The Hsc/Hsp70 co-chaperones of the BAG (Bcl-2-associated athanogene) protein family are modulators of protein quality control. We examined the specific roles of BAG1 and BAG3 in protein degradation during the aging process. We show that BAG1 and BAG3 regulate proteasomal and macroautophagic pathways, respectively, for the degradation of polyubiquitinated proteins. Moreover, using models of cellular aging, we find that a switch from BAG1 to BAG3 determines that aged cells use more intensively the macroautophagic system for turnover of polyubiquitinated proteins. This increased macroautophagic flux is regulated by BAG3 in concert with the ubiquitin-binding protein p62/SQSTM1. The BAG3/BAG1 ratio is also elevated in neurons during aging of the rodent brain, where, consistent with a higher macroautophagy activity, we find increased levels of the autophagosomal marker LC3-II as well as a higher cathepsin activity. We conclude that the BAG3-mediated recruitment of the macroautophagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro-oxidant and aggregation-prone milieu characteristic of aging.
BAG1; p62; proteasome; SQSTM1; ubiquitination
Constitutive macroautophagy involved in the turnover of defective long-lived proteins and organelles is crucial for neuronal homeostasis. We hypothesized that macroautophagic dysregulation in selective brain regions was associated with memory impairment in aged mice. We used the single-trial object recognition test to measure short-term memory in 18 aged mice compared to 22 young mice and employed immunohistochemistry to assess cellular distribution of proteins involved in the selective degradation of ubiquitinated proteins via macroautophagy. Values of the discrimination ratio (DR, a measure of short-term recognition memory performance) in aged mice were significantly lower than those in young mice (median, 0.54 vs. 0.67; p = 0.005, U test). Almost exclusively in aged mice, there were clusters of puncta immunoreactive for microtubule-associated protein 1 light chain 3 (LC3), ubiquitin- and LC3-binding protein p62, and ubiquitin in neuronal processes predominantly in the hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex. The hippocampal burden of clustered puncta immunoreactive for LC3 and p62 exhibited inverse linear correlations with DR in aged mice (ρ = −0.48 and −0.55, p = 0.044 and 0.018, respectively, Spearman’s rank correlation). These findings suggest that increased accumulation of autophagosomes within neuronal processes in selective brain regions is characteristic of aging. The dysregulation of macroautophagy can adversely affect the turnover of aggregate-prone proteins and defective organelles, which may contribute to memory impairment in aged mice.
Autophagy; Brain aging; MAP1LC3; Object recognition test; p62; Ubiquitin
Macroautophagy is a cellular process whereby the cell sequesters and recycles cytosolic constituents in a lysosome-dependent manner. It has also been implicated in a number of disorders, including cancer and neurodegeneration. Although a previous report that AGS3 over-expression promotes macroautophagy suggests a stimulatory role of AGS3 in this process, we have found that knock-down of AGS3, unexpectedly, also induces macroautophagy, indicating an inhibitory function of endogenous AGS3 in macroautophagy. Interestingly, AGS3 phosphorylation is decreased upon induction of mammalian target of rapamycin (mTOR)-dependent macroautophagy. Moreover, unlike wild-type AGS3, over-expression of an AGS3 mutant lacking this modification fails to enhance macroautophagic activity. These observations imply that AGS3 phosphorylation may participate in the modulation of macroautophagy.
Macroautophagy involves lysosomal/vacuolar elimination of long-lived proteins and entire organelles from the cytosol. The process begins with formation of a double-membrane vesicle that sequesters bulk cytoplasm, or a specific cargo destined for lysosomal/vacuolar delivery. The completed vesicle fuses with the lysosome/vacuole limiting membrane, releasing its content into the organelle lumen for subsequent degradation and recycling of the resulting macromolecules. A majority of the autophagy-related (Atg) proteins are required at the step of vesicle formation. The integral membrane protein Atg9 cycles between certain intracellular compartments and the vesicle nucleation site, presumably to supply membranes necessary for macroautophagic vesicle formation. In this study we have tracked the movement of Atg9 over time in living cells by using real-time fluorescence microscopy. Our results reveal that an actin-related protein, Arp2, briefly colocalizes with Atg9 and directly regulates the dynamics of Atg9 movement. We propose that proteins of the Arp2/3 complex regulate Atg9 transport for specific types of autophagy.
