The plasma membrane contributes to formation of autophagosomes, the double-membrane vesicles that sequester cytosolic cargo and deliver it to lysosomes for degradation during autophagy. In this study, we have identified a regulatory role for connexins (Cx), main components of plasma membrane gap junctions, in autophagosome formation. We have found that plasma membrane-localised Cx proteins constitutively downregulate autophagy via a direct interaction with several autophagy-related proteins involved in the initial steps of autophagosome formation such as Atg16 and components of the PI3K autophagy initiation complex (Vps34, Beclin-1 and Vps15). On nutrient starvation, this inhibitory effect is released by the arrival of Atg14 to the Cx-Atg complex. This promotes the internalization of Cx-Atg along with Atg9, which is also recruited to the plasma membrane in response to starvation. Maturation of the Cx-containing pre-autophagosomes into autophagosomes leads to degradation of these endogenous inhibitors, allowing for sustained activation of autophagy.
Autophagy; Connexins; Gap junctions; Lysosomes; Plasma membrane
Far from now are the days when investigators raced to identify the proteolytic system responsible for the degradation of their favorite protein. Nowadays, it is well accepted that the same protein can be degraded by different systems depending on factors such as cell type, cellular conditions, or functionality of each proteolytic pathway. The realization of this sharing of substrates among pathways has also helped to unveil deeper levels of communication among the different proteolytic systems. Thus, cells often respond to blockage of one degradative mechanism by upregulating any of the other available pathways. In addition, effectors and regulators of one proteolytic system can be degraded by a different proteolytic pathway that exerts, in this way, a regulatory function. In this mini-review, we describe the different levels of cross-talk among autophagic pathways and the ubiquitin/proteasome system. We also provide examples of how this proteolytic communication is used for compensatory purposes in different pathological conditions and discuss the possible therapeutic potential of targeting the modulators of the cross-talk among proteolytic pathways.
The importance of the cellular quality control systems in the maintenance of neuronal homeostasis and in the defense against neurodegeneration is well recognized. Chaperones and proteolytic systems, main components of these cellular surveillance mechanisms, are key in the fight against the proteotoxicity that is often associated with severe neurodegenerative diseases. However, in recent years, a new theme has emerged that suggests that components of the protein quality control pathways are often targets of the toxic effects of pathogenic proteins and that their failure to function properly contributes to pathogenesis and disease progression. In this mini-review, we describe this dual role as “savior” and “victim” in the context of neurodegeneration for chaperone-mediate autophagy, a cellular pathway involved in the selective degradation of cytosolic proteins in lysosomes.
aging; autophagy; chaperones; lysosomes; neurodegeneration; proteotoxicity
Autophagy is essential for neuronal homeostasis and its dysfunction has been directly linked to a growing number of neurodegenerative disorders. The reasons behind autophagic failure in degenerating neurons can be very diverse because of the different steps required for autophagy and the characterization of the molecular players involved in each of them. Understanding the step(s) affected in the autophagic process in each disorder could explain differences in the course of these pathologies and will be essential to develop targeted therapeutic approaches for each disease based on modulation of autophagy. In this review we present examples of different types of autophagic dysfunction described in common neurodegenerative disorders, and discuss the prospect of exploring some of the recently identified autophagic variants and the interactions among autophagic and non-autophagic proteolytic systems as possible future therapeutic targets.
Autophagy is a process traditionally known to contribute to cellular cleaning through the removal of intracellular components in lysosomes. In recent years, the intensive scrutiny that autophagy has been subjected to at the molecular level, has also contributed to expand our understanding of the physiological role of this pathway. Added to the well-characterized role in quality control, autophagy has proven important in the maintenance of cellular homeostasis and of the energetic balance, in cellular and tissue remodeling and in the cellular defense against extracellular insults and pathogens. It is not a surprise that in light of this growing number of physiological functions, connections between autophagic malfunctioning and human pathologies have also been strengthened. In this review, we focus on several pathological conditions associated to primary or secondary defects in autophagy, and comment on a recurring theme for many of them, that is the fact that autophagy can often exert both beneficial and aggravating effects on the progression of disease. Elucidating the factors that determine the switch between these dual functions of autophagy in disease has become a priority when considering the potential therapeutic implications of the pharmacological modulation of autophagy in many of these pathological conditions.
