Artophagy: The art of autophagy--macroautophagy. Interview by Daniel J. Klionsky.

Many of us have sketched (by hand or on the computer) depictions of macroautophagy; however, how often have we considered which elements in the drawing are key to illustrating the process? These types of illustrations are easily modified and/or discarded. On the other hand, if you plan to depict the process of macroautophagy in a more permanent medium you need to be more thoughtful about the composition. What items must be included? How should they be situated? What should be the size of each component? Here, we consider one example of an artist's approach to depicting macroautophagy in a mixed-medium sculpture.

Chaperone-mediated autophagy: the heretofore untold story of J. Fred "Paulo" Dice. Interview by Daniel J. Klionsky.

The best-characterized process of autophagy is macroautophagy. Many an article or talk has started with the phrase "...macroautophagy, hereafter referred to as autophagy." This one will be different because we are going to learn more about the person most responsible for increasing our understanding of chaperone-mediated autophagy, or CMA, J. Fred Dice.

Chaperone-mediated autophagy is defective in mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin-1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two-hybrid and co-immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone-mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex- and age-matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal-associated membrane protein type 2A (LAMP-2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly-Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is a lysosomal pathway of proteolysis that is responsible for the degradation of 30% of cytosolic proteins under conditions of prolonged nutrient deprivation. Molecular chaperones in the cytosol and in the lysosomal lumen stimulate this proteolytic pathway. The molecular chaperones in the cytosol unfold substrate proteins prior to their translocation across the lysosomal membrane, while the chaperone in the lysosomal lumen is probably required to pull the substrate protein across the lysosomal membrane. A critical component for CMA is a receptor in the lysosomal membrane, the lysosome-associated membrane protein (LAMP) type 2A. LAMP-2A levels in the lysosomal membrane can be increased by reduced degradation and/or redistribution from the lysosomal lumen to the lysosomal membrane. Recent results show that CMA is also activated by oxidative stress, and in this case LAMP-2A is increased due to transcriptional regulation. CMA can be reduced by inhibitors of glucose-6-phosphate dehydrogenase and of the heat shock protein of 90 kDa. Reduction of levels of LAMP-2A using RNAi strategies reduces CMA activity, but macroautophagy is activated as a result. The decrease in CMA causes cells to be more susceptibile to oxidative and other stresses. LAMP-2A in the lysosomal membrane can be sequestered into cholesterol-rich microdomains where it is inactive. When CMA is activated, LAMP-2A moves out of these domains. The reduced CMA in aging is due to reduced LAMP-2A in the lysosomal membrane. This reduction is caused by an age-related increased degradation of LAMP-2A and an age-related reduced ability of LAMP-2A to reinsert into the lysosomal membrane. These findings reveal a rich complexity of mechanisms to control CMA activity.

Phagocytosis and remodeling of collagen matrices.

The biodegradation of collagen and the deposition of new collagen-based extracellular matrices are of central importance in tissue remodeling and function. Similarly, for collagen-based biomaterials used in tissue engineering, the degradation of collagen scaffolds with accompanying cellular infiltration and generation of new extracellular matrix is critical for the integration of in vitro grown tissues in vivo. In earlier studies we observed significant impact of collagen structure on primary lung fibroblast behavior in vitro in terms of collagen uptake and matrix remodeling. Therefore, in the present work, the response of human fibroblasts (IMR-90) to the structural state of collagen was studied with respect to phagocytosis in the presence and absence of inhibitors. Protein content and transcript levels for collagen I (Col-1), matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), tissue inhibitor of matrix metalloproteinase 2 (TIMP-2), and heat shock protein 70 (HSP-70) were characterized as a function of collagen matrix concentration, structure and cell culture time to assess effects on cellular collagen matrix remodeling processes. Phagocytosis of collagen was assessed quantitatively by the uptake of collagen-coated fluorescent beads incorporated into the collagen matrices. Significantly higher levels of collagen phagocytosis were observed for the cells grown on the denatured collagen versus native collagen matrices. Significant reduction in collagen phagocytosis was observed by blocking several phagocytosis pathways when the cells were grown on denatured collagen versus non-denatured collagen. Collagen phagocytosis inhibition effects were significantly greater for PDL57 IMR-90 cells versus PDL48 cells, reflecting a reduced number of collagen processing pathways available to the older cells. Transcript levels related to the deposition of new extracellular matrix proteins varied as a function of the structure of the collagen matrix presented to the cells. A four-fold increase in transcript level of Col-1 and a higher level of collagen matrix incorporation were observed for cells grown on denatured collagen versus cells grown on non-denatured collagen. The data suggest that biomaterial matrices incorporating denatured collagen may promote more active remodeling toward new extracellular matrices in comparison to cells grown on non-denatured collagen. A similar effect of cellular action toward denatured (wound-related) collagen in the remodeling of tissues in vivo may have significant impact on tissue regeneration as well as the progression of collagen-related diseases.

