Structural basis of Atg conjugation systems essential for autophagy

Autophagy is an evolutionarily-conserved, intracellular degradation system for which two ubiquitin-like modifiers, Atg8 and Atg12, play essential roles. After processed by Atg4, the exposed C-terminal glycine of Atg8 is activated by Atg7 (E1) and is then transferred to Atg3 (E2), and is finally conjugated with a phospholipid, phosphatidylethanolamine (PE) through an amide bond. Whereas, Atg12 is activated by the same E1, Atg7, without processing, and is then transferred to Atg10 (E2), and is finally conjugated with Atg5 through an isopeptide bond. Atg12-Atg5 conjugates, together with Atg16, function as an E3-like enzyme to facilitate the conjugation reaction between Atg8 and PE. During autophagy, Atg8-PE conjugates play a critical role in selective cargo recognition in addition to autophagosome formation. We determined the structures of all these Atg proteins and their complexes mainly by X-ray crystallography, and performed structure-based biochemical analyses on them [1,2]. These studies established the molecular mechanisms of Atg8 and Atg12 modification reactions that have many unique features compared with canonical ubiquitin-like systems. Furthermore, we found a conserved motif named the Atg8-family interacting motif (AIM), through which Atg8 recognizes specific cargoes and selectively incorporates them into autophagosomes for degradation [3].

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Structural catalog of core Atg proteins opens new era of autophagy research

The Journal of Biochemistry ◽

10.1093/jb/mvab017 ◽

2021 ◽

Author(s):

Kazuaki Matoba ◽

Nobuo N Noda

Keyword(s):

De Novo ◽

Structural Information ◽

Membrane Dynamics ◽

Autophagosome Formation ◽

New Era ◽

Intracellular Degradation ◽

Atg Proteins ◽

Biological Studies ◽

Evolutionarily Conserved ◽

Biological Methods

Summary Autophagy, which is an evolutionarily conserved intracellular degradation system, involves de novo generation of autophagosomes that sequester and deliver diverse cytoplasmic materials to the lysosome for degradation. Autophagosome formation is mediated by approximately 20 core autophagy-related (Atg) proteins, which collaborate to mediate complicated membrane dynamics during autophagy. To elucidate the molecular functions of these Atg proteins in autophagosome formation, many researchers have tried to determine the structures of Atg proteins by using various structural biological methods. Although not sufficient, the basic structural catalog of all core Atg proteins was established. In this review article, we summarize structural biological studies of core Atg proteins, with an emphasis on recently unveiled structures, and describe the mechanistic breakthroughs in autophagy research that have derived from new structural information.

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Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets

Molecular Biology of the Cell ◽

10.1091/mbc.e11-09-0785 ◽

2012 ◽

Vol 23 (5) ◽

pp. 896-909 ◽

Cited By ~ 214

Author(s):

Anoop Kumar G. Velikkakath ◽

Taki Nishimura ◽

Eiko Oita ◽

Naotada Ishihara ◽

Noboru Mizushima

Keyword(s):

Lipid Droplet ◽

Lipid Droplets ◽

Autophagic Flux ◽

Autophagosome Formation ◽

Independent Manner ◽

Intracellular Degradation ◽

Atg Proteins ◽

High Conservation ◽

Droplet Morphology

Macroautophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by the autophagosome and delivered to the lysosome. Autophagosome formation is considered to take place on the endoplasmic reticulum and involves functions of autophagy-related (Atg) proteins. Here, we report the identification and characterization of mammalian Atg2 homologues Atg2A and Atg2B. Simultaneous silencing of Atg2A and Atg2B causes a block in autophagic flux and accumulation of unclosed autophagic structures containing most Atg proteins. Atg2A localizes on the autophagic membrane, as well as on the surface of lipid droplets. The Atg2A region containing amino acids 1723–1829, which shows relatively high conservation among species, is required for localization to both the autophagic membrane and lipid droplet and is also essential for autophagy. Depletion of both Atg2A and Atg2B causes clustering of enlarged lipid droplets in an autophagy-independent manner. These data suggest that mammalian Atg2 proteins function both in autophagosome formation and regulation of lipid droplet morphology and dispersion.

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In vitro reconstitution of autophagic processes

Biochemical Society Transactions ◽

10.1042/bst20200130 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2003-2014

Author(s):

Jahangir Md. Alam ◽

Nobuo N. Noda

Keyword(s):

Molecular Mechanisms ◽

De Novo ◽

Membrane Structures ◽

Autophagosome Formation ◽

The Past ◽

Atg Proteins ◽

In Vitro Reconstitution ◽

Complicated Process

Autophagy is a lysosomal degradation system that involves de novo autophagosome formation. A lot of factors are involved in autophagosome formation, including dozens of Atg proteins that form supramolecular complexes, membrane structures including vesicles and organelles, and even membraneless organelles. Because these diverse higher-order structural components cooperate to mediate de novo formation of autophagosomes, it is too complicated to be elaborated only by cell biological approaches. Recent trials to regenerate each step of this phenomenon in vitro have started to elaborate on the molecular mechanisms of such a complicated process by simplification. In this review article, we outline the in vitro reconstitution trials in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations.

