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1.  Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3 
The EMBO Journal  2009;28(7):889-901.
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.
doi:10.1038/emboj.2009.29
PMCID: PMC2647772  PMID: 19229298
BAG1; p62; proteasome; SQSTM1; ubiquitination
2.  Activation of the macroautophagic system in scrapie-infected experimental animals and human genetic prion diseases 
Autophagy  2012;8(11):1604-1620.
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.
doi:10.4161/auto.21482
PMCID: PMC3494590  PMID: 22874564
transmissible spongiform encephalopathies; autophagy; neurodegenerative diseases
3.  The selective macroautophagic degradation of aggregated proteins requires the phosphatidylinositol 3-phosphate binding protein Alfy 
Molecular cell  2010;38(2):265-279.
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.
doi:10.1016/j.molcel.2010.04.007
PMCID: PMC2867245  PMID: 20417604
4.  Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice 
Age  2011;34(2):305-316.
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.
doi:10.1007/s11357-011-9234-4
PMCID: PMC3312638  PMID: 21431350
Autophagy; Brain aging; MAP1LC3; Object recognition test; p62; Ubiquitin
5.  An Inhibitory Role of the G-Protein Regulator AGS3 in mTOR-Dependent Macroautophagy 
PLoS ONE  2010;5(1):e8877.
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.
doi:10.1371/journal.pone.0008877
PMCID: PMC2811177  PMID: 20126274
6.  Arp2 Links Autophagic Machinery with the Actin Cytoskeleton 
Molecular Biology of the Cell  2008;19(5):1962-1975.
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.
doi:10.1091/mbc.E07-09-0892
PMCID: PMC2366845  PMID: 18287533
7.  Alternative Macroautophagic Pathways 
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.
doi:10.1155/2012/189794
PMCID: PMC3320029  PMID: 22536246
8.  The Intriguing Life of Autophagosomes 
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.
doi:10.3390/ijms13033618
PMCID: PMC3317731  PMID: 22489171
autophagosome; degradation; lysosome; macroautophagy; mammals; membrane; organelle; yeast
9.  Specific Distribution of the Autophagic Protein GABARAPL1/GEC1 in the Developing and Adult Mouse Brain and Identification of Neuronal Populations Expressing GABARAPL1/GEC1 
PLoS ONE  2013;8(5):e63133.
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.
doi:10.1371/journal.pone.0063133
PMCID: PMC3655077  PMID: 23690988
10.  Anthrax Protective Antigen Cleavage and Clearance from the Blood of Mice and Rats▿  
Infection and Immunity  2007;75(11):5175-5184.
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.
doi:10.1128/IAI.00719-07
PMCID: PMC2168306  PMID: 17724066
11.  Interrelations between the maturation of a 100 kDa nucleolar protein and pre rRNA synthesis in CHO cells. 
Nucleic Acids Research  1984;12(7):3025-3035.
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.
Images
PMCID: PMC318727  PMID: 6562463
12.  Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets 
Molecular Biology of the Cell  2012;23(5):896-909.
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.
doi:10.1091/mbc.E11-09-0785
PMCID: PMC3290647  PMID: 22219374
13.  Identification of Genome-Scale Metabolic Network Models Using Experimentally Measured Flux Profiles 
PLoS Computational Biology  2006;2(7):e72.
Genome-scale metabolic network models can be reconstructed for well-characterized organisms using genomic annotation and literature information. However, there are many instances in which model predictions of metabolic fluxes are not entirely consistent with experimental data, indicating that the reactions in the model do not match the active reactions in the in vivo system. We introduce a method for determining the active reactions in a genome-scale metabolic network based on a limited number of experimentally measured fluxes. This method, called optimal metabolic network identification (OMNI), allows efficient identification of the set of reactions that results in the best agreement between in silico predicted and experimentally measured flux distributions. We applied the method to intracellular flux data for evolved Escherichia coli mutant strains with lower than predicted growth rates in order to identify reactions that act as flux bottlenecks in these strains. The expression of the genes corresponding to these bottleneck reactions was often found to be downregulated in the evolved strains relative to the wild-type strain. We also demonstrate the ability of the OMNI method to diagnose problems in E. coli strains engineered for metabolite overproduction that have not reached their predicted production potential. The OMNI method applied to flux data for evolved strains can be used to provide insights into mechanisms that limit the ability of microbial strains to evolve towards their predicted optimal growth phenotypes. When applied to industrial production strains, the OMNI method can also be used to suggest metabolic engineering strategies to improve byproduct secretion. In addition to these applications, the method should prove to be useful in general for reconstructing metabolic networks of ill-characterized microbial organisms based on limited amounts of experimental data.
