ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407–4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434–1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.     

Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.Subject terms: Mechanisms of disease, Amyotrophic lateral sclerosis     

Selective autophagy mediated by autophagic adapter proteins

Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated substrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.     

The role of autophagy in the pathogenesis of glycogen storage disease type II (GSDII)

Regulated removal of proteins and organelles by autophagy-lysosome system is critical for muscle homeostasis. Excessive activation of autophagy-dependent degradation contributes to muscle atrophy and cachexia. Conversely, inhibition of autophagy causes accumulation of protein aggregates and abnormal organelles, leading to myofiber degeneration and myopathy. Defects in lysosomal function result in severe muscle disorders such as Pompe (glycogen storage disease type II (GSDII)) disease, characterized by an accumulation of autophagosomes. However, whether autophagy is detrimental or not in muscle function of Pompe patients is unclear. We studied infantile and late-onset GSDII patients and correlated impairment of autophagy with muscle wasting. We also monitored autophagy in patients who received recombinant α-glucosidase. Our data show that infantile and late-onset patients have different levels of autophagic flux, accumulation of p62-positive protein aggregates and expression of atrophy-related genes. Although the infantile patients show impaired autophagic function, the late-onset patients display an interesting correlation among autophagy impairment, atrophy and disease progression. Moreover, reactivation of autophagy in vitro contributes to acid α-glucosidase maturation in both healthy and diseased myotubes. Together, our data suggest that autophagy protects myofibers from disease progression and atrophy in late-onset patients.     

Autophagy and p62/sequestosome 1 generate neo-antimicrobial peptides (cryptides) from cytosolic proteins

In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.Key words: autophagy, tuberculosis, ribosome, ubiquitin, antimicrobial peptidesAutophagy is an evolutionarily conserved cytoplasm-homeostatic process with a multitude of functions supporting, for the most part, cellular viability. During autophagy, cytoplasmic targets ranging from protein aggregates to whole organelles such as mitochondria and intracellular microbes are sequestered into a double-membrane bound organelle called the autophagosome. Autophagosomes mature into autolysosomes through fusion with lysosomes or their transport intermediates, bringing about acidification and acquisition of hydrolases leading to the digestion of the captured substrates. It is generally assumed that autophagy produces terminal degradative products such as free amino acids that are then used by the cell or the body as nutrients at times of starvation. Recently, we have discovered that autophagy generates, by proteolysis of captured cytosolic proteins, a mixture of peptides conferring potential cryptic biological functions, termed “cryptides.” Some of the cryptides with thus far assigned biological functions are the neo-antimicrobial peptides liberated from innocuous cytoplasmic proteins such as the ribosomal protein precursor FAU and ubiquitin.Our study was motivated by the search for factors or ingredients that make autophagic organelles particularly mycobactericidal, as Mycobacterium tuberculosis can survive the environment of the conventional phagolysosome. This was shown in the 1970s by the classical work of Armstrong and D''Arcy Hart at the same time when these authors established the more broadly appreciated and well-ingrained reputation of the tubercle bacillus as inhibiting the conventional phagosome-lysosome fusion. The approach to identifying such hypothetical ingredients was to first examine the steps of the autophagic pathway that are necessary for the mycobactericidal nature of macrophages induced for autophagy by, for example, starvation. We have found that not only are all stages of autophagy (initiation, elongation/closure and maturation) required for full mycobactericidal potency, but that p62, the first autophagic adapter characterized by the Johansen group, and also known as sequestosome 1, is absolutely required for autophagic elimination of M. tuberculosis. Sequestosome 1/p62 recognizes ubiquitinated protein aggregates and possibly ubiquitinated depolarized mitochondria and other targets, and delivers them to nascent autophagosomes; p62 also binds to the mammalian Atg8 paralog LC3 via its LC3-interaction region (LIR), thus conveniently bridging the targets with forming phagophores.At first blush, it may seem that mycobacteria follow the same fate demonstrated for several other bacteria, whereby p62 or another autophagic adapter, NDP52, capture cytosolic microbes and deliver them to autophagosomes. For example, the fraction of Salmonellae that are no longer retained within phagosomes and are free in the cytosol, or Shigella and Listeria that actively escape into the cytosol, are associated with ubiquitinated material or become otherwise recognized by p62 or NDP52, and end up being sequestered into autophagosomes. However, we found no evidence for p62 acting directly to transfer intraphagosomal mycobacteria into autophagic vacuoles. Instead, we observed p62-positive organelles as periodically fusing with mycobacterial phagosomes. At the same time, we found by imaging and biochemical means that proteins recruited by p62 from the cytosol into conventional autophagic organelles are subsequently transferred to model (latex bead phagosomes formed upon feeding 1 µm beads to macrophages) or mycobacterial phagosomes, as they gradually acquire autolysosomal characteristics. Next, we established that p62-captured cytosolic proteins (ribosomal protein rpS30 precursor FAU and ubiquitin) are proteolytically degraded into smaller peptides, and that specific peptides from these complex mixtures show antimycobacterial activity. Thus, the emerging model posits that autophagy captures cytosolic proteins and converts them into neo-antimycobacterial peptides that can then kill M. tuberculosis upon delivery to mycobacteria-containing phagosomes, which in turn gradually acquire autolysosomal properties (Fig. 1).Open in a separate windowFigure 1Elimination of M. tuberculosis by autophagy and p62. Mycobacteria are phagocytosed by macrophages and at least for some time reside within phagosomes. Upon induction of autophagy, p62, as a bifunctional agent interacting with autophagic substrates and with LC3, recruits into autophagosomes pre-antimicrobicidal cytosolic substrates. Autophagosome maturation including acquisition of lysosomal hydrolases leads to the proteolytic cleavage of p62 substrates and their conversion into peptides (cryptides) that can act as antimicrobial peptides.In contrast to the direct mechanism of capturing bacteria employed in some instances described above, in the case of M. tuberculosis, an organism that resides within the phagosomes, the adapter molecule p62 exerts its anti-microbial action through an indirect, but rather sophisticated mechanism. By sequestering into autophagosomes the initially harmless cytosolic components and by proteolytically processing them within maturing autophagosomes, p62 and autophagy liberate antimicrobial peptides from the otherwise innocuous substrates. This amounts to a resourceful utilization by the cell of otherwise spent or to-be-discarded cytoplasmic proteins and gives them an after-function upon completion of their “day jobs” that they performed as whole proteins.Our studies have uncovered a previously unappreciated function for autophagy in generating neo-antimicrobial peptides, and perhaps also opened the prospect for other biological functions potentially engendered by the products of autophagic proteolysis. Given that autophagy has the capacity to capture en masse and subject to digestion large sections of the cytoplasm, most cellular proteins are undergoing, or can undergo, processing into peptides or peptide intermediates within autophagic organelles. We postulate that the antimicrobial peptide production revealed in our studies thus far is only one manifestation of a spectrum of potential biological functions of cryptides generated by autophagy.     