cancer; cardiomyopathy; immunity; lysosomes; neurodegeneration; protein aggregation; proteolysis; proteotoxicity; T-cell function
Turnover of cellular components in lysosomes or autophagy is an essential mechanism for cellular quality control. Added to this cleaning role, autophagy has recently been shown to participate in the dynamic interaction of cells with the surrounding environment by acting as a point of integration of extracellular cues. In this review, we focus on the relationship between autophagy and two types of environmental factors: nutrients and pathogens. We describe their direct effect on autophagy and discuss how the autophagic reaction to these stimuli allows cells to accommodate the requirements of the cellular response to stress, including those specific to the immune responses.
aging; antigen presentation; chaperones; lipids; lysosomes; starvation; T-cells
Cells continuously turn over proteins through cycles of synthesis and degradation in order to maintain a functional proteome and to exert a tight control in the levels of regulatory proteins. Selective degradation of proteins was initially thought to be an exclusive function of the ubiquitin-proteasome system however, over the years, the contribution of lysosomes to this selective degradation, through the process of autophagy, has become consolidated. In this context, molecular chaperones, classically associated with protein folding, unfolding and assembling, have been revealed as important modulators of selectivity during the autophagic process. Here, we review this relatively new role of chaperones in mediating selective autophagy and comment on how alterations of this function can lead to human pathologies associated to proteotoxicity.
Aging; chaperones; lysosomes; membrane proteins; protein degradation
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD). In this work, we demonstrate that LRRK2 can be degraded in lysosomes by chaperone-mediated autophagy (CMA), whereas the most common pathogenic mutant form of LRRK2, G2019S, is poorly degraded by this pathway. In contrast to typical CMA substrates, lysosomal binding of both wild-type and several pathogenic mutant LRRK2 proteins is enhanced in the presence of other CMA substrates, which interferes with the organization of the CMA translocation complex, resulting in defective CMA. Cells respond to such LRRK2-mediated CMA compromise by increasing levels of the CMA lysosomal receptor as seen in neuronal cultures and brains of LRRK2 transgenic mice, iPSC-derived dopaminergic neurons, and brains of mutant LRRK2 PD patients. This novel LRRK2 self-perpetuating inhibitory effect on CMA could underlie toxicity in PD by compromising the degradation of alpha-synuclein, another PD-related protein degraded by this pathway.
chaperones; lysosomal membrane proteins; lysosomes; neurodegeneration; Parkinson’s disease; proteotoxicity; induced pluripotent stem cells
Chaperone-mediated autophagy (CMA) is a selective form of autophagy whose distinctive feature is the fact that substrate proteins are translocated directly from the cytosol across the lysosomal membrane for degradation inside lysosomes. CMA substrates are cytosolic proteins bearing a pentapeptide motif in their sequence that, when recognized by the cytosolic chaperone HSPA8/HSC70, targets them to the surface of the lysosomes. Once there, substrate proteins bind to the lysosome-associated membrane protein type 2 isoform A (LAMP2A), inducing assembly of this receptor protein into a higher molecular weight protein complex that is used by the substrate proteins to reach the lysosomal lumen. CMA is constitutively active in most cells but it is maximally activated under conditions of stress.
cholesterol; diet; lipid microdomains; lipidomic analysis; lysosomes; membrane proteins; proteolysis
Protein quality control is essential for cellular survival. Failure to eliminate pathogenic proteins leads to their intracellular accumulation in the form of protein aggregates. Autophagy can recognize protein aggregates and degrade them in lysosomes. However, some aggregates escape the autophagic surveillance. Here we analyze the autophagic degradation of different types of aggregates of synphilin-1 (Sph1), a protein often found in pathogenic protein inclusions. We show that small Sph1 aggregates and large aggresomes are differentially targeted by constitutive and inducible autophagy. Furthermore, we identify a region in Sph1 necessary for its own basal and inducible aggrephagy, and sufficient for the degradation of other pro-aggregating proteins. Although the presence of this peptide is sufficient for basal aggrephagy, inducible aggrephagy requires its ubiquitination, which diminishes protein mobility on the surface of the aggregate and favors the recruitment and assembly of the protein complexes required for autophagosome formation. Our study reveals different mechanisms for cells to cope with aggregate proteins via autophagy and supports the idea that autophagic susceptibility of prone-to-aggregate proteins may not depend on the nature of the aggregating proteins per se but on their dynamic properties in the aggregate.