Extracellular matrix remodeling--methods to quantify cell-matrix interactions.

Tissue turnover during wound healing, regeneration or integration of biomedical materials depends on the rate and extent of materials trafficking into and out of cells involved in extracellular matrix (ECM) remodeling. To exploit these processes, we report the first model for matrix trafficking in which these issues are quantitatively assessed for cells grown on both native collagen (normal tissue) and denatured collagen (wound state) substrates. Human fibroblasts more rapidly remodeled denatured versus normal collagen type I to form new ECM. Fluxes to and from the cells from the collagen substrates and the formation of new ECM were quantified using radioactively labeled substrates. The model can be employed for the systematic and quantitative study of the impact of a broad range of physiological factors and disease states on tissue remodeling, integrating extracellular matrix structures and cell biology.

Akt and Mammalian target of rapamycin regulate separate systems of proteolysis in renal tubular cells.

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Proteolytic and lipolytic responses to starvation.

Mammals survive starvation by activating proteolysis and lipolysis in many different tissues. These responses are triggered, at least in part, by changing hormonal and neural statuses during starvation. Pathways of proteolysis that are activated during starvation are surprisingly diverse, depending on tissue type and duration of starvation. The ubiquitin-proteasome system is primarily responsible for increased skeletal muscle protein breakdown during starvation. However, in most other tissues, lysosomal pathways of proteolysis are stimulated during fasting. Short-term starvation activates macroautophagy, whereas long-term starvation activates chaperone-mediated autophagy. Lipolysis also increases in response to starvation, and the breakdown of triacylglycerols provides free fatty acids to be used as an energy source by skeletal muscle and other tissues. In addition, glycerol released from triacylglycerols can be converted to glucose by hepatic gluconeogenesis. During long-term starvation, oxidation of free fatty acids by the liver leads to the production of ketone bodies that can be used for energy by skeletal muscle and brain. Tissues that cannot use ketone bodies for energy respond to these small molecules by activating chaperone-mediated autophagy. This is one form of interaction between proteolytic and lipolytic responses to starvation.

Unifying nomenclature for the isoforms of the lysosomal membrane protein LAMP-2.

The present nomenclature of the splice variants of the lysosome-associated membrane protein type 2 (LAMP-2) is confusing. The LAMP-2a isoform is uniformly named in human, chicken, and mouse, but the LAMP-2b and LAMP-2c isoforms are switched in human as compared with mouse and chicken. We propose to change the nomenclature of the chicken and mouse b and c isoforms to agree with that currently used for the human isoforms. To avoid confusion in the literature, we further propose to adopt the use of capital letters for the updated nomenclature of all the isoforms in all three species: LAMP-2A, LAMP-2B, and LAMP-2C.

Ketone bodies stimulate chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is a selective lysosomal protein degradative process that is activated in higher organisms under conditions of prolonged starvation and in cell culture by the removal of serum. Ketone bodies are comprised of three compounds (beta-hydroxybutyrate, acetoacetate, and acetone) that circulate during starvation, especially during prolonged starvation. Here we have investigated the hypothesis that ketone bodies induce CMA. We found that physiological concentrations of beta-hydroxybutyrate (BOH) induced proteolysis in cells maintained in media with serum and without serum; however, acetoacetate only induced proteolysis in cells maintained in media with serum. Lysosomes isolated from BOH-treated cells displayed an increased ability to degrade both glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, substrates for CMA. Isolated lysosomes from cells maintained in media without serum also demonstrated an increased ability to degrade glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A when the reaction was supplemented with BOH. Such treatment did not affect the levels of lysosome-associated membrane protein 2a or lysosomal heat shock cognate protein of 70 kDa, two rate-limiting proteins in CMA. However, pretreatment of glyceraldehyde-3-phosphate and ribonuclease A with BOH increased their rate of degradation by isolated lysosomes. Lysosomes pretreated with BOH showed no increase in proteolysis, suggesting that BOH acts on the substrates to increase their rates of proteolysis. Using OxyBlot analysis to detect carbonyl formation on proteins, one common marker of protein oxidation, we showed that treatment of substrates with BOH increased their oxidation. Neither glycerol, another compound that increases in circulation during prolonged starvation, nor butanol or butanone, compounds closely related to BOH, had an effect on CMA. The induction of CMA by ketone bodies may provide an important physiological mechanism for the activation of CMA during prolonged starvation.

