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


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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    • "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.
    BioMed Research International 06/2014; 2014(1):832704. DOI:10.1155/2014/832704 · 2.71 Impact Factor
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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.
    05/2014; 2(1):50. DOI:10.1186/2051-5960-2-50
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    • "Continuous basal autophagy is usually difficult to be visualized directly, due to its low levels. For this reason, assessing basal autophagic activity by looking at levels of ref(2)P has become a standard test, similar to mammals [14, 19, 22, 41, 42]. Expression of either of the two Atg101 silencing constructs results in large-scale accumulation of ref(2)P aggregates in GFP-marked cells, when compared to surrounding wild-type tissue in well-fed larvae (Figures 5(a), 5(b), and 5(c)), indicating defects in selective basal autophagy. "
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    ABSTRACT: The large-scale turnover of intracellular material including organelles is achieved by autophagy- mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13, and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes.
    BioMed Research International 03/2014; 2014(4). DOI:10.1155/2014/470482 · 2.71 Impact Factor
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