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Autophagy in Alcohol-Induced Multiorgan Injury: Mechanisms and Potential Therapeutic Targets

BioMed Research International ◽

10.1155/2014/498491 ◽

2014 ◽

Vol 2014 ◽

pp. 1-20 ◽

Cited By ~ 25

Author(s):

Yuan Li ◽

Shaogui Wang ◽

Hong-Min Ni ◽

Heqing Huang ◽

Wen-Xing Ding

Keyword(s):

Molecular Mechanisms ◽

Tissue Injury ◽

Current Knowledge ◽

Therapeutic Targets ◽

Degradation Pathway ◽

Protective Mechanism ◽

Intracellular Degradation ◽

Injury Mechanisms ◽

Evolutionarily Conserved

Autophagy is a genetically programmed, evolutionarily conserved intracellular degradation pathway involved in the trafficking of long-lived proteins and cellular organelles to the lysosome for degradation to maintain cellular homeostasis. Alcohol consumption leads to injury in various tissues and organs including liver, pancreas, heart, brain, and muscle. Emerging evidence suggests that autophagy is involved in alcohol-induced tissue injury. Autophagy serves as a cellular protective mechanism against alcohol-induced tissue injury in most tissues but could be detrimental in heart and muscle. This review summarizes current knowledge about the role of autophagy in alcohol-induced injury in different tissues/organs and its potential molecular mechanisms as well as possible therapeutic targets based on modulation of autophagy.

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Interaction of caveolin-1 with ATG12-ATG5 system suppresses autophagy in lung epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00268.2013 ◽

2014 ◽

Vol 306 (11) ◽

pp. L1016-L1025 ◽

Cited By ~ 41

Author(s):

Zhi-Hua Chen ◽

Jiao-Fei Cao ◽

Jie-Sen Zhou ◽

Hui Liu ◽

Luan-Qing Che ◽

...

Keyword(s):

Epithelial Cells ◽

Molecular Mechanisms ◽

Lung Epithelial Cells ◽

Major Constituent ◽

Binding Motif ◽

Conjugation System ◽

Autophagosome Formation ◽

Caveolin 1 ◽

Lung Epithelial ◽

Biochemical Analyses

Autophagy plays a pivotal role in cellular homeostasis and adaptation to adverse environments, although the regulation of this process remains incompletely understood. We have recently observed that caveolin-1 (Cav-1), a major constituent of lipid rafts on plasma membrane, can regulate autophagy in cigarette smoking-induced injury of lung epithelium, although the underlying molecular mechanisms remain incompletely understood. In the present study we found that Cav-1 interacted with and regulated the expression of ATG12-ATG5, an ubiquitin-like conjugation system crucial for autophagosome formation, in lung epithelial Beas-2B cells. Deletion of Cav-1 increased basal and starvation-induced levels of ATG12-ATG5 and autophagy. Biochemical analyses revealed that Cav-1 interacted with ATG5, ATG12, and their active complex ATG12-ATG5. Overexpression of ATG5 or ATG12 increased their interactions with Cav-1, the formation of ATG12-ATG5 conjugate, and the subsequent basal levels of autophagy but resulted in decreased interactions between Cav-1 and another molecule. Knockdown of ATG12 enhanced the ATG5-Cav-1 interaction. Mutation of the Cav-1 binding motif on ATG12 disrupted their interaction and further augmented autophagy. Cav-1 also regulated the expression of ATG16L, another autophagy protein associating with the ATG12-ATG5 conjugate during autophagosome formation. Altogether these studies clearly demonstrate that Cav-1 competitively interacts with the ATG12-ATG5 system to suppress the formation and function of the latter in lung epithelial cells, thereby providing new insights into the molecular mechanisms by which Cav-1 regulates autophagy and suggesting the important function of Cav-1 in certain lung diseases via regulation of autophagy homeostasis.

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Structural Biology and Electron Microscopy of the Autophagy Molecular Machinery

Cells ◽

10.3390/cells8121627 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1627 ◽

Cited By ~ 2

Author(s):

Louis Tung Faat Lai ◽

Hao Ye ◽

Wenxin Zhang ◽

Liwen Jiang ◽

Wilson Chun Yu Lau

Keyword(s):

Electron Microscopy ◽

Single Particle ◽

Molecular Mechanisms ◽

De Novo ◽

Critical Role ◽

Degradation Process ◽

Particle Analysis ◽

Single Particle Analysis ◽

Environmental Stress Response ◽

Atg Proteins

Autophagy is a highly regulated bulk degradation process that plays a key role in the maintenance of cellular homeostasis. During autophagy, a double membrane-bound compartment termed the autophagosome is formed through de novo nucleation and assembly of membrane sources to engulf unwanted cytoplasmic components and targets them to the lysosome or vacuole for degradation. Central to this process are the autophagy-related (ATG) proteins, which play a critical role in plant fitness, immunity, and environmental stress response. Over the past few years, cryo-electron microscopy (cryo-EM) and single-particle analysis has matured into a powerful and versatile technique for the structural determination of protein complexes at high resolution and has contributed greatly to our current understanding of the molecular mechanisms underlying autophagosome biogenesis. Here we describe the plant-specific ATG proteins and summarize recent structural and mechanistic studies on the protein machinery involved in autophagy initiation with an emphasis on those by single-particle analysis.