Effects of small molecules on chaperone-mediated autophagy.

Autophagy, including macroautophagy (MA), chaperone-mediated autophagy (CMA), crinophagy, pexophagy and microautophagy, are processes by which cells select internal components such as proteins, secretory vesicles, organelles, or foreign bodies, and deliver them to lysosomes for degradation. MA and CMA are activated during conditions of serum withdrawal in cell culture and during short-term and prolonged starvation in organisms, respectively. Although MA and CMA are activated under similar conditions, they are regulated by different mechanisms. We used pulse/chase analysis under conditions in which most intracellular proteolysis is due to CMA to test a variety of compounds for effects on this process. We show that inhibitors of MA such as 3-methyladenine, wortmannin, and LY294002 have no effect on CMA. Protein degradation by MA is sensitive to microtubule inhibitors such as colcemide and vinblastine, but protein degradation by CMA is not. Activators of MA such as rapamycin also have no effect on CMA. We demonstrate that CMA, like MA, is inhibited by protein synthesis inhibitors anisomycin and cycloheximide. CMA is also partially inhibited when the p38 mitogen activated protein kinase is blocked. Finally we demonstrate that the glucose-6-phophate dehydrogenase inhibitor, 6-aminonicotinamide, and heat shock protein of 90 kilodaltons inhibitor, geldanamycin, have the ability to activate CMA.

Mechanisms of chaperone-mediated autophagy.

Chaperone-mediated autophagy is one of several lysosomal pathways of proteolysis. This pathway is activated by physiological stresses such as prolonged starvation. Cytosolic proteins with particular peptide sequence motifs are recognized by a complex of molecular chaperones and delivered to lysosomes. No vesicular traffic is required for this protein degradation pathway, so it differs from microautophagy and macroautophagy. Protein substrates bind to a receptor in the lysosomal membrane, the lysosome-associated membrane protein (lamp) type 2a. Levels of lamp2a in the lysosomal membrane are controlled by alterations in the lamp2a half-life as well as by the dynamic distribution of the protein between the lysosomal membrane and the lumen. Substrate proteins are unfolded before transport into the lysosome lumen, and the transport of substrate proteins requires a molecular chaperone within the lysosomal lumen. The exact roles of this lysosomal chaperone remain to be defined. The mechanisms of chaperone-mediated autophagy are similar to mechanisms of protein import into mitochondria, chloroplasts, and the endoplasmic reticulum.

Famotidine for infant gastro-oesophageal reflux: a multi-centre, randomized, placebo-controlled, withdrawal trial.

BACKGROUND: Gastro-oesophageal reflux afflicts up to 7% of all infants. Histamine-2 receptor antagonists are the most commonly prescribed medications for this disorder, but few controlled studies support this practice. AIM: To evaluate the safety and efficacy of famotidine for infant gastro-oesophageal reflux disease. METHODS: Thirty-five infants, 1.3-10.5 months of age, entered an 8-week, multi-centre, randomized, placebo-controlled, two-phase trial: first 4 weeks, observer-blind comparison of famotidine 0.5 mg/kg and famotidine 1.0 mg/kg; second 4 weeks, double-blind withdrawal comparison (safety and efficacy) of each dose with placebo. RESULTS: No serious adverse events were reported. Eleven patients had 16 non-serious, possibly drug-related adverse experiences: 6 patients with agitation or irritability (manifested as head-rubbing in two), 3 patients with somnolence, 2 patients with anorexia, 2 with headache, 1 patient with vomiting, 1 patient with hiccups, and 1 patient with candidiasis. Of the 35 infants, 27 completed Part I. There were significant score improvements for famotidine 0.5 mg/kg in regurgitation frequency (P = 0.04), and for famotidine 1.0 mg/kg in crying time (P = 0.027) and regurgitation frequency (P = 0.004) and volume (P = 0.01). Eight infants completed Part II on double-blind treatment, which was insufficient for meaningful comparisons. CONCLUSIONS: Histamine-2 receptor antagonists may cause agitation and headache in infants. A possibly efficacious famotidine dose for infants is 0.5 mg/kg (frequency adjusted for age). As 1.0 mg/kg may be more efficacious in some, the dosage may require individualization based on response. Further sizeable placebo-controlled evaluations of histamine-2 receptor antagonists in infants with gastro-oesophageal reflux disease are warranted.

Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor.

Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of unknown physiological function. We now demonstrate that this protease activity triggers the degradation of the lysosome-associated membrane protein type 2a (lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of lamp2a is important because its level in the lysosomal membrane is a rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a degradation, higher levels of lamp2a and higher rates of CMA. Restoration of PPCA protease activity increases rates of lamp2a degradation, reduces levels of lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the lysosomal membrane and cleaves lamp2a near the boundary between the luminal and transmembrane domains. In addition to the well-studied role of PPCA in targeting and protecting two lysosomal glycosidases, we have defined a role for the proteolytic activity of this multifunctional protein.

A molecular chaperone complex at the lysosomal membrane is required for protein translocation.

A group of cytosolic proteins are targeted to lysosomes for degradation in response to serum withdrawal or prolonged starvation by a process termed chaperone-mediated autophagy. In this proteolytic pathway little is known about how proteins are translocated across lysosomal membranes. We now show that an isoform of the constitutively expressed protein of the heat shock family of 70 kDa (Hsc70) is associated with the cytosolic side of the lysosomal membrane where it binds to substrates of this proteolytic pathway. Results from coimmunoprecipitation and colocalization studies indicate that this molecular chaperone forms complexes with other molecular chaperones and cochaperones, including Hsp90, Hsp40, the Hsp70-Hsp90 organizing protein (Hop), the Hsp70-interacting protein (Hip), and the Bcl2-associated athanogene 1 protein (BAG-1). Antibodies against Hip, Hop, Hsp40 and Hsc70 block transport of protein substrates into purified lysosomes.

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.

Unique properties of lamp2a compared to other lamp2 isoforms.

Lamp2a acts as a receptor in the lysosomal membrane for substrate proteins of chaperone-mediated autophagy. Using antibodies specific for the cytosolic tail of lamp2a and others recognizing all lamp2 isoforms, we found that in rat liver lamp2a represents 25% of lamp2s in the lysosome. We show that lamp2a levels in the lysosomal membrane in rat liver and fibroblasts in culture directly correlate with rates of chaperone-mediated autophagy in a variety of physiological and pathological conditions. The concentration of other lamp2s in the lysosomal membrane show no correlation under the same conditions. Furthermore, substrate proteins bind to lamp2a but not to other lamp2s. Four positively-charged amino acids uniquely present in the cytosolic tail of lamp2a are required for the binding of substrate proteins. Lamp2a also distributes to an unique subpopulation of perinuclear lysosomes in cultured fibroblasts in response to serum withdrawal, and lamp2a, more than other lamp2s, tends to multimerize. These characteristics may be important for lamp2a to act as a receptor for chaperone-mediated autophagy.

Selective degradation of annexins by chaperone-mediated autophagy.

Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. Analysis of the sequences of the different members of the annexin family revealed the presence of a pentapeptide biochemically related to KFERQ in some annexins but not in others. Such sequences have been proposed to be a targeting sequence for chaperone-mediated autophagy, a lysosomal pathway of protein degradation that is activated in confluent cells in response to removal of serum growth factors. We demonstrate that annexins II and VI, which contain KFERQ-like sequences, are degraded more rapidly in response to serum withdrawal, while annexins V and XI, without such sequences, are degraded at the same rate in the presence and absence of serum. Using isolated lysosomes, only the annexins containing KFERQ-like sequences are degraded by chaperone mediated-autophagy. Annexins V and XI could associate with lysosomes but did not enter the lysosomes and were not proteolytic substrates. Furthermore, four annexins containing KFERQ-like sequences, annexins I, II, IV, and VI, are enriched in lysosomes with high chaperone-mediated autophagy activity as expected for substrate proteins. These results provide striking evidence for the importance of KFERQ motifs in substrates of chaperone-mediated autophagy.

Age-related decline in chaperone-mediated autophagy.

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.