Synopsis
One of the major uses of in silico models in biology is to identify discrepancies between model predictions and experimental data and use these discrepancies to drive discovery of novel biological mechanisms. However, models only allow for identification of the discrepancies; they do not necessarily provide any assistance in discovering what are the missing or incorrect functionalities in the model that cause these discrepancies. Herrgård et al. describe a new in silico method, optimal metabolic network identification, or OMNI, that performs this discovery process in an efficient and systematic manner for genome-scale metabolic networks. Given a preliminary metabolic network model and experimentally determined metabolic flux data, OMNI finds the changes that need to be made to the model so that its predictions match the experimental data as well as possible. Herrgård et al. apply the method to identify metabolic bottlenecks in experimentally evolved Escherichia coli strains and to diagnose problems in strains designed through metabolic engineering strategies to overproduce specific desirable byproducts. The OMNI method can also be adapted to number of other settings, including identification of novel biochemical pathways in ill-characterized organisms based on limited amounts of experimental data.
doi:10.1371/journal.pcbi.0020072
PMCID: PMC1487183  PMID: 16839195
14.  Identification of Genome-Scale Metabolic Network Models Using Experimentally Measured Flux Profiles 
PLoS Computational Biology  2006;2(7):e72.
Genome-scale metabolic network models can be reconstructed for well-characterized organisms using genomic annotation and literature information. However, there are many instances in which model predictions of metabolic fluxes are not entirely consistent with experimental data, indicating that the reactions in the model do not match the active reactions in the in vivo system. We introduce a method for determining the active reactions in a genome-scale metabolic network based on a limited number of experimentally measured fluxes. This method, called optimal metabolic network identification (OMNI), allows efficient identification of the set of reactions that results in the best agreement between in silico predicted and experimentally measured flux distributions. We applied the method to intracellular flux data for evolved Escherichia coli mutant strains with lower than predicted growth rates in order to identify reactions that act as flux bottlenecks in these strains. The expression of the genes corresponding to these bottleneck reactions was often found to be downregulated in the evolved strains relative to the wild-type strain. We also demonstrate the ability of the OMNI method to diagnose problems in E. coli strains engineered for metabolite overproduction that have not reached their predicted production potential. The OMNI method applied to flux data for evolved strains can be used to provide insights into mechanisms that limit the ability of microbial strains to evolve towards their predicted optimal growth phenotypes. When applied to industrial production strains, the OMNI method can also be used to suggest metabolic engineering strategies to improve byproduct secretion. In addition to these applications, the method should prove to be useful in general for reconstructing metabolic networks of ill-characterized microbial organisms based on limited amounts of experimental data.
Synopsis
One of the major uses of in silico models in biology is to identify discrepancies between model predictions and experimental data and use these discrepancies to drive discovery of novel biological mechanisms. However, models only allow for identification of the discrepancies; they do not necessarily provide any assistance in discovering what are the missing or incorrect functionalities in the model that cause these discrepancies. Herrgård et al. describe a new in silico method, optimal metabolic network identification, or OMNI, that performs this discovery process in an efficient and systematic manner for genome-scale metabolic networks. Given a preliminary metabolic network model and experimentally determined metabolic flux data, OMNI finds the changes that need to be made to the model so that its predictions match the experimental data as well as possible. Herrgård et al. apply the method to identify metabolic bottlenecks in experimentally evolved Escherichia coli strains and to diagnose problems in strains designed through metabolic engineering strategies to overproduce specific desirable byproducts. The OMNI method can also be adapted to number of other settings, including identification of novel biochemical pathways in ill-characterized organisms based on limited amounts of experimental data.
doi:10.1371/journal.pcbi.0020072
PMCID: PMC1487183  PMID: 16839195
15.  Clonal viability measurements on Plasmodium falciparum to assess in vitro schizonticidal activity of leupeptin, chloroquine, and 5-fluoroorotate. 
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.
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PMCID: PMC187909  PMID: 8517698
16.  MURF2B, a Novel LC3-Binding Protein, Participates with MURF2A in the Switch between Autophagy and Ubiquitin Proteasome System during Differentiation of C2C12 Muscle Cells 
PLoS ONE  2013;8(10):e76140.
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.
doi:10.1371/journal.pone.0076140
PMCID: PMC3790703  PMID: 24124537
17.  MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins 
Autophagy  2012;8(12):1724-1740.
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.
doi:10.4161/auto.21857
PMCID: PMC3541284  PMID: 22948227
MAP kinases; signal transduction; autophagy; LC3B; GABARAP; SQSTM1
18.  Disruption of Protein Quality Control in Parkinson’s Disease 
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.
doi:10.1101/cshperspect.a009423
PMCID: PMC3331692  PMID: 22553500
19.  Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion 
Virology  2011;413(2):265-274.