There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation, and can occur selectively for substrates including aggregated proteins. It remains unclear, however, if starvation-induced and selective macroautophagy share identical adapter molecules to capture their cargo. Here we report that Alfy, a phosphatidylinositol 3-phosphate binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner, and likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy, by bridging cargo to the molecular machinery that builds autophagosomes.
The synthesis of preribosomal RNA is inhibited "in vivo" and "in vitro" by the protease inhibitor leupeptin. "In vivo" leupeptin decreases by 74% the incorporation of labeled uridine into 45S pre rRNA while the synthesis of other RNA species is only slightly decreased. "In vitro", the elongation of already initiated pre rRNA chains that is achieved by incubation of isolated nucleoli is blocked by leupeptin. On the other hand, "in vitro" leupeptin has no direct effect on RNA polymerase I, tested in a nonspecific transcriptional system with Calf thymus DNA as template and in run off experiments with a cloned DNA containing the initiation site of the rDNA gene. A 100 kDa nucleolar protein which has been shown to be endoproteolytic cleaved "in vivo" (1) acts as an inhibitor of rDNA transcription in presence of leupeptin but produces little effect on the nonspecific transcription. In absence of the drug, the 100 kDa protein is processed in specific peptides which appeared to be similar to the "in vivo" maturation products. The possible role of the 100 kDa maturation process in the regulation of rDNA transcription is discussed.
Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrPSc in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrPSc in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrPSc in a prion-infected cell line after treatment with bafilomycin A1. These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.
transmissible spongiform encephalopathies; autophagy; neurodegenerative diseases
Macroautophagy is a bulk degradation process that mediates the clearance of long-lived proteins, aggregates, or even whole organelles. This process includes the formation of autophagosomes, double-membrane structures responsible for delivering cargo to lysosomes for degradation. Currently, other alternative autophagy pathways have been described, which are independent of macroautophagic key players like Atg5 and Beclin 1 or the lipidation of LC3. In this review, we highlight recent insights in indentifying and understanding the molecular mechanism responsible for alternative autophagic pathways.
The ubiquitin proteasome system and macroautophagy are proteolytic pathways essential in the maintenance of cellular homeostasis during differentiation and remodelling of skeletal muscle. In both pathways, proteins to be degraded are tagged with polyubiquitin. In skeletal muscles, the MURF2 proteins display E3 ubiquitin ligase structure suggesting that they may covalently attach ubiquitin polypeptides to still unknown target proteins. So far only MURF2A isoforms were studied and shown to interact with p62/SQSTM1, a protein implicated in macroautophagic and ubiquitin proteasome system degradations. Here, we analyzed the MURF2B and MURF2A proteins and show that the ratio of the isoforms changes during differentiation of muscle C2C12 cells and that the shift of the isoforms expression follows the sequential activation of autophagic or proteasomal degradation. We also show that MURF2B has a functional domain needed for its interaction with LC3, a protein needed for autophagic vesicles formation. Using specific MURF2 RNAi cells we observed that MURF2A and MURF2B are both needed for the formation of autophagosomes and that in the absence of MURF2B, the cells expressing MURF2A display an activated ubiquitin proteasome system implicated in the degradation of p62/SQSTM1 by UPS. Altogether, our results indicate that MURF2A and MURF2B proteins could participate in the molecular switch between the two ubiquitin degradative pathways.
