HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.     

Drp1-dependent mitochondrial fission regulates p62-mediated autophagy in LPS-induced activated microglial cells

Microglial activation is known to be an important event during innate immunity, but microglial inflammation is also thought to play a role in the etiology of neurodegenerative diseases. Recently, it was reported that autophagy could influence inflammation and activation of microglia. However, little is known about the regulation of autophagy during microglial activation. In this study, we demonstrated that mitochondrial fission-induced ROS can promote autophagy in microglia. Following LPS-induced autophagy, GFP-LC3 puncta were increased, and this was suppressed by inhibiting mitochondrial fission and mitochondrial ROS. Interestingly, inhibition of mitochondrial fission and mitochondrial ROS also resulted in decreased p62 expression, but Beclin1 and LC3B were unaffected. Taken together, these results indicate that ROS induction due to increased LPS-stimulated mitochondrial fission triggers p62 mediated autophagy in microglial cells. Our findings provide the first important clues towards understanding the correlation between mitochondrial ROS and autophagy.

Abbreviations: Drp1; Dynamin related protein 1, LPS; Lipopolysaccharide, ROS; Reactive Oxygen Species, GFP; Green Fluorescent Protein, CNS; Central Nervous System, AD; Alzheimer’s Disease, PD; Parkinson’s Disease, ALIS; Aggresome-like induced structures, iNOS; inducible nitric oxide synthase, Cox-2; Cyclooxygenase-2, MAPK; Mitogen-activated protein kinase; SODs; Superoxide dismutase, GPXs; Glutathione Peroxidase, Prxs; Peroxiredoxins     

Persistent expression of Nqo1 by p62-mediated Nrf2 activation facilitates p53-dependent mitotic catastrophe

Prolonged mitosis due to aberrant chromosome segregation permits cells to enter the G1 phase without cytokinesis and subsequently triggers the p53-dependent cell death program, known as mitotic catastrophe. Cells which fail to go through mitotic catastrophe create aneuploidy, posing a risk of oncogenesis. In the present report, we show that p62-mediated non-canonical activation of Nrf2 leads to the persistent expression of Nqo1, which plays a critical role for p53 stabilization during mitotic catastrophe. With prolonged exposure to nocodazole, a microtubule-depolymerizing agent, p62-deficient HCT116 cells exhibited an accumulation of a polyploid population with a limited appearance of apoptotic cells, which was attributable to the attenuated stabilization of p53. Combinatorial gene manipulation analysis verified that the regulatory cascade with a hierarchy of p62–Keap1–Nrf2–Nqo1 is required for p53 stabilization for mitotic catastrophe. This is consistent with the role of Nqo1 as a gatekeeper for proteasomal degradation of p53. Thus, we demonstrate for the first time the functional connection between the non-canonical Nrf2 pathway and p53-dependent cell death program upon prolonged mitosis.     

SQSTM1/p62 (sequestosome 1) senses cellular ubiquitin stress through E2-mediated ubiquitination

The alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. We recently reported that the macroautophagy/autophagy receptor SQSTM1/p62, functions as a novel Ub sensor to activate autophagy upon Ub⁺ stress (upregulation of the Ub level). First, SQSTM1 was found to undergo extensive ubiquitination and activate autophagy under Ub⁺ stress induced by prolonged Bortezomib (BTZ) treatment, Ub overexpression or by heat shock. Mechanistically, Ubiquitination of SQSTM1 disrupts its dimerization of the UBA domain, switching it from an auto-inhibitory conformation to recognize poly-ubiquitinated cargoes, promoting autophagic flux. Interestingly, Ub⁺ stress-responsive SQSTM1 ubiquitination is mediated by Ub conjugating enzymes, UBE2D2/3, in a unique E2-dependent manner. Our work has thus revealed a novel mechanism for how SQSTM1 senses cellular Ub stress conditions and regulates selective autophagy in response to diverse intrinsic or extrinsic challenges.     