Phosphodiesterase 4 inhibitor regulates the TRANCE/OPG ratio via COX-2 expression in a manner similar to PTH in osteoblasts

Phosphodiesterase 4 (PDE4) inhibitors stimulate osteoclast formation by increasing the TRANCE/OPG mRNA ratio via cAMP-mediated pathways in a manner similar to parathyroid hormone (PTH) in osteoblasts. We investigated the role of cyclooxygenase-2 (COX-2) in osteoclast formation induced by the PDE4 inhibitor rolipram. Rolipram induced COX-2 expression in mRNA and protein levels, followed by increased prostaglandin E(2) production in osteoblasts. PKA, ERK, and p38 MAPK pathways regulate COX-2 mRNA expression induced by rolipram, in which PKA is a central regulator of the ERK and p38 MAPK pathways. A COX-2 inhibitor reversed the up-regulation of the TRANCE/OPG mRNA ratio induced by rolipram in osteoblasts, resulting in decreased osteoclast formation. These data suggest that COX-2 mediates rolipram induced osteoclast formation by regulating the TRANCE/OPG mRNA ratio in osteoblasts. Furthermore, the effects of the PDE4 inhibitor on osteoblasts were very similar to those of PTH, indicating that the PDE4 inhibitor largely shares the biological actions of PTH in osteoblasts.     

Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

The accumulation of ubiquitin-positive protein aggregates has been implicated in the pathogenesis of neurodegenerative diseases, heart disease and diabetes. Emerging evidence indicates that the autophagy lysosomal pathway plays a critical role in the clearance of ubiquitin aggregates, a process that is mediated by the ubiquitin binding protein p62. In addition to binding ubiquitin, p62 also interacts with LC3 and transports ubiquitin conjugates to autophagosomes for degradation. The exact regulatory mechanism of this process is still largely unknown. Here we report the identification of Keap1 as a binding partner for p62 and LC3. Keap1 inhibits Nrf2 by sequestering it in the cytosol and preventing its translocation to the nucleus and activation of genes involved in the oxidative stress response. In this study, we found that Keap1 interacts with p62 and LC3 in a stress-inducible manner, and that Keap1 colocalizes with LC3 and p62 in puromycin-induced ubiquitin aggregates. Moreover, p62 serves as a bridge between Keap1 and ubiquitin aggregates and autophagosomes. Finally, genetic ablation of Keap1 leads to the accumulation of ubiquitin aggregates, increased cytotoxicity of misfolded protein aggregates, and defective activation of autophagy. Therefore, this study assigns a novel positive role of Keap1 in upregulating p62-mediated autophagic clearance of ubiquitin aggregates.     

Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process

In cells transfected to express wild-type PDE4A4 cAMP phosphodiesterase (PDE), the PDE4 selective inhibitor rolipram caused PDE4A4 to relocalise so as to form accretion foci. This process was followed in detail in living cells using a PDE4A4 chimera formed with Green Fluorescent Protein (GFP). The same pattern of behaviour was also seen in chimeras of PDE4A4 formed with various proteins and peptides, including LimK, RhoC, FRB and the V5-6His tag. Maximal PDE4A4 foci formation, occurred over a period of about 10 h, was dose-dependent on rolipram and was reversible upon washout of rolipram. Inhibition of protein synthesis, using cycloheximide, but not PKA activity with H89, inhibited foci generation. Foci formation was elicited by Ro20-1724 and RS25344 but not by either Ariflo or RP73401, showing that not all PDE4 selective inhibitors had this effect. Ariflo and RP73401 dose-dependently antagonised rolipram-induced foci formation and dispersed rolipram pre-formed foci as did the adenylyl cyclase activator, forskolin. Foci formation showed specificity for PDE4A4 and its rodent homologue, PDE4A5, as it was not triggered in living cells expressing the PDE4B2, PDE4C2, PDE4D3 and PDE4D5 isoforms as GFP chimeras. Altered foci formation was seen in the Deltab-LR2-PDE4A4 construct, which deleted a region within LRZ, showing that appropriate linkage between the N-terminal portion of PDE4A4 and the catalytic unit of PDE4A4 was needed for foci formation. Certain single point mutations within the PDE4A4 catalytic site (His505Asn, His506Asn and Val475Asp) were shown to ablate foci formation but still allow rolipram inhibition of PDE4A4 catalytic activity. We suggest that the binding of certain, but not all, PDE4 selective inhibitors to PDE4A4 induces a conformational change in this isoform by 'inside-out' signalling that causes it to redistribute in the cell. Displacing foci-forming inhibitors with either cAMP or inhibitors that do not form foci can antagonise this effect. Specificity of this effect for PDE4A4 and its homologue PDE4A5 suggests that interplay between the catalytic site and the unique N-terminal region of these isoforms is required. Thus, certain PDE4 selective inhibitors may exert effects on PDE4A4 that extend beyond simple catalytic inhibition. These require protein synthesis and may lead to redistribution of PDE4A4 and any associated proteins. Foci formation of PDE4A4 may be of use in probing for conformational changes in this isoform and for sub-categorising PDE4 selective inhibitors.     

p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.     

Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain

P62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.     

NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets

Autophagy is an evolutionary conserved cell survival process for degradation of long-lived proteins, damaged organelles and protein aggregates. The mammalian proteins p62 and NBR1 are selectively degraded by autophagy and can act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates. Despite differing in size and primary sequence, both proteins share a similar domain architecture containing an N-terminal PB1 domain, a LIR motif interacting with ATG8 family proteins, and a C-terminal UBA domain interacting with ubiquitin. The LIR motif is essential for their autophagic degradation, indicating that ATG8 family proteins are responsible for the docking of p62 and NBR1 to nucleating autophagosomes. p62 and NBR1 co-operate in the sequestration of misfolded and ubiquitinated proteins in p62 bodies and are both required for their degradation by autophagy. Here we discuss the role of p62 and NBR1 in degradation of ubiquitinated cargoes and the putative role of LIR as a general motif for docking of proteins to ATG8 family proteins.     

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.     

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Deltavps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Deltavps15 larvae. Fluorescence microscopy showed that Deltavps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.     

Autophagy and p62/sequestosome 1 generate neo-antimicrobial peptides (cryptides) from cytosolic proteins

In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.     

The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes

PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C.?elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function?causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of?protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for?omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides?a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.     

Effect of phoshpodiesterase 4 (PDE4) inhibibtors on eotaxin expression in humen bronchial epithelial cells

The increasing number of eosinophils into bronchoaelvolar space is observed during noninfectious inflammatory lung diseases. Eotaxins (eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26) are the strongest chemotactic agents for eosinophils. Inhibitors of phosphodiesterase 4 (PDE4), the enzyme decomposing cAMP, are anti-inflammatory agents which act through cAMP elevation and inhibit numerous steps of allergic inflammation. The effect of PDE4 inhibitors on eotaxin expression is not known in details. The aim of our study was to evaluate the influence of PDE4 inhibitors: rolipram and RO-20-1724 on expression of eotaxins in bronchial epithelial cell line BEAS-2B. Cells were preincubated with PDE4 inhibitors or dexamethasone for 1 hour and then stimulated with IL-4 or IL-13 alone or in combination with TNF-α. After 48 hours eotaxin protein level was measured by ELISA and mRNA level by real time PCR. Results: PDE4 inhibitors decreased CCL11 and CCL26 expression only in cultures co-stimulated with TNF-α. In cultures stimulated with IL-4 and TNF-α rolipram and RO-20-1724 diminished CCL11 mRNA expression by 34 and 37%, respectively, and CCL26 by 43 and 47%. In cultures stimulated with IL-13 and TNF-α rolipram and RO-20-1724 decreased expression of both eotaxins by about 50%. These results were confirmed at the protein level. The effect of PDE4 inhibitors on eotaxin expression in BEAS-2B cells, in our experimental conditions, depends on TNF-α contribution.     

Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant

Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum(ER)fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells.However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3-or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.     

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.