Bacillus anthracis protective antigen (PA) is an 83-kDa (PA83) protein that is cleaved to the 63-kDa protein (PA63) as an essential step in binding and internalizing lethal factor (LF). To assess in vivo receptor saturating PA concentrations, we injected mice with PA variants and measured the PA remaining in the blood at various times using PA83- and PA63-specific enzyme-linked immunosorbent assays. We found that both wild-type PA (WT-PA) and a receptor-binding-defective mutant (Ub-PA) were cleaved to PA63 independent of their ability to bind cells. This suggested a PA-acting protease activity in the blood. The protease cleaved PA at the furin cleavage sequence because furin site-modified PA mutants were not cleaved. Cleavage measured in vitro was leupeptin sensitive and dependent on calcium. Cell surface cleavage was important for toxin clearance, however, as Ub-PA and uncleavable PA mutants were cleared at slower rates than WT-PA. The cell binding-independent cleavage of PA was also verified by using Ub-PA (which is still cleaved) to rescue mice from toxin challenge by competitively binding circulating LF. This mutant was able to rescue mice even when given 12 h before toxin challenge. Its therapeutic ability was comparable to that of dominant-negative PA, which binds cells but does not allow LF translocation, and to the protection afforded through receptor clearance by WT-PA and uncleavable receptor binding-competent mutants. The PA cleavage and clearance observed in mice did not appear to have a role in the differential mouse susceptibility as it occurred similarly in lethal toxin (LT)-resistant DBA/2J and LT-sensitive BALB/cJ mice. Interestingly, PA63 was not found in LT-resistant or -sensitive rats and PA83 clearance was slower in rats than in mice. Finally, to determine the minimum amount of PA required in circulation for LT toxicity in mice, we administered time-separated injections of PA and LF and showed that lethality of LF for mice after PA was no longer measurable in circulation, suggesting active PA sequestration at tissue surfaces.
Autophagosomes are double-membrane vesicles characteristic of macroautophagy, a degradative pathway for cytoplasmic material and organelles terminating in the lysosomal or vacuole compartment for mammals and yeast, respectively. This highly dynamic, multi-step process requires significant membrane reorganization events at different stages of the macroautophagic process. Such events include exchange and flow of lipids and proteins between membranes and vesicles (e.g., during initiation and growth of the phagophore), vesicular positioning and trafficking within the cell (e.g., autophagosome location and movement) and fusion of autophagosomes with the boundary membranes of the degradative compartment. Here, we review current knowledge on the contribution of different organelles to the formation of autophagosomes, their trafficking and fate within the cell. We will consider some of the unresolved questions related to the molecular mechanisms that regulate the “life and death” of the autophagosome.
autophagosome; degradation; lysosome; macroautophagy; mammals; membrane; organelle; yeast
Macroautophagy is a highly conserved cellular degradation process, regulated by autophagy-related (atg) factors, in which a double membrane autophagosome engulfs cytoplasmic components to target them for degradation. In yeast, the Atg8 protein is indispensable for autophagosome formation. In mammals, this is complicated by the presence of six Atg8 homologues grouped into the GABARAP and MAP1LC3 subfamilies. Although these proteins share a high similarity, their transcript expression, regulation and protein interactions differ, suggesting they may display individual properties and specific functions. GABARAPL1/GEC1 is a member of the GABARAP subfamily and its mRNA is the most highly expressed Atg8 homologue in the central nervous system. Consequently, we performed an in depth study of GABARAPL1 distribution in the developing and adult murine brain. Our results show that GABARAPL1 brain expression is visible as early as embryonic day 11 and progressively increases to a maximum level in the adult. Immunohistochemical staining was detected in both fibers and immature neurons in embryos but was restrained to neurons in adult tissue. By E17, intense punctate-like structures were visible and these accumulated in cortical primary neurons treated with the autophagosome/lysosome fusion inhibitor Bafilomycin A1 (Baf A1), suggesting that they represent autophagosomes. Finally, GABARAPL1 expression was particularly intense in motoneurons in the embryo and in neurons involved in somatomotor and neuroendocrine functions in the adult, particularly in the substantia nigra pars compacta, a region affected in Parkinson's disease. Our study of cerebral GABARAPL1 protein expression provides insight into its role in the development and homeostasis of the mouse brain.
Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants.
Rat liver secretory component is synthesized as an integral membrane protein (mSC) and cleaved to an 80-kD soluble form (fSC) sometime during transcellular transport from the sinusoidal to the bile canalicular plasma membrane domain of hepatocytes. We have used 24-h monolayer cultures of rat hepatocytes to characterize the conversion of mSC to fSC. Cleavage of mSC in cultured hepatocytes is inhibited by the thiol protease inhibitors leupeptin, antipain, and E-64, but not by other inhibitors, including disopropylfluorophosphate, pepstatin, N- ethylmalemide, p-chloromercuribenzoic acid, and chloroquine. Leupeptin- mediated inhibition of cleavage is concentration dependent and reversible. In the presence or absence of leupeptin, only 10-20% of mSC is accessible at the cell surface. To characterize the behavior of surface as opposed to intracellular mSC, cell surface mSC was labeled with 125I by lactoperoxidase-catalyzed iodination at 4 degrees C. Cell surface 125I-mSC was converted to extracellular fSC at 4 degrees C in the absence of detectable internalization. Cleavage was inhibited by leupeptin and by anti-secretory component antiserum. Cleavage also occurred at 4 degrees C after cell disruption. In contrast, 125I-mSC that had been internalized from the cell surface was not converted to fSC at 4 degrees C in either intact or disrupted cells. Hepatocytes metabolically labeled with [35S]cys also released small quantities of fSC into the medium at 4 degrees C. The properties of fSC production indicate that cleavage occurs on the surface of cultured rat hepatocytes and not intracellularly. Other features of the cleavage reaction suggest that the mSC-cleaving protease is segregated from the majority of cell surface mSC, possibly within a specialized plasma membrane domain.