Patulin induces pro-survival functions via autophagy inhibition and p62 accumulation

Patulin (PAT) is one of the most common mycotoxins found in moldy fruits. Skin contact is one of the most likely exposure routes of PAT. Investigation of dermal toxicity of PAT is clearly needed and has been highlighted by WHO. In the present study, using human keratinocyte HaCaT cells as a model, we found that treatment with PAT caused an increased autophagosome accumulation. Measurements of autophagic flux demonstrated that the accumulation of autophagosomes by PAT was not directly due to enhanced autophagosome formation but due to inhibition of autophagosome degradation. Reductions in the activities of the lysosomal enzymes cathepsin B and cathepsin D by PAT might contribute to this inhibitory effect. Consistent with this, inhibition of autophagosome degradation by PAT resulted in accumulation of p62 that functioned as a pro-survival signal. The pro-survival function of p62 was found to be attributed to reactive oxygen species-mediated cytoprotective endoplasmic reticulum (ER) stress response. ER stress exerted cytoprotective effect via extracellular signal-regulated kinase1/2-dependent B-cell CLL/lymphoma 2-associated agonist of cell death inhibitory phosphorylation. Given the critical role of autophagy and its substrate p62 in carcinogenesis, our findings may have important implications in PAT-induced skin carcinogenesis.     

pH-dependent regulation of SQSTM1/p62 during autophagy

During macroautophagy/autophagy, SQSTM1/p62 plays dual roles as a key mediator of cargo selection and as an autophagic substrate. SQSTM1 links N-degrons and/or ubiquitinated cargoes to the autophagosome by forming homo- or hetero-oligomers, although its N-degron recognition and oligomerization mechanisms are not well characterized. We recently found that SQSTM1 is a novel type of N-recognin whose ZZ domain provides a negatively-charged binding pocket for Arg-charged N-degron (Nt-Arg), a prototype type-1 substrate. Although differences in binding affinity exist for each N-degron, SQSTM1 also interacts with type-2 N-degrons, such as Nt-Tyr and Nt-Trp. Intriguingly, interactions between SQSTM1’s ZZ domain and various N-degrons are greatly influenced by pH-dependent SQSTM1 oligomerization via its PB1 domain. Because cellular pH conditions vary from neutral to acidic depending on the stage of autophagy, the pH-dependent regulation of SQSTM1’s oligomerization must be tightly coupled with the autophagic process.     

Lectin-mediated retention of p62 facilitates p62-E1 heterodimerization in endoplasmic reticulum of Semliki Forest virus-infected cells

The Semliki Forest virus (SFV) spike subunits p62 and E1 are made from a common coding unit in the order p62-E1. The proteins are separated by the host signal peptidase upon translocation into the endoplasmic reticulum (ER). Shortly thereafter, p62 and E1 form heterodimers. Heterodimerization preferentially occurs between subunits derived from the same translation product, so-called cis heterodimerization. As the p62 protein has the capacity to leave the ER in the absence of E1, it has been postulated that there exists a retention mechanism for the p62 protein, putatively at or near the translocon, in the ER in order to promote cis heterodimerization (B. U. Barth and H. Garoff, J. Virol. 71:7857-7865, 1997). Here we show that there exists such a mechanism, that it is at least in part mediated by the ER chaperones calnexin and calreticulin, and that the retention is important for efficient cis heterodimerization.     