autophagy; protein aggregates; aggresomes; synphilin-1; protein mobility; ubiquitination
Connexins modulate intercellular communication when assembled in gap junctions. Compromised macroautophagy increases cellular communication due to failure to degrade connexins at gap junctions. Nedd4-mediated ubiquitinylation of the connexin molecule is required to trigger its autophagy-dependent internalization and degradation.
Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.
Autophagy contributes to the removal of prone-to-aggregate proteins, but in several instances these pathogenic proteins have been shown to interfere with autophagic activity. In the case of Huntington’s disease (HD), a congenital neurodegenerative disorder resulting from mutation in the huntingtin protein, we have previously described that the mutant protein interferes with the ability of autophagic vacuoles to recognize cytosolic cargo. Growing evidence supports the existence of cross-talk among autophagic pathways, suggesting the possibility of functional compensation when one of them is compromised. In this study, we have identified a compensatory upregulation of chaperone-mediated autophagy (CMA) in different cellular and mouse models of HD. Components of CMA, namely the lysosome-associated membrane protein type 2A (LAMP-2A) and lysosomal-hsc70, are markedly increased in HD models. The increase in LAMP-2A is achieved through both an increase in the stability of this protein at the lysosomal membrane and transcriptional upregulation of this splice variant of the lamp-2 gene. We propose that CMA activity increases in response to macroautophagic dysfunction in the early stages of HD, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to cellular failure and the onset of pathological manifestations.
chaperones; lysosomal membrane proteins; lysosomes; neurodegeneration; proteotoxicity; proteolysis
Autophagy mediates the degradation of cellular components in lysosomes, assuring removal of altered or dysfunctional proteins and organelles. Autophagy is not only activated in response to cellular damage, but in fact, one of its strongest and better-characterized stimuli is starvation. Activation of autophagy when nutrients are scarce allows cells to reutilize their own constituents for energy. Besides protein breakdown, autophagy also contributes to the mobilization of diverse cellular energy stores. This recently discovered interplay between autophagy and lipid and carbohydrate metabolism reveals the existence of a dynamic feedback between autophagy and cellular energy balance.
glycogen; energy; lysosomes; lipid stores; lipolysis; proteolysis
All cells count on precise mechanisms that regulate protein homeostasis to maintain a stable and functional proteome. A progressive deterioration in the ability of cells to preserve the stability of their proteome occurs with age and contributes to the functional loss characteristic of old organisms. Molecular chaperones and the proteolytic systems are responsible for this cellular quality control by assuring continuous renewal of intracellular proteins. When protein damage occurs, such as during cellular stress, the coordinated action of these cellular surveillance systems allows detection and repair of the damaged structures or, in many instances, leads to the complete elimination of the altered proteins from inside cells. Dysfunction of the quality control mechanisms and intracellular accumulation of abnormal proteins in the form of protein inclusions and aggregates occur in almost all tissues of an aged organism. Preservation or enhancement of the activity of these surveillance systems until late in life improves their resistance to stress and is sufficient to slow down aging. In this work, we review recent advances on our understanding of the contribution of chaperones and proteolytic systems to the maintenance of cellular homeostasis, the cellular response to stress and ultimately to longevity.