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Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471400947x ◽

2014 ◽

Vol 70 (7) ◽

pp. 1944-1953 ◽

Cited By ~ 35

Author(s):

Eunha Hwang ◽

Hae-Kap Cheong ◽

Ameeq Ul Mushtaq ◽

Hye-Yeon Kim ◽

Kwon Joo Yeo ◽

...

Keyword(s):

Structural Information ◽

Critical Role ◽

Three Dimensional ◽

Helical Structure ◽

Structural Features ◽

Signalling Pathway ◽

Structural Basis ◽

X Ray Crystallography ◽

Hippo Signalling

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1–RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1–RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432–Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1–RASSF5 complex showed a longer helical structure (Ser438–Lys480) than that in the MST1 homodimer (Val441–Lys480). Moreover, extensive polar and nonpolar contacts in the MST1–RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST–RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1–RASSF5 SARAH domain in apoptosis signalling.

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Structural basis for an exceptionally strong preference for asparagine residue at the S2 subsite of Stenotrophomonas maltophilia dipeptidyl peptidase 7

Scientific Reports ◽

10.1038/s41598-021-86965-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Akihiro Nakamura ◽

Yoshiyuki Suzuki ◽

Yasumitsu Sakamoto ◽

Saori Roppongi ◽

Chisato Kushibiki ◽

...

Keyword(s):

Stenotrophomonas Maltophilia ◽

Molecular Mechanisms ◽

Structural Basis ◽

Resistant Bacteria ◽

Drug Resistant Bacteria ◽

The Family ◽

Hydrophobic Amino Acids ◽

Biochemical Analyses ◽

Strict Specificity ◽

S2 Subsite

AbstractThe emergence of drug-resistant bacteria has become a major problem worldwide. Bacterial dipeptidyl peptidases 7 and 11 (DPP7s and DPP11s), belonging to the family-S46 peptidases, are important enzymes for bacterial growth and are not present in mammals. Therefore, specific inhibitors for these peptidases are promising as potential antibiotics. While the molecular mechanisms underlining strict specificity at the S1 subsite of S46 peptidases have been well studied, those of relatively broad preference at the S2 subsite of these peptidases are unknown. In this study, we performed structural and biochemical analyses on DPP7 from Stenotrophomonas maltophilia (SmDPP7). SmDPP7 showed preference for the accommodation of hydrophobic amino acids at the S2 subsite in general, but as an exception, also for asparagine, a hydrophilic amino acid. Structural analyses of SmDPP7 revealed that this exceptional preference to asparagine is caused by a hydrogen bonding network at the bottom of the S2 subsite. The residues in the S2 subsite are well conserved among S46 peptidases as compared with those in the S1 subsite. We expect that our findings will contribute toward the development of a universal inhibitor of S46 peptidases.

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On the molecular mechanisms of mitotic kinase activation

Open Biology ◽

10.1098/rsob.120136 ◽

2012 ◽

Vol 2 (11) ◽

pp. 120136 ◽

Cited By ~ 74

Author(s):

Richard Bayliss ◽

Andrew Fry ◽

Tamanna Haq ◽

Sharon Yeoh

Keyword(s):

Protein Interactions ◽

Molecular Mechanisms ◽

Critical Role ◽

Significant Proportion ◽

Structural Basis ◽

Kinase Activation ◽

Mitotic Kinases ◽

Mitotic Kinase ◽

Disease Biology ◽

Activation Mechanisms

During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients.

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Structural basis of glycerophosphodiester recognition by the Mycobacterium tuberculosis substrate-binding protein UgpB

10.1101/529602 ◽

2019 ◽

Author(s):

Jonathan Fenn ◽

Ridvan Nepravishta ◽

Collette S Guy ◽

James Harrison ◽

Jesus Angulo ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Potential Role ◽

Substrate Binding ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

X Ray Crystallography ◽

Biochemical Analyses ◽

Substrate Binding Domain ◽

Substrate Binding Protein

AbstractMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833) is the substrate binding domain of the UgpABCE transporter that recognises glycerophosphocholine (GPC) indicating a potential role in glycerophospholipid recycling. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB is promiscuous for other glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC therefore optimising the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites during intracellular infection.

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