (Ams1) are selectively transported to the vacuole through the cytoplasm-to-vacuole targeting pathway under vegetative conditions and through autophagy under starvation conditions. Atg19 plays a central role in these processes by linking Ams1 and prApe1 to Atg8 and Atg11. However, little is known about the molecular mechanisms of cargo recognition by Atg19. Here, we report structural and functional analyses of Atg19 and its paralog, Atg34. A protease-resistant domain was identified in the C-terminal region of Atg19, which was also conserved in Atg34. In vitro pulldown assays showed that the C-terminal domains of both Atg19 and Atg34 are responsible for Ams1 binding; these domains are hereafter referred to as Ams1-binding domains (ABDs). The transport of Ams1, but not prApe1, was blocked in atg19Δatg34Δ cells expressing Atg19^ΔABD, indicating that ABD is specifically required for Ams1 transport. We then determined the solution structures of the ABDs of Atg19 and Atg34 using NMR spectroscopy. Both ABD structures have a canonical immunoglobulin fold consisting of eight β-strands with highly conserved loops clustered at one side of the fold. These facts, together with the results of a mutational analysis, suggest that ABD recognizes Ams1 using these conserved loops.     

iPS cells—Alternative pluripotent cells to embryo stem cells

<正>Since the first murine and human embryonic stem cell lines were established by Drs. Evans and Kaufman [1] and Thomson et al. [2], respectively, great progress has been make in the field of     

Murine Müller cells are progenitor cells for neuronal cells and fibrous tissue cells

Mammalian Müller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Müller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Müller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Müller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Müller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.     

Glial cells more than support cells?

Glial cells are the most abundant cells in the human brain and have long been considered as passive supporting cells for neurons. In contrast to the extensive studies on various neuronal functions in the nervous system, we still have limited knowledge about glial cells. Recently a number of pioneering studies have provided convincing evidence that glia play active roles in development and function of the central nervous system. This review discusses recent advances in our understanding of the molecular mechanisms underlying glial cell differentiation. We then highlight some of the novel findings about glial function, i.e. the role of glia in synaptogenesis and the intricate relationship between astrocytes and adult neural stem cells. Finally, we summarize the emerging studies that implicate abnormalities in the formation or maintenance of glia leading to severe brain diseases, such as Alexander disease, glioblastoma and multiple sclerosis, and potential therapeutic strategies to tackle these diseases.     

A role for Atg8–PE deconjugation in autophagosome biogenesis

Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8–PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8–PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8–PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.     

鳞翅目昆虫中自噬相关分子Atg8的研究进展

自噬是细胞通过溶酶体自主降解以实现细胞内物质循环利用的过程,在昆虫细胞分化和个体发育中起着重要作用。鳞翅目昆虫属于完全变态昆虫,会通过自噬和凋亡完成蜕变重建过程,是研究自噬机制的模式生物。自噬相关蛋白Atg8是哺乳动物微管相关蛋白1轻链3的同系物,是自噬相关蛋白的核心蛋白家族,对自噬小体形成、膜的延伸、特定物质识别等具有重要意义。文中就鳞翅目昆虫Atg8在自噬信号通路中的作用、Atg8结构特点、Atg8表达分布及Atg8-PE/Atg8水平与自噬活性关系进行了综述。Atg8-PE是自噬信号通路中两个类泛素结合系统之一,在自噬中起着关键作用。序列分析表明,鳞翅目昆虫Atg8与其他真核生物同源蛋白的整体结构相似,尤其与其他昆虫同源蛋白的氨基酸序列高度一致,体现了Atg8的高度保守性。鳞翅目昆虫发育不同阶段,Atg8在中肠、唾液腺、卵巢、脂肪体、丝腺等器官中的表达分布各不相同。并且,Atg8在核质中分布也存在差异,Atg8在细胞核与细胞质之间的穿梭可能存在蛹化前阶段的某些细胞中。通过检测Atg8-PE在细胞内的表达水平或Atg8含量的变化,可以评价细胞自噬的发生程度。     

Are mesenchymal stromal cells immune cells?

Mesenchymal stromal cells (MSCs) are considered to be promising agents for the treatment of immunological disease. Although originally identified as precursor cells for mesenchymal lineages, in vitro studies have demonstrated that MSCs possess diverse immune regulatory capacities. Pre-clinical models have shown beneficial effects of MSCs in multiple immunological diseases and a number of phase 1/2 clinical trials carried out so far have reported signs of immune modulation after MSC infusion. These data indicate that MSCs play a central role in the immune response. This raises the academic question whether MSCs are immune cells or whether they are tissue precursor cells with immunoregulatory capacity. Correct understanding of the immunological properties and origin of MSCs will aid in the appropriate and safe use of the cells for clinical therapy. In this review the whole spectrum of immunological properties of MSCs is discussed with the aim of determining the position of MSCs in the immune system.     

Atg4b-Dependent Autophagic Flux Alleviates Huntington’s Disease Progression

The accumulation of aggregated mutant huntingtin (mHtt) inclusion bodies is involved in Huntigton’s disease (HD) progression. Medium sized-spiny neurons (MSNs) in the corpus striatum are highly vulnerable to mHtt aggregate accumulation and degeneration, but the mechanisms and pathways involved remain elusive. Here we have developed a new model to study MSNs degeneration in the context of HD. We produced organotypic cortico-striatal slice cultures (CStS) from HD transgenic mice mimicking specific features of HD progression. We then show that induction of autophagy using catalytic inhibitors of mTOR prevents MSNs degeneration in HD CStS. Furthermore, disrupting autophagic flux by overexpressing Atg4b in neurons and slice cultures, accelerated mHtt aggregation and neuronal death, suggesting that Atg4b-dependent autophagic flux influences HD progression. Under these circumstances induction of autophagy using catalytic inhibitors of mTOR was inefficient and did not affect mHtt aggregate accumulation and toxicity, indicating that mTOR inhibition alleviates HD progression by inducing Atg4b-dependent autophagic flux. These results establish modulators of Atg4b-dependent autophagic flux as new potential targets in the treatment of HD.     

