RESUMEN
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.
Asunto(s)
Autofagia , Fagosomas/ultraestructura , Escultura , HumanosRESUMEN
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.
Asunto(s)
Autofagia , Biología Celular/historia , Chaperonas Moleculares/metabolismo , Animales , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Estados UnidosRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Chaperonas Moleculares/metabolismo , Mucolipidosis/metabolismo , Mucolipidosis/fisiopatología , Canales Catiónicos TRPM/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Fibroblastos/citología , Fibroblastos/fisiología , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ionomicina/metabolismo , Ionóforos/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Lisosomas/ultraestructura , Chaperonas Moleculares/genética , Mucolipidosis/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales Catiónicos TRPM/genética , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Técnicas de Laboratorio Clínico , Interpretación Estadística de Datos , Células Eucariotas/fisiología , Guías como Asunto , Animales , Familia de las Proteínas 8 Relacionadas con la Autofagia , Humanos , Microscopía Fluorescente/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Fagosomas/metabolismo , Fagosomas/fisiología , Plantas/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Chaperonas Moleculares/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de Choque Térmico/fisiología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas , Lisosomas/metabolismo , Modelos Biológicos , Proteínas/fisiologíaRESUMEN
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.
Asunto(s)
Colágeno/farmacología , Matriz Extracelular/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Proliferación Celular , Colágeno/química , Relación Dosis-Respuesta a Droga , Matriz Extracelular/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Proteínas del Choque Térmico HSP72/metabolismo , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Fragmentos de Péptidos/metabolismo , Fagocitosis/fisiología , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
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.
Asunto(s)
Comunicación Celular/fisiología , Colágeno/química , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Modelos Biológicos , Línea Celular , Tejido Conectivo/metabolismo , Fibroblastos/enzimología , HumanosRESUMEN
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.
Asunto(s)
Túbulos Renales/fisiología , Péptido Hidrolasas/metabolismo , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagia/efectos de los fármacos , Línea Celular , Cromonas/farmacología , Factor de Crecimiento Epidérmico/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Túbulos Renales/citología , Leupeptinas/farmacología , Lisosomas/efectos de los fármacos , Metilaminas/farmacología , Morfolinas/farmacología , Factor de Transcripción PAX2/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TORRESUMEN
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.
Asunto(s)
Lipólisis , Péptido Hidrolasas/metabolismo , Inanición/metabolismo , Acilcoenzima A/metabolismo , Autofagia , Activación Enzimática , Ácidos Grasos no Esterificados/metabolismo , Humanos , Cuerpos Cetónicos/metabolismo , Lisosomas/metabolismo , Chaperonas Moleculares/fisiología , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Triglicéridos/metabolismo , Ubiquitina/metabolismoRESUMEN
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.
Asunto(s)
Lisosomas/clasificación , Terminología como Asunto , Empalme Alternativo/genética , Animales , Bases de Datos Genéticas , Humanos , Lisosomas/química , Lisosomas/genética , Lisosomas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
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.
Asunto(s)
Autofagia/efectos de los fármacos , Autofagia/fisiología , Fibroblastos/citología , Cuerpos Cetónicos/farmacología , Chaperonas Moleculares/metabolismo , Antígenos CD/metabolismo , Proteínas Sanguíneas/farmacología , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Fibroblastos/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas , Lisosomas/metabolismo , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Chaperonas Moleculares/fisiología , 6-Aminonicotinamida/farmacología , Autofagia/efectos de los fármacos , Benzoquinonas/farmacología , Línea Celular , Fibroblastos/citología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Lisosomas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Moduladores de Tubulina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Chaperonas Moleculares/fisiología , Proteínas/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Antígenos CD/fisiología , Citosol/fisiología , Proteínas del Choque Térmico HSC70 , Proteínas HSP70 de Choque Térmico/fisiología , Humanos , Proteínas de Membrana de los Lisosomas , Lisosomas/fisiologíaRESUMEN
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.