Dataset Information

Proximity proteomics on RPE1 stable cell lines with miniTurbo-p62 and ATG5 Knock-down to investigate p62 filaments and calcium-rich p62-enwrapped lipid droplets

ABSTRACT: The selective autophagy receptor p62/SQSTM1 (from hereon p62) is known to form higher-order filaments in vitro and to undergo liquid-liquid phase separation when mixed with poly-ubiquitin. We determined the full-length cryo-EM structure of p62 and elucidated a structured double helical filament scaffold composed of the PB1-domain associated with the flexible C-terminal part residing in the lumen and the solvent-accessible major groove. At different pH values and upon binding to soluble LC3, LC3-conjugated membranes and poly-ubiquitin, we observed p62 filament re-arrangements in the form of structural unwinding, disassembly, lateral association and bundling, respectively. In the cellular environment, under conditions of ATG5 knockdown leading to stalled autophagy, we imaged high-contrast layers consisting of p62 oligomers enwrapping lipid droplets by cryogenic electron tomography in cellulo, which we identified as calcium as well as phosphorus by compositional spectroscopy analysis. Together, we visualized the cellular ultrastructure of p62 oligomers with high calcium content as a potential early stage of autophagy. Therefore, we performed proximity biotinylation and proteomics to identify calcium transporters and binding proteins in proximity to the p62 encapsulated lipid droplets.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Maya Marita Dierig

LAB HEAD: Pitter Florian Huesgen

PROVIDER: PXD066163 | Pride | 2025-12-08

REPOSITORIES: Pride

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Publications

Structural organization of p62 filaments and the cellular ultrastructure of calcium-rich p62-enwrapped lipid droplet cargo.

Berkamp Sabrina S Jungbluth Lisa L Katranidis Alexandros A Mostafavi Siavash S Korculanin Olivera O Lu Peng-Han PH Ickert Lotte L Dierig Maya M MM Sharma Lokesh L Thukral Lipi L Huesgen Pitter F PF Kononenko Natalia L NL Fitter Jörg J Dunin-Borkowski Rafal E RE Sachse Carsten C

Nature communications 20251128 1

The selective autophagy receptor p62/SQSTM1 is known to form higher-order filaments in vitro and to undergo liquid-liquid phase separation when mixed with poly-ubiquitin. Here, we determine the full-length cryo-EM structure of p62 and elucidate a structured double helical filament scaffold composed of the PB1-domain associated with the flexible C-terminal part and the solvent-accessible major groove. At different pH values and upon binding to soluble LC3, LC3-conjugated membranes and poly-ubiqui ...[more]

PMID: 41315362

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Project description:Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer lifespans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyglutamine length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions. Gene expression profiles were analyzed to examine the effects of p62 depletion in mouse with or without mutant huntingtin exon 1 To examine the effect of p62 depletion on the transcriptome of Huntington's disease model mice, we crossed p62 knockout mice with HD model mice. We extracted total RNA from the striatum of these mice at 8 weeks and used for a microaaray analysis. The samples are HD transgenic mice with p62 knockout (HD_p62KO), HD mice with normal p62 (HD_p62WT), non-HD-transgenic mice with p62 knockout (NT_p62KO), and non-HD-transgenic mice with normal p62 (NT_p62WT).

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