NBR1 co-operates with p62 in selective autophagy of ubiquitinated targets

Selective degradation of intracellular targets, such as misfolded proteins and damaged organelles, is an important homeostatic function that autophagy has acquired in addition to its more general role in restoring the nutrient balance during stress and starvation. Although the exact mechanism underlying selection of autophagic substrates is not known, ubiquitination is a candidate signal for autophagic degradation of misfolded and aggregated proteins. p62/SQSTM1 was the first protein shown to bind both target-associated ubiquitin (Ub) and LC3 conjugated to the phagophore membrane, thereby effectively acting as an autophagic receptor for ubiquitinated targets. Importantly, p62 not only mediates selective degradation but also promotes aggregation of ubiquitinated proteins that can be harmful in some cell types. Is p62 the only autophagic receptor for selective autophagy? Looking for proteins that interact with ATG8 family proteins, we identified NBR1 (neighbor of BRCA1 gene 1) as an additional LC3- and Ub-binding protein. NBR1 is degraded by autophagy depending on its LC3-interacting region (LIR) but does not strictly require p62 for this process. Like p62, NBR1 accumulates and aggregates when autophagy is inhibited and is a part of pathological inclusions. We propose that NBR1 together with p62 promotes autophagic degradation of ubiquitinated targets and simultaneously regulates their aggregation when autophagy becomes limited.     

p62/SQSTM1 protects against cisplatin-induced oxidative stress in kidneys by mediating the cross talk between autophagy and the Keap1-Nrf2 signalling pathway

Acute kidney injury (AKI) is a major kidney disease associated with poor clinical outcomes. Oxidative stress is predominantly involved in the pathogenesis of AKI. Autophagy and the Keap1-Nrf2 signalling pathway are both involved in the oxidative-stress response. However, the cross talk between these two pathways in AKI remains unknown. Here, we found that autophagy is upregulated during cisplatin-induced AKI. In contrast with previous studies, we observed a marked increase in p62. We also found that p62 knockdown reduces autophagosome formation and the expression of LC3II. To explore the cross talk between p62 and the Keap1-Nrf2 signalling pathway, HK-2 cells were transfected with siRNA targeting Nrf2, and we found that Nrf2 knockdown significantly reduced cisplatin-induced p62 expression. Moreover, p62 knockdown significantly decreased the protein expression of Nrf2, as well as Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1), whereas the expression of kelch-like ECH-associated protein 1 (Keap1) was upregulated. These results indicate that p62 creates a positive feedback loop in the Keap1-Nrf2 signalling pathway. Finally, we examined the role of p62 in cell protection during cisplatin-induced oxidative stress, and we found that p62 silencing in HK-2 cells increases apoptosis and reactive oxygen species (ROS) levels, which further indicates the protective role of p62 under oxidative stress and suggests that the cytoprotection 62 mediated is in part by regulating autophagic activity or the Keap1-Nrf2 signalling pathway. Taken together, our results have demonstrated a reciprocal regulation of p62, autophagy and the Keap1-Nrf2 signalling pathway under oxidative stress, which may be a potential therapeutic target against AKI.     

TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS)

Autophagy plays an evolutionarily conserved role in host defense against pathogens. Autophagic protection

mechanisms against microbes range from regulating immune signaling responses to directly targeting the

pathogens for lysosomal degradation. Toll-like receptors (TLRs) that detect conserved molecular features shared by pathogens regulate several innate immune responses including autophagy. Our recent study demonstrates that autophagy reported in response to TLR4-stimulation in macrophages is selective

autophagy of aggresome-like induced structures (ALIS), and p62 (also known as SQSTM1) plays an essential role in this process. Treatment of macrophages with either Escherichia coli or lipopolysaccharide (LPS) results in the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), leading to an increase in the levels of p62 mRNA and protein, assembly of ALIS and their autophagic degradation. This study revealed a signaling

role for p62, distinct from its known function as a bacterial-targeting factor, which might be critical for cellular stress response during infection.     

TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS)

Autophagy plays an evolutionarily conserved role in host defense against pathogens. Autophagic protection mechanisms against microbes range from regulating immune signaling responses to directly targeting the pathogens for lysosomal degradation. Toll-like receptors (TLRs) that detect conserved molecular features shared by pathogens regulate several innate immune responses including autophagy. Our recent study demonstrates that autophagy reported in response to TLR4-stimulation in macrophages is selective autophagy of aggresome-like induced structures (ALIS), and p62 (also known as SQSTM1) plays an essential role in this process. Treatment of macrophages with either Escherichia coli or lipopolysaccharide (LPS) results in the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), leading to an increase in the levels of p62 mRNA and protein, assembly of ALIS and their autophagic degradation. This study revealed a signaling role for p62, distinct from its known function as a bacterial-targeting factor, which might be critical for cellular stress response during infection.     