基于自噬探讨Atg5参与肝脏疾病调控的研究进展

肝脏疾病作为危害人类健康的常见病和多发性疾病,在国内外对其的防治与治疗仍是个严峻的问题。细胞自噬是细胞体内自我调节的重要方式,细胞生长、分化、存活和自我平衡都依赖于此,同时在对抗慢性肝炎病毒感染、酒精性肝病、脂肪肝等肝脏疾病时发挥重要作用。研究表明,细胞自噬受到自噬相关基因-5(autophagy related gene-5,Atg5)的调控,通过Atg5调控细胞自噬来对抗肝脏疾病也引起越来越多的关注。例如在肝细胞中,上调Atg5的表达量可促进细胞自噬,从而减少内质网应激(ER)介导的损伤,这是治疗脂肪性肝炎的一个新策略。本文就Atg5通过调控自噬参与常见肝脏疾病的内在机制做一总结。     

Sinoatrial node pacemaker cells:cardiomyocyte-or neuron-like cells?

The sinoatrial node(SAN)is the headquarter of heartbeat throughout our lifetime(Lakatta et al.,2010;Cingolani et al.,2018;Peters et al.,2020).Every beat of the heart is triggered by a bioelectric pulse spontaneously released by SAN pacemaker cells(SANPCs)(Yaniv et al.,2014;Yavari et al.,2017).In adult human heart,the SAN is a crescent-shaped structure of 1-2 cm long and 0.5 cm wide,which is located at the junction of the superior vena cava and the right atrium and lies along the sulcus terminalis(John et al.,2016).However,the nature of SANPCs remains incompletely known.In general,SANPCs have long been considered as specialized cardiomyocytes(Van Eif et al.,2018;Linscheid et al.,2019;Galang et al.,2020;).However,SANPCs do not have myofibril and T-tube,thus not sharing the contractility property of cardiomyocytes(Satoh,2003;Protze et al.,2017).Interestingly,SANPCs share some electrophysiolog-ical characteristics with neurons:excitability and conductiv-ity.In addition,SANPCs have their intrinsic autonomic rhythm,while neurons also possess the intrinsic ability to generate spontaneous electrical impulses(Lisman et al.,2018).Whether SANPCs are neuron-like cells that reside in the heart remains enigmatic in the field.     

Are folliculo-stellate cells in the anterior pituitary gland supportive cells or organ-specific stem cells?

Folliculo-stellate cells (FS-cells) in the anterior pituitary gland are star-shaped cells and form tiny follicles. FS-cells are positive for S-100 protein and produce many cytokines or growth factors, such as interleukin-6 (IL-6), leukemia inhibitory factor (LIF), basic fibroblastic growth factor (bFGF) and vascular endothelial cell growth factor (VEGF). Therefore, it is generally accepted that FS-cells regulate endocrine cells through these growth factors. FS-cells also exhibit a phagocytotic activity and are known to work as scavenger cells. In addition to these functions, FS-cells are considered to have some unknown functions. In order to reveal the biological significance of FS-cells in the anterior pituitary gland, we performed a morphological study and obtained some new findings. First, we were interested in the colloid formation in the senescent porcine pituitary gland. We analyzed the colloids and found that clusterin is a major protein in them. We also found that the accumulation of clusterin in the colloids is related to the phagocytotic activity of FS-cells. In our next study, we found that FS-cells have the potential to differentiate into striated muscle cells. From FS-cells show multi-potent cell character and other cytological evidence, we propose that FS-cells are candidate of organ-specific stem cells in the anterior pituitary gland.     

Atg9 reservoirs, a new organelle of the yeast endomembrane system?

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is: intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.     

细胞自噬基因Atg6在涡虫中枢神经系统再生中的功能研究

细胞自噬基因Atg6在细胞自噬过程中发挥重要作用,其功能缺陷影响神经发生。涡虫是研究中枢神经系统(central nervous system, CNS)再生的良好模型,其头部切除后1周就能再生出一个新的头部。因此,研究Atg6基因在涡虫CNS再生中的作用对探究自噬调控神经发生具有重要意义。本研究首次报道了日本三角涡虫(Dugesia japonica) Atg6基因(DjAtg6)的分子特征,并利用RNAi技术研究了其在涡虫CNS再生中作用。结果显示:DjAtg6 cDNA全长1366 bp,编码423个氨基酸。DjATG6含有ATG6/Beclin 1蛋白家族的Coil-Coil结构域和β折叠α螺旋自噬功能结构域。涡虫沿咽前咽后切割后,DjAtg6表达量显著增加,其转录本主要在新再生的脑神经节表达。RNAi-DjAtg6引起涡虫头部再生迟缓、脑神经结构偏小,并下调神经相关基因的表达。此外,本研究还发现,RNAi-DjAtg6不影响涡虫干细胞的增殖,但下调细胞迁移相关基因mmp1和mmp2的表达,且干扰mmp1和mmp2的表达影响涡虫头再生。因此,本研究结果表明,DjAtg6在涡虫CNS再生的组织重构中发挥重要作用,干扰DjAtg6影响涡虫CNS再生可能与细胞迁移有关,其详细的分子机制尚需进行深入研究。