Parkinson's disease (PD) is a prevalent neurodegenerative disorder. Recent identification of genes linked to familial forms of PD such as Parkin and PINK1 (PTEN-induced putative kinase 1) has ...revealed that ubiquitylation and mitochondrial integrity are key factors in disease pathogenesis. However, the exact mechanism underlying the functional interplay between Parkin-catalyzed ubiquitylation and PINK1-regulated mitochondrial quality control remains an enigma. In this study, we show that PINK1 is rapidly and constitutively degraded under steady-state conditions in a mitochondrial membrane potential-dependent manner and that a loss in mitochondrial membrane potential stabilizes PINK1 mitochondrial accumulation. Furthermore, PINK1 recruits Parkin from the cytoplasm to mitochondria with low membrane potential to initiate the autophagic degradation of damaged mitochondria. Interestingly, the ubiquitin ligase activity of Parkin is repressed in the cytoplasm under steady-state conditions; however, PINK1-dependent mitochondrial localization liberates the latent enzymatic activity of Parkin. Some pathogenic mutations of PINK1 and Parkin interfere with the aforementioned events, suggesting an etiological importance. These results provide crucial insight into the pathogenic mechanisms of PD.
The Keap1-Nrf2 system and autophagy are both involved in the oxidative-stress response, metabolic pathways, and innate immunity, and dysregulation of these processes is associated with pathogenic ...processes. However, the interplay between these two pathways remains largely unknown. Here, we show that phosphorylation of the autophagy-adaptor protein p62 markedly increases p62’s binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2. Thus, p62 phosphorylation induces expression of cytoprotective Nrf2 targets. p62 is assembled on selective autophagic cargos such as ubiquitinated organelles and subsequently phosphorylated in an mTORC1-dependent manner, implying coupling of the Keap1-Nrf2 system to autophagy. Furthermore, persistent activation of Nrf2 through accumulation of phosphorylated p62 contributes to the growth of human hepatocellular carcinomas (HCCs). These results demonstrate that selective autophagy and the Keap1-Nrf2 pathway are interdependent, and that inhibitors of the interaction between phosphorylated p62 and Keap1 have potential as therapeutic agents against human HCC.
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•S351-phosphorylated p62 has higher affinity for Keap1 and activates Nrf2•Phosphorylation of p62 mainly occurs on selective autophagic cargos•Cytotoxic cargos, along with phosphorylated p62 and Keap1, are degraded by autophagy•Hepatocellular carcinoma cells utilize p62 phosphorylation for their growth
Genome-wide association studies of inflammatory bowel diseases identified susceptible loci containing an autophagy-related gene. However, the role of autophagy in the colon, a major affected area in ...inflammatory bowel diseases, is not clear. Here, we show that colonic epithelial cell-specific autophagy-related gene 7 (Atg7) conditional knock-out (cKO) mice showed exacerbation of experimental colitis with more abundant bacterial invasion into the colonic epithelium. Quantitative PCR analysis revealed that cKO mice had abnormal microflora with an increase of some genera. Consistently, expression of antimicrobial or antiparasitic peptides such as angiogenin-4, Relmβ, intelectin-1, and intelectin-2 as well as that of their inducer cytokines was significantly reduced in the cKO mice. Furthermore, secretion of colonic mucins that function as a mucosal barrier against bacterial invasion was also significantly diminished in cKO mice. Taken together, our results indicate that autophagy in colonic epithelial cells protects against colitis by the maintenance of normal gut microflora and secretion of mucus.
Background: The role of autophagy in colonic homeostasis is not clear.
Results: Colonic epithelial cell-specific and autophagy-deficient mice showed exacerbation of colitis and abnormal gut microflora with less abundant antimicrobial peptide production and secretion of mucus.
Conclusion: Autophagy protects against colitis by the maintenance of normal gut microflora and secretion of mucus.
Significance: These findings may ultimately lead to an efficient intervention for inflammatory bowel diseases.
•Ubiquitin-chain binding of p62 is required for its translocation to invasive Salmonella.•Localization of p62 on invasive Salmonella precedes its phosphorylation of Ser351.•Oligomerization of p62 is ...required for phosphorylation of Ser351.•p62 is essential for Nrf2 activation during xenophagy but not for xenophagy itself.