Selective turnover of p62/A170/SQSTM1 by autophagy

Loss of autophagy causes liver injury, cardiomyopathy, and neurodegeneration, associated with the formation of ubiquitin-positive inclusion bodies. However, the pathogenic mechanism and molecular machinery involved in inclusion formation are not fully understood. We recently identified a ubiquitin-binding protein, p62/A170/SQSTM1, as a molecule involved in inclusion formation. p62 interacts with LC3 which regulates autophagosome formation, through an 11 amino acid sequence rich in acidic and hydrophobic residues, named the LC3-recognition sequence (LRS), and the LC3-p62 complex is degraded by autophagy. Furthermore, structural analysis reveals an interaction of Trp-340 and Leu-343 of p62 with different hydrophobic pockets in the ubiquitin-fold of LC3. p62 mutants, defective in binding the LRS, escape efficient turnover by autophagy, forming ubiquitin- and p62-positive inclusions. Importantly, such ubiquitin- and p62-positive inclusions are identified in various human diseases, implying the involvement of autophagy in their pathogenic mechanisms. Our reports identify an important role for autophagy in the selective turnover of p62, and demonstrate that in addition to the essential role of LC3 in autophagosome formation, LC3 is also involved in sorting autophagy-specific substrate(s).

Addendum to: Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, Mizushima N, Iwata JI, Ezaki J, Murata S, Hamazaki J, Nishito Y, Iemura SI, Natsume T, Yanagawa T, Uwayama J, Warabi E, Yoshida H, Ishii T, Kobayashi A, Yamamoto M, Yue Z, Uchiyama Y, Kominami E, Tanaka K. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 2007; 131:1149-63.

and

Ichimura Y, Kumanomidou T, Sou YS, Mizushima T, Ezaki J, Ueno T, Kominami E, Yamane T, Tanaka K, Komatsu M. Structural basis for sorting mechanism of p62 in selective autophagy. J Biol Chem 2008; 283:22847-57.     

Lysine 63-linked polyubiquitin potentially partners with p62 to promote the clearance of protein inclusions by autophagy

Although protein inclusions associated with neurodegenerative diseases are typically enriched with ubiquitin, it is currently unclear whether the topology of ubiquitin linkage plays a role in their biogenesis. In an attempt to clarify this, our recent work identified K63-linked polyubiquitin as a key regulator of inclusion dynamics. We found in the setting of ectopic overexpression of different ubiquitin species in cultured cells that K63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions linked to several major neurodegenerative diseases. Further supporting this, we report here a similar phenomenon in cells co-expressing Ubc13 and Uev1a but not those expressing UbcH7 or UbcH8. Notably, Ubc13 in association with Uev1a is known to promote K63-linked ubiquitination. In exploring how K63-linked ubiquitination could promote the clearance of inclusions by autophagy, we also found in our current study that K63-linked polyubiquitin interacts with p62, a ubiquitin-binding protein previously demonstrated by others to facilitate autophagy-mediated clearance of inclusions. Further, K63 ubiquitin-positive inclusions were found to be enriched with p62. Given the observed intimate relationship between p62 and K63 polyubiquitin, our results suggest that p62 and K63-linked polyubiquitin may function as key partners involved in directing clearance of protein inclusions by autophagy.

Addendum to: Tan JMM, Wong ESP, Kirkpatrick DS, Pletnikova O, Ko HS, Tay S-P, Ho M.W.L., Troncoso J, Gygi SP, Lee MK, Dawson VL, Dawson TM, Lim K-L. Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Human Mol Genet; In press.     