Autophagy; chaperones; proteases; proteasome; proteolysis; ubiquitin
Chaperone-mediated autophagy is a selective mechanism for degradation of soluble cytosolic proteins in lysosomes that distinguishes itself from other autophagic pathways by the selectivity with which CMA substrates are targeted for degradation. The recent molecular dissection of this autophagic pathway and the development of experimental models with compromised CMA have unveiled the important contribution of this pathway to protein quality control. In fact, CMA activation seems to be a common mechanism of cellular defense against proteotoxicity.
chaperones; lysosomes; oxidative stress; proteases; selective autophagy
Lipid droplets (LDs), initially considered “inert” lipid deposits, have gained during the last decade the classification of cytosolic organelles due to their defined composition and the multiplicity of specific cellular functions in which they are involved. The classification of LD as organelles brings along the need for their regulated turnover and recent findings support the direct contribution of autophagy to this turnover through a process now described as lipophagy. This paper focuses on the characteristics of this new type of selective autophagy and the cellular consequences of the mobilization of intracellular lipids through this process. Lipophagy impacts the cellular energetic balance directly, through lipid breakdown and, indirectly, by regulating food intake. Defective lipophagy has been already linked to important metabolic disorders such as fatty liver, obesity and atherosclerosis, and the age-dependent decrease in autophagy could underline the basis for the metabolic syndrome of aging.
Autophagy delivers cytosolic components to lysosomes for their degradation. The delivery of autophagic cargo to late endosomes for complete or partial degradation has also been described. In this report, we present evidence that distinct autophagic mechanisms control cytosolic protein delivery to late endosomes and identify a microautophagy-like process that delivers soluble cytosolic proteins to the vesicles of late endosomes/multivesicular bodies (MVB). This microautophagy-like process has selectivity and is distinct from chaperone-mediated autophagy that occurs in lysosomes. Endosomal microautophagy occurs during MVB formation, relying on the ESCRT I and III systems for formation of the vesicles in which the cytosolic cargo is internalized. Protein cargo selection is mediated by the chaperone hsc70 and requires the cationic domain of hsc70 for electrostatic interactions with the endosomal membrane. Therefore, we propose that endosomal microautophagy shares molecular components with both the endocytic and autophagic pathways.
Chaperone-mediated autophagy (CMA) is a selective lysosomal pathway for the degradation of cytosolic proteins. We review in this work some of the recent findings on this pathway regarding the molecular mechanisms that contribute to substrate targeting, binding and translocation across the lysosomal membrane. We have placed particular emphasis on the critical role that changes in the lipid composition of the lysosomal membrane play in the regulation of CMA, as well as the modulatory effect of other novel CMA components. In the second part of this review, we describe the physiological relevance of CMA and its role as one of the cellular mechanisms involved in the response to stress. Changes with age in CMA activity and the contribution of failure of CMA to the phenotype of aging and to the pathogenesis of several age-related pathologies are also described.
chaperones; lysosomes; membrane proteins; protein translocation; proteases; proteolysis
Continuous renewal of intracellular components is required to preserve cellular functionality. In fact, failure to timely turnover proteins and organelles leads often to cell death and disease. Different pathways contribute to the degradation of intracellular components in lysosomes or autophagy. In this review, we focus on chaperone-mediated autophagy (CMA), a selective form of autophagy that modulates the turnover of a specific pool of soluble cytosolic proteins. Selectivity in CMA is conferred by the presence of a targeting motif in the cytosolic substrates that, upon recognition by a cytosolic chaperone, determines delivery to the lysosomal surface. Substrate proteins undergo unfolding and translocation across the lysosomal membrane before reaching the lumen, where they are rapidly degraded. Better molecular characterization of the different components of this pathway in recent years, along with the development of transgenic models with modified CMA activity and the identification of CMA dysfunction in different severe human pathologies and in aging, are all behind the recent regained interest in this catabolic pathway.
aging; lysosomes; membrane proteins; proteases; protein translocation
Chaperone-mediated autophagy (CMA) is a selective type of autophagy responsible for the lysosomal degradation of soluble cytosolic proteins. In contrast to other forms of autophagy where cargo is sequestered and delivered to lysosomes through membrane fusion/excision, CMA substrates reach the lysosomal lumen after direct translocation across the lysosomal membrane. CMA is part of the cellular quality control systems and as such, essential for the cellular response to stress. CMA activity decreases with age, likely contributing to the accumulation of altered proteins characteristic in tissues from old organisms. Furthermore, impairment of CMA underlies the pathogenesis of certain human pathologies such as neurodegenerative disorders. These findings have drawn renewed attention to CMA and a growing interest in the measurement of changes in CMA activity under different physiological and pathological conditions. In this chapter we review the different experimental approaches utilized to assess CMA activity both in cells in culture and in different organs from animals.