Upon infection of a cell by Salmonella, p62/Sqstm1 assembles on the microbes; simultaneously, p62/Sqstm1 is phosphorylated at Ser351, leading to inactivation of Keap1, which is responsible for degrading Nrf2. Thus, cytoprotective Nrf2 targets are induced at the same time that autophagosomes entrap the microbes (xenophagy). However, the detailed role of p62/Sqstm1 during xenophagy has remained unclear. Here we show that translocation of p62/Sqstm1 to invasive Salmonella precedes Ser351 phosphorylation. Furthermore, in addition to Ser351 phosphorylation, oligomerization of p62/Sqstm1 is also required for localization of Keap1 onto microbes, which is followed by Nrf2 activation. Our data reveal the sequential dynamics of p62/Sqstm1 in response to bacterial infection.
Nrf2 is a transcription factor responsible for gene expression of a series of anti-oxidant proteins and detoxifying enzymes. Keap1, an adaptor protein of Cullin-3 ubiquitin ligase, senses ...electrophilic or oxidative stresses and then arrests ubiquitination of Nrf2, leading to Nrf2 activation. In addition to this canonical pathway, one Nrf2 target (p62/SQSTM1) competitively binds to Keap1 to activate Nrf2. The p62/SQSTM1-Keap1-Nrf2 axis is linked to selective autophagy and regulated by post-translational modifications such as sequential phosphorylation and ubiquitination of p62/SQSTM1. Importantly, this non-canonical pathway is hyper-activated in autophagy-deficient mouse livers and tissues of hepatocellular carcinoma. In this review, we delineate a molecular mechanism of Nrf2-activation by p62/SQSTM1, and describe its physiological role as well as the pathophysiological significance in autophagy-knockout livers and human hepatocellular carcinoma.
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Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurons, but the relationship between impaired autophagy and Lewy bodies (LBs) as well as the in vivo ...process of formation remains unknown. Synuclein, a component of LBs, is the defining characteristic of Parkinson's disease (PD). Here, we characterize dopamine (DA) neuron-specific autophagy-deficient mice and provide in vivo evidence for LB formation. Synuclein deposition is preceded by p62 and resulted in the formation of inclusions containing synuclein and p62. The number and size of these inclusions were gradually increased in neurites rather than soma with aging. These inclusions may facilitate peripheral failures. As a result, DA neuron loss and motor dysfunction including the hindlimb defect were observed in 120-week-old mice. P62 aggregates derived from an autophagic defect might serve as "seeds" and can potentially be cause of LB formation.
A key antioxidant pathway, the Keap1-Nrf2 system, is regulated by p62/Sqstm1 via multiple mechanisms, including gene expression, posttranslational modifications (such as ubiquitination and ...phosphorylation), and autophagic degradation of p62/Sqstm1 and Keap1. Here we demonstrate a novel mode of regulation of the Keap1-Nrf2 system, mediated by a splicing variant of p62/Sqstm1 pre-mRNA. Ensembl database searches and subsequent biochemical analyses of mice revealed the presence of an mRNA that encodes a p62/Sqstm1 protein lacking the Keap1-interacting region (KIR), which is essential for the interaction with Keap1. Like full-length p62, the variant was induced under conditions in which Nrf2 was activated (e.g., impairment of autophagy), formed oligomers with itself and/or the full-length protein, and was degraded by autophagy. However, the variant failed to interact with Keap1 and sequester it in variant-positive aggregates. Remarkably, while full-length p62 stabilized Nrf2 and induced the gene expression of Nrf2 targets, the variant increased the amount of Keap1 and enhanced ubiquitination of Nrf2, thereby suppressing the induction of Nrf2 targets. Hepatocytes isolated from genetically modified mice that express full-length p62, but not the variant, were susceptible to activation of Nrf2 in response to stress. Collectively, our results suggest that splicing of p62/Sqstm1 pre-mRNA negatively regulates the Keap1-Nrf2 pathway.
Chondrogenesis is accompanied by not only cellular renovation, but also metabolic stress. Therefore, macroautophagy/autophagy is postulated to be involved in this process. Previous reports have shown ...that suppression of autophagy during chondrogenesis causes mild growth retardation. However, the role of autophagy in chondrocyte differentiation still largely remains unclear. Here, we show the important role of autophagy on chondrogenesis. The transition of mesenchymal cells to chondrocytes was severely impaired by ablation of
, a gene essential for autophagy. Mice lacking
after the transition exhibited phenotypes severer than mutant mice in which
was removed before the transition.
-deficient chondrocytes accumulated large numbers of glycogen granules, hardly proliferate and died specifically in the proliferative zone without any ER-stress signal. Our results suggest that the suppression of autophagy in prechondrogenic cells drives compensatory mechanism(s) that mitigate defective chondrogenesis, and that autophagy participates in glycogenolysis to supply glucose in avascular growth plates.
DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; SQSTM1/p62: sequestosome 1; STBD1: starch-binding domain-containing protein 1.
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