Selective turnover of p62/A170/SQSTM1 by autophagy

Loss of autophagy causes liver injury, cardiomyopathy and neurodegeneration, associated with the formation of ubiquitin-positive inclusion bodies. However, the pathogenic mechanism and molecular machinery involved in inclusion formation are not fully understood. We recently identified a ubiquitin-binding protein, p62/A170/SQSTM1, as a molecule involved in inclusion formation. p62 interacts with LC3 which regulates autophagosome formation, through an 11 amino acid sequence rich in acidic and hydrophobic residues, named the LC3-recognition sequence (LRS), and the LC3-p62 complex is degraded by autophagy. Furthermore, structural analysis reveals an interaction of Trp-340 and Leu-343 of p62 with different hydrophobic pockets in the ubiquitin-fold of LC3. p62 mutants, defective in binding the LRS, escape efficient turnover by autophagy, forming ubiquitin- and p62-positive inclusions. Importantly, such ubiquitin- and p62-positive inclusions are identified in various human diseases, implying the involvement of autophagy in their pathogenic mechanisms. Our reports identify an important role for autophagy in the selective turnover of p62, and demonstrate that in addition to the essential role of LC3 in autophagosome formation, LC3 is also involved in sorting autophagy-specific substrate(s).     

Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as ubiquitin E3 ligases in ubiquitin-mediated muscle protein turnover. Despite their well-characterised roles in muscle atrophy, the dynamics of MURF expression in the development and early postnatal adaptation of striated muscle is largely unknown. Here, we show that MURF2 is expressed at the very onset of mouse cardiac differentiation at embryonic day 8.5, and represents a sensitive marker for differentiating myocardium. During cardiac development, expression shifts from the 50 kDa to the 60 kDa A-isoform, which dominates postnatally. In contrast, MURF1 shows strong postnatal upregulation and MURF3 is not significantly expressed before birth. MURF2 expression parallels that of the autophagy-associated proteins LC3, p62/SQSTM1 and nbr1. SiRNA knockdown of MURF2 in neonatal rat cardiomyocytes disrupts posttranslational microtubule modification and myofibril assembly, and is only partly compensated by upregulation of MURF3 but not MURF1. Knockdown of both MURF2 and MURF3 severely disrupts the formation of ordered Z- and M-bands, likely by perturbed tubulin dynamics. These results suggest that ubiquitin-mediated protein turnover and MURF2 in particular play an unrecognised role in the earliest steps of heart muscle differentiation, and that partial complementation of MURF2 deficiency is afforded by MURF3.     

A reporter cell system to monitor autophagy based on p62/SQSTM1

Macroautophagy (hereafter referred to as autophagy) is a catabolic pathway to isolate and transport cytosolic components to the lysosome for degradation. Recently, autophagy receptors, like p62/SQSTM1 and NBR1, which physically link autophagic cargo to ATG8/MAP1-LC3/GABARAP family members located on the forming autophagic membranes, have been identified. To identify conditions or compounds that affect autophagy cell systems that efficiently report on autophagic flux are required. Here we describe reporter cell systems based on induced expression of GFP-p62, GFP-NBR1 or GFP-LC3B. The degradation of the fusion proteins was followed after promoter shut off by flow cytometry of live cells. All three fusion proteins were degraded at a basal rate by autophagy. Surprisingly, the basal degradation rate varied for the three reporter fusion proteins. GFP-LC3B was the most stable protein. GFP-NBR1 was most efficiently degraded under basal conditions while degradation of GFP-p62 displayed the strongest response to amino acid starvation. GFP-p62 was found to perform best of the tested reporters. Single cell analysis of autophagic flux by flow cytometry allows estimates of heterogeneous cell populations. The feasibility of this approach was demonstrated using transient overexpression of a dominant negative ULK1 kinase and siRNA-mediated knock-down of LC3B to inhibit autophagic degradation of GFP-p62. The inducible GFP-p62 cell system allows quantification by several approaches and will be useful in screening for compounds or conditions that affect the rate of autophagy. Inducers of autophagy can be identified using rich medium whereas inhibitors are identified under starvation conditions.