Autophagy is an intracellular degradation process that is mediated by autophagosomes. Mammalian Atg2 proteins Atg2A and Atg2B are identified and characterized as essential for autophagy. They are also present on lipid droplets and are involved in regulation of lipid droplet volume and distribution.
Macroautophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by the autophagosome and delivered to the lysosome. Autophagosome formation is considered to take place on the endoplasmic reticulum and involves functions of autophagy-related (Atg) proteins. Here, we report the identification and characterization of mammalian Atg2 homologues Atg2A and Atg2B. Simultaneous silencing of Atg2A and Atg2B causes a block in autophagic flux and accumulation of unclosed autophagic structures containing most Atg proteins. Atg2A localizes on the autophagic membrane, as well as on the surface of lipid droplets. The Atg2A region containing amino acids 1723–1829, which shows relatively high conservation among species, is required for localization to both the autophagic membrane and lipid droplet and is also essential for autophagy. Depletion of both Atg2A and Atg2B causes clustering of enlarged lipid droplets in an autophagy-independent manner. These data suggest that mammalian Atg2 proteins function both in autophagosome formation and regulation of lipid droplet morphology and dispersion.
Until now, the in vitro activity of potential antimalarial agents has been evaluated primarily by monitoring decreases in parasite proliferation. These traditional assays do not distinguish between compounds that arrest proliferation of parasites and compounds that kill them. In this report, a more complex in vitro cytocidal assay for Plasmodium falciparum is described. This assay measures the clonal viability of P. falciparum after the parasites have been treated with an antimalarial agent. The new assay was used to assess cytocidal activities of three antimalarial agents that work through unrelated mechanisms. Leupeptin, a protease inhibitor, arrested the proliferation of W2 clones of P. falciparum at a MIC of 50 microM, but at least 80% of leupeptin-treated cells were viable as judged by the cytocidal assay. On the other hand, chloroquine at 1 microM, its MIC for W2 cells, not only arrested parasite proliferation but also killed more than 99% of the cells. Earlier studies had shown that treatment of P. falciparum with 100 nM 5-fluoroorotate for 48 h was sufficient to inhibit parasite proliferation and parasite thymidylate synthase but not enough to cause significant incorporation of 5-fluoropyrimidines in parasite nucleic acids. By using the new schizonticidal assay, these conditions were found to be necessary and sufficient to kill all parasites in culture. Results of these studies are consistent with the hypothesis that 5-fluoroorotate-based inactivation of P. falciparum thymidylate synthase triggers a lethal mechanism against malarial parasites.
Macroautophagy is a highly conserved mechanism of lysosomal mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced following effector T cell activation. Engagement of the T cell receptor and CD28 results in enhanced LC3 processing, increased numbers of LC3-containing vesicles and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional knockout mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector T helper cells have defective IL-2 and INFγ production and reduced proliferation following stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T-cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to T cell receptor engagement to accommodate the bioenergetic requirements of activated T cells.
Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.
SARS coronavirus; spike protein; proteolytic cleavage; cathepsin L; furin
Parkinson’s disease (PD), like a number of neurodegenerative diseases associated with aging, is characterized by the abnormal accumulation of protein in a specific subset of neurons. Although researchers have recently elucidated the genetic causes of PD, much remains unknown about what causes increased protein deposition in the disease. Given that increased protein aggregation may result not only from an increase in production, but also from decreased protein clearance, it is imperative to investigate both possibilities as potential PD culprits. This article provides a review of the systems that regulate protein clearance, including the ubiquitin proteasome system (UPS) and the autophagy-lysosomal pathway. Literature implicating failure of these mechanisms—such as UPS dysfunction resulting from environmental toxins and mutations in α-synuclein and parkin, as well as macroautophagic pathway failure because of oxidative stress and aging—in the pathogenesis of PD is also discussed.