The cellular process of autophagy (literally “self-eating”) is important for maintaining the homeostasis and bioenergetics of mammalian cells. Two of the best-studied mechanisms of autophagy are macroautophagy and chaperone-mediated autophagy (CMA). Changes in macroautophagy activity have been described in cancer cells and in solid tumors, and inhibition of macroautophagy promotes tumorigenesis. Because normal cells respond to inhibition of macroautophagy by up-regulation of the CMA pathway, we aimed to characterize the CMA status in different cancer cells and to determine the contribution of changes in CMA to tumorigenesis. Here, we show consistent up-regulation of CMA in different types of cancer cells regardless of the status of macroautophagy. We also demonstrate an increase in CMA components in human cancers of different types and origins. CMA is required for cancer cell proliferation in vitro because it contributes to the maintenance of the metabolic alterations characteristic of malignant cells. Using human lung cancer xenografts in mice, we confirmed the CMA dependence of cancer cells in vivo. Inhibition of CMA delays xenograft tumor growth, reduces the number of cancer metastases, and induces regression of existing human lung cancer xenografts in mice. The fact that similar manipulations of CMA also reduce tumor growth of two different melanoma cell lines suggests that targeting this autophagic pathway may have broad antitumorigenic potential.
Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signaling motivated us to explore the possible functional interactions between this signaling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signaling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. While abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signaling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie the basis of some common ciliopathies.
primary cilia; intraflagellar transport proteins; lysosomes; autophagosomes; vesicular trafficking
Chaperone-mediated autophagy (CMA) contributes to cellular quality control and the cellular response to stress through the selective degradation of cytosolic proteins in lysosomes. Decrease in CMA activity occurs in aging and in age-related disorders (for example, neurodegenerative diseases and diabetes). Although prevention of this age-dependent decline through genetic manipulation in mouse has proven beneficial, chemical modulation of CMA is not currently possible, due in part to the lack of information on the signaling mechanisms that modulate this pathway. In this work, we report that signaling through the retinoic acid receptor alpha (RARα) inhibits CMA and apply structure-based chemical design to develop synthetic derivatives of all-trans-retinoic acid (ATRA) to specifically neutralize this inhibitory effect. We demonstrate that chemical enhancement of CMA protects cells from oxidative stress and from proteotoxicity, supporting a potential therapeutic opportunity when reduced CMA contributes to cellular dysfunction and disease.
all-trans-retinoic acid; lysosomes; oxidative stress; proteotoxicity; retinoic acid receptor
All cellular proteins undergo continuous synthesis and degradation. This permanent renewal is necessary to maintain a functional proteome and to allow for rapid changes in levels of specific proteins with regulatory purposes. Although for a long time lysosomes were considered unable to contribute to the selective degradation of individual proteins, the discovery of chaperone-mediated autophagy (CMA) changed this notion. Here, we review the characteristics that set CMA apart from other types of lysosomal degradation and the subset of molecules that confer cells the capability to identify individual cytosolic proteins and direct them across the lysosomal membrane for degradation.
aging; cancer; chaperones; lysosomes; membrane proteins; neurodegeneration; protein degradation
Cells maintain a healthy proteome through continuous evaluation of the quality of each of their proteins. Quality control requires the coordinated action of chaperones and proteolytic systems. Chaperones identify abnormal or unstable conformations in proteins and often assist them to regain stability. However, if repair is not possible, the aberrant protein is eliminated from the cellular cytosol to prevent undesired interactions with other proteins or its organization into toxic multimeric complexes. Autophagy and the ubiquitin/proteasome system mediate the complete degradation of abnormal protein products. In this article, we describe each of these proteolytic systems and their contribution to cellular quality control. We also comment on the cellular consequences resulting from the dysfunction of these systems in common human protein conformational disorders and provide an overview on current therapeutic interventions based on the modulation of the proteolytic systems.
Lysosomes and proteasomes are coordinated quality-control mechanisms that degrade abnormal proteins. In each case, the targets must be selected and tagged appropriately, and malfunctions can cause severe protein-conformation disorders.