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.
doi:10.1016/j.virol.2011.02.020
PMCID: PMC3086175  PMID: 21435673
SARS coronavirus; spike protein; proteolytic cleavage; cathepsin L; furin
20.  Changes in the Plasmodial Surface Anion Channel Reduce Leupeptin Uptake and Can Confer Drug Resistance in Plasmodium falciparum-Infected Erythrocytes▿  
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.
doi:10.1128/AAC.00057-08
PMCID: PMC2443925  PMID: 18443109
21.  Cleavage of membrane secretory component to soluble secretory component occurs on the cell surface of rat hepatocyte monolayers 
The Journal of Cell Biology  1987;104(6):1725-1733.
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.
PMCID: PMC2114513  PMID: 3294861
22.  Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13C Metabolic Flux Analysis 
Journal of Bacteriology  2012;194(19):5413-5422.
Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-13C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter−1. To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation.
doi:10.1128/JB.00713-12
PMCID: PMC3457242  PMID: 22865845
23.  Biogenesis of Multilamellar Bodies via Autophagy 
Molecular Biology of the Cell  2000;11(1):255-268.
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.
PMCID: PMC14772  PMID: 10637306
24.  Sensitivity Analysis of Flux Determination in Heart by H218O -provided Labeling Using a Dynamic Isotopologue Model of Energy Transfer Pathways 
PLoS Computational Biology  2012;8(12):e1002795.
To characterize intracellular energy transfer in the heart, two organ-level methods have frequently been employed: inversion and saturation transfer, and dynamic labeling. Creatine kinase (CK) fluxes obtained by following oxygen labeling have been considerably smaller than the fluxes determined by saturation transfer. It has been proposed that dynamic labeling determines net flux through CK shuttle, whereas saturation transfer measures total unidirectional flux. However, to our knowledge, no sensitivity analysis of flux determination by oxygen labeling has been performed, limiting our ability to compare flux distributions predicted by different methods. Here we analyze oxygen labeling in a physiological heart phosphotransfer network with active CK and adenylate kinase (AdK) shuttles and establish which fluxes determine the labeling state. A mathematical model consisting of a system of ordinary differential equations was composed describing enrichment in each phosphoryl group and inorganic phosphate. By varying flux distributions in the model and calculating the labeling, we analyzed labeling sensitivity to different fluxes in the heart. We observed that the labeling state is predominantly sensitive to total unidirectional CK and AdK fluxes and not to net fluxes. We conclude that measuring dynamic incorporation of into the high-energy phosphotransfer network in heart does not permit unambiguous determination of energetic fluxes with a higher magnitude than the ATP synthase rate when the bidirectionality of fluxes is taken into account. Our analysis suggests that the flux distributions obtained using dynamic labeling, after removing the net flux assumption, are comparable with those from inversion and saturation transfer.
Author Summary
In heart, the movement of energy metabolites between force-producing myosin, other ATPases, and mitochondria is vital for its function and closely related to heart pathologies. In addition to diffusion, transport of ATP, ADP, Pi, and phosphocreatine occurs along parallel pathways such as the adenylate kinase and creatine kinase shuttles. Two organ-level methods have been developed to study the relative flux through these pathways. However, their results differ. It was recently demonstrated that studies often suffer from the exclusion of compartmentation from their metabolic models. One study overcame this limitation by using compartmental models and statistical methods on multiple experiments. Here, we analyzed the sensitivity of the other method - dynamic labeling of phosphoryl groups and inorganic phosphate. For that, we composed a mathematical model tracking enrichment of the metabolites and evaluated sensitivity of labeling to different flux distribution scenarios. Our study shows that the dynamic method provides a measure of total flux, and not net flux as presumed previously, making the fluxes predicted from both methods consistent. Importantly, conclusions derived on the basis of labeling analysis, particularly those regarding the net flux through the shuttles in control and pathological cases, need to be reevaluated.
doi:10.1371/journal.pcbi.1002795
PMCID: PMC3516558  PMID: 23236266
25.  About Merging Threshold and Critical Flux Concepts into a Single One: The Boundary Flux 
The Scientific World Journal  2014;2014:656101.
In the last decades much effort was put in understanding fouling phenomena on membranes. One successful approach to describe fouling issues on membranes is the critical flux theory. The possibility to measure a maximum value of the permeate flux for a given system without incurring in fouling issues was a breakthrough in membrane process design. However, in many cases critical fluxes were found to be very low, lower than the economic feasibility of the process. The knowledge of the critical flux value must be therefore considered as a good starting point for process design. In the last years, a new concept was introduced, the threshold flux, which defines the maximum permeate flow rate characterized by a low constant fouling rate regime. This concept, more than the critical flux, is a new practical tool for membrane process designers. In this paper a brief review on critical and threshold flux will be reported and analyzed. And since the concepts share many common aspects, merged into a new concept, called the boundary flux, the validation will occur by the analysis of previously collected data by the authors, during the treatment of olive vegetation wastewater by ultrafiltration and nanofiltration membranes.
doi:10.1155/2014/656101
PMCID: PMC3925542  PMID: 24592177

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