Article

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

BioScience Center, San Diego State University, CA, USA.
Autophagy (Impact Factor: 11.75). 06/2011; 7(6):572-83. DOI: 10.4161/auto.7.6.14943
Source: PubMed

ABSTRACT

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.

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    • "P62, also known as sequestosome 1, is a shuttle protein transporting polyubiquitinated proteins for both the proteasomal and autophagy/lysosomal dependent degradation (Komatsu et al., 2007; Pankiv et al., 2007; Seibenhener et al., 2004; Wooten et al., 2008). P62 and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects (Bartlett et al., 2011). In particular, p62 was commonly detected in ubiquitinated protein aggregates in neuronal diseases including LBs in PD, neurofibrillary tangles in Alzheimer's disease, Huntington aggregates in Huntington's disease, and skein-like inclusions in amyotrophic lateral sclerosis (Lowe et al., 1988; Rue et al., 2013; Seibenhener et al., "
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    ABSTRACT: Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.
    Preview · Article · Jan 2016 · Protein & Cell
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    • "Knockout studies in mice and Drosophila revealed that p62 is required for the aggregation of ubiquitinylated proteins and thus plays essential roles for their autophagic clearance [44, 45]. The levels of p62 usually inversely correlate with autophagic degradation, as the loss of Atg genes or factors required for the fusion of autophagosomes with lysosomes all result in a marked increase of p62-positive aggregates [46, 47]. p62 can also deliver ubiquitinylated cargos to the proteasome, although they are mainly degraded by autophagy [48, 49]. "
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    ABSTRACT: The ubiquitin-proteasome system and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. By now we know that these degradation pathways share certain substrates and regulatory molecules and show coordinated and compensatory function. Two ubiquitin-like protein conjugation pathways were discovered that are required for autophagosome biogenesis: the Atg12-Atg5-Atg16 and Atg8 systems. Autophagy has been considered to be essentially a nonselective process, but it turned out to be at least partially selective. Selective substrates of autophagy include damaged mitochondria, intracellular pathogens, and even a subset of cytosolic proteins with the help of ubiquitin-binding autophagic adaptors, such as p62/SQSTM1, NBR1, NDP52, and Optineurin. These proteins selectively recognize autophagic cargo and mediate its engulfment into autophagosomes by binding to the small ubiquitin-like modifiers that belong to the Atg8/LC3 family.
    Full-text · Article · Jun 2014 · BioMed Research International
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    • "Importantly, we showed that S349 on p62 is in fact phosphorylated in the brains of patients with AD and that the level was significantly increased in AD compared with controls. Consistent with previous results [9, 32], we also demonstrated that the total p62 level was significantly increased in AD relative to controls. The ratio of P-S349 levels to total p62 is significantly increased in AD relative to controls. "
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    ABSTRACT: Background Extensive research on p62 has established its role in oxidative stress, protein degradation and in several diseases such as Paget’s disease of the bone, frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Importantly, previous studies showed that p62 binds directly to Keap1, which is a ubiquitin E3 ligase responsible for degrading Nrf2. Indeed, colocalisation of p62 and Keap1 occurs in tumorigenesis and neurodegeneration. A serine (S) residue in the Keap1-interacting region of p62 is phosphorylated in hepatocellular carcinoma, and this phosphorylation contributes to tumour growth through the higher affinity of p62 to Keap1. However, it remains largely unknown whether p62 is phosphorylated in the Keap1-interacting region under neurodegenerative conditions. Results To answer this question, we generated an antibody against phosphorylated S349 (P-S349) of p62 and showed that S349 is phosphorylated following disruption of protein degradation. In particular, the ratio of P-S349 to total p62 levels was significantly increased in the brains with Alzheimer’s disease (AD) compared with controls. We also compared the reactivity of the P-S349 antibody with P-S403 of p62 and showed that these two phosphorylated sites on p62 cause different responses with proteasome inhibition and show distinct localisation patterns in AD brains. In addition to disruption of protein degradation systems, activation of oxidative stress can induce P-S349. Conclusion These results support the hypothesis that disruption of protein degradation systems and sustained activation of the Keap1-Nrf2 system occur in the brains with AD. Electronic supplementary material The online version of this article (doi:10.1186/2051-5960-2-50) contains supplementary material, which is available to authorized users.
    Full-text · Article · May 2014
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