The LIR motif - crucial for selective autophagy.
TL;DR: Comment on new insights on the interactions of LIR-containing proteins with members of the ATG8 protein family on the interaction of autophagy receptors to LC3-interacting region proteins anchored in the phagophore membrane.
Abstract: (Macro)autophagy is a fundamental degradation process for macromolecules and organelles of vital importance for cell and tissue homeostasis. Autophagy research has gained a strong momentum in recent years because of its relevance to cancer, neurodegenerative diseases, muscular dystrophy, lipid storage disorders, development, ageing and innate immunity. Autophagy has traditionally been thought of as a bulk degradation process that is mobilized upon nutritional starvation to replenish the cell with building blocks and keep up with the energy demand. This view has recently changed dramatically following an array of papers describing various forms of selective autophagy. A main driving force has been the discovery of specific autophagy receptors that sequester cargo into forming autophagosomes (phagophores). At the heart of this selectivity lies the LC3-interacting region (LIR) motif, which ensures the targeting of autophagy receptors to LC3 (or other ATG8 family proteins) anchored in the phagophore membrane. LIR-containing proteins include cargo receptors, members of the basal autophagy apparatus, proteins associated with vesicles and of their transport, Rab GTPase-activating proteins (GAPs) and specific signaling proteins that are degraded by selective autophagy. Here, we comment on these new insights and focus on the interactions of LIR-containing proteins with members of the ATG8 protein family.
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TL;DR: In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.
Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.
For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
5,187 citations
TL;DR: How AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology is discussed.
Abstract: Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.
1,873 citations
TL;DR: Observations suggest Nrf2 directs metabolic reprogramming during stress, which would enable the factor to orchestrate adaptive responses to diverse forms of stress.
1,482 citations
Daniel J. Klionsky1, Amal Kamal Abdel-Aziz2, Sara Abdelfatah3, Mahmoud Abdellatif4 +2980 more•Institutions (777)
TL;DR: In this article, the authors present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes.
Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
1,129 citations
TL;DR: It is proposed that the isolation membrane forms from the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and the role of ATG proteins and the vesicular trafficking machinery in autophagosome formation is proposed.
Abstract: Autophagosome biogenesis starts at the isolation membrane (also called the phagophore). Our understanding of the molecular processes that initiate the isolation membrane, the membrane sources from which this membrane originates and how it is expanded to the autophagosome membrane by autophagy-related (ATG) proteins and the vesicular trafficking machinery, is increasing.
1,117 citations
References
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TL;DR: This Review summarizes recent advances in understanding the physiological functions of autophagy and its possible roles in the causation and prevention of human diseases.
6,301 citations
"The LIR motif - crucial for selecti..." refers background in this paper
...…autophagy is highly relevant for a number of diseases, including cancer, neurodegenerative diseases, muscular dystrophy, lipidstorage disorders and processes such as development, ageing and innate immunity (Levine and Kroemer, 2008; Levine et al., 2011; Mizushima and Komatsu, 2011; Deretic, 2012)....
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...Because of its fundamental importance in cellular homeostasis and cellular signaling, autophagy is highly relevant for a number of diseases, including cancer, neurodegenerative diseases, muscular dystrophy, lipidstorage disorders and processes such as development, ageing and innate immunity (Levine and Kroemer, 2008; Levine et al., 2011; Mizushima and Komatsu, 2011; Deretic, 2012)....
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TL;DR: It is explored how recent mouse models in combination with advances in human genetics are providing key insights into how the impairment or activation of autophagy contributes to pathogenesis of diverse diseases, from neurodegenerative diseases such as Parkinson disease to inflammatory disorders such as Crohn disease.
4,529 citations
"The LIR motif - crucial for selecti..." refers background in this paper
...…autophagy is highly relevant for a number of diseases, including cancer, neurodegenerative diseases, muscular dystrophy, lipidstorage disorders and processes such as development, ageing and innate immunity (Levine and Kroemer, 2008; Levine et al., 2011; Mizushima and Komatsu, 2011; Deretic, 2012)....
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...Because of its fundamental importance in cellular homeostasis and cellular signaling, autophagy is highly relevant for a number of diseases, including cancer, neurodegenerative diseases, muscular dystrophy, lipidstorage disorders and processes such as development, ageing and innate immunity (Levine and Kroemer, 2008; Levine et al., 2011; Mizushima and Komatsu, 2011; Deretic, 2012)....
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TL;DR: It is demonstrated that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation and p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.
3,676 citations
"The LIR motif - crucial for selecti..." refers background or methods in this paper
...The structures of p62 and Atg19 peptides bound to LC3B and Atg8, respectively, revealed a common W-x-x-L motif (x5any amino acid) (Ichimura et al., 2008b; Noda et al., 2008) (Fig....
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...Different strategies have been used to identify proteins that interact with ATG8 proteins through LIR motifs, including candidate approaches (Pankiv et al., 2007; Noda et al., 2008; Sancho et al., 2012), bioinformatics searches (Kraft et al....
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...Detailed deletion mapping and point mutation analyses, together with X-ray crystallography and NMR lead to the elucidation of the LC3-interacting region (LIR) motifs of p62 and of the Cvt cargo receptor Atg19 (Pankiv et al., 2007; Ichimura et al., 2008b; Noda et al., 2008)....
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...J o u rn a l o f C e ll S c ie n c e Box 1), and the importance of the acidic residues N-terminal to the core of the DDDWTHL LIR motif of p62 was verified by alanine substitutions (Pankiv et al., 2007; Ichimura et al., 2008a; Noda et al., 2008)....
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...The first selective autophagy receptor to be identified was p62 [also known as sequestosome-1 (SQSTM1)] (Bjørkøy et al., 2005; Komatsu et al., 2007; Pankiv et al., 2007)....
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01 Jan 2007
TL;DR: In this article, the authors showed that the polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy by using a 22-residue sequence of p62 containing an evolutionarily conserved motif.
Abstract: Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related -aminobutyrate receptor-associated protein and-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 m diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.
3,172 citations
TL;DR: Crosstalk between multiple Rab GTPases through shared effectors, or through effectors that recruit selective Rab activators, ensures the spatiotemporal regulation of vesicle traffic.
Abstract: Rab GTPases control intracellular vesicle traffic by acting as regulatable switches that recruit effector molecules when in their GTP-bound form. The functional coupling between multiple Rab GTPases ensures the spatiotemporally coordinated regulation of vesicle traffic. Membrane trafficking between organelles by vesiculotubular carriers is fundamental to the existence of eukaryotic cells. Central in ensuring that cargoes are delivered to their correct destinations are the Rab GTPases, a large family of small GTPases that control membrane identity and vesicle budding, uncoating, motility and fusion through the recruitment of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors. Crosstalk between multiple Rab GTPases through shared effectors, or through effectors that recruit selective Rab activators, ensures the spatiotemporal regulation of vesicle traffic. Functional impairments of Rab pathways are associated with diseases, such as immunodeficiencies, cancer and neurological disorders.
2,893 citations
"The LIR motif - crucial for selecti..." refers background in this paper
...The Rab GTPases (a large family of monomeric, small GTPases) in their active form are spatially organized into distinct membrane regions, where they recruit effectors to regulate intracellular vesicle trafficking events (Stenmark, 2009)....
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