Protein misfolding, as well as dysfunction in the protein degradation systems, may play a pivotal role in the cascade of deleterious events implicated in the neurodegenerative process of Parkinson’s disease.
Transfection of Mv1Lu mink lung type II alveolar cells with β1–6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the β1–6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome–MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2–positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.
Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes. Although the induction of autophagy by starvation has been extensively studied, we still understand very little about how autophagy is regulated under normal nutritional conditions. Here we describe a study using a small molecule autophagy inducer, fluspirilene, as a tool to explore the mechanism of autophagy induction in normal living cells. We confirm the activity of fluspirilene in inhibiting Ca2+ flux. Furthermore, we show that reducing intracellular Ca2+ prevents the cleavage of ATG5, which in turn increases the levels of full length ATG5 and ATG12-ATG5 conjugate. Using siRNA mediated gene silencing, we demonstrate that inhibiting calpain1 is sufficient to induce autophagy in living cells. We conclude that calpain1 plays an important role in controlling the levels of autophagy in normal living cells by regulating the levels of a key signaling molecule, ATG12-ATG5 conjugate.
autophagy; Fluspirilene; calpain; ATG5; Ca2+; cleavage; ATG12-ATG5
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.
MAP kinases; signal transduction; autophagy; LC3B; GABARAP; SQSTM1
Over this past decade, macroautophagy has gained prominence in the field of adult-onset neurodegeneration: from sporadic disorders such as Alzheimer's and Parkinson's disease, to genetic disorders such as Huntington's disease and frontotemporal dementia, the influence of this fundamental pathway has become an important topic of discussion. While there has been particular emphasis on the potential benefits of macroautophagy, there is growing literature that also suggests that macroautophagy contributes towards neurotoxicity. In this review, we discuss the molecular mechanism of macroautophagy and the currently available pharmacological tools, with special emphasis on mammalian macroautophagy in adult brain. Studies indicate that neuronal context strongly influences the role macroautophagy plays in maintaining cellular health, reflecting an ongoing need for better understanding of how macroautophagic regulation is achieved in the heavily differentiated and polarized neurons if we are to effectively manipulate it to treat neurodegenerative disease.
It has been shown that Porphyromonas gingivalis 381, a suspected periodontopathogen, possesses fimbriae on its cell surface. The organism is also known to produce proteases which can degrade the host cell surface matrix proteins. In this study, we investigated the effect of protease on the binding of the purified P. gingivalis fimbriae to cultured fibroblasts or matrix proteins. A protease that can hydrolyze benzoyl-L-arginine p-nitroanilide was obtained from P. gingivalis 381 cells by sonication in phosphate-buffered 0.2% Triton X-100 and was purified by column chromatography. The molecular size of the protease was estimated to be 55 kDa by gel filtration or 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity was markedly inhibited by sulfhydryl reagents, antipain, and leupeptin. The protease degraded various host proteins, including collagen and fibronectin, and cleaved the COOH terminus of the arginine residue in peptides such as benzoyl-L-arginine p-nitroanilide. However, P. gingivalis fimbriae were not degraded by protease activity. The enzyme activity was enhanced in the presence of reducing agents or CaCl2. When cultured fibroblasts were partially treated with the protease, the binding of the purified P. gingivalis fimbriae to the fibroblast monolayer was increased significantly. However, this enhancing effect was suppressed upon the addition of antipain and leupeptin. Similarly, binding of the fimbriae to the collagen or fibronectin immobilized on the microtiter wells was also enhanced. Addition of these host matrix proteins efficiently inhibited the binding of fimbriae to the fibroblast monolayer. The binding assay of fimbriae using dipeptidyl ligand affinity column chromatography demonstrated a clear interaction between fimbriae and the arginine residue. Taken together, these results indicate that the P. gingivalis protease at least partially degrades the host matrix proteins, which, in turn, may lead to an increased exposure of the cryptic ligands that can result in enhanced fimbria-mediated binding of this organism to periodontal tissues.
Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.