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Filamentous Aggregates Are Fragmented by the Proteasome Holoenzyme.

ABSTRACT: Filamentous aggregates (fibrils) are regarded as the final stage in the assembly of amyloidogenic proteins and are formed in many neurodegenerative diseases. Accumulation of aggregates occurs as a result of an imbalance between their formation and removal. Here we use single-aggregate imaging to show that large fibrils assembled from full-length tau are substrates of the 26S proteasome holoenzyme, which fragments them into small aggregates. Interestingly, although degradation of monomeric tau is not inhibited by adenosine 5'-(3-thiotriphosphate) (ATP?S), fibril fragmentation is predominantly dependent on the ATPase activity of the proteasome. The proteasome holoenzyme also targets fibrils assembled from ?-synuclein, suggesting that its fibril-fragmenting function may be a general mechanism. The fragmented species produced by the proteasome shows significant toxicity to human cell lines compared with intact fibrils. Together, our results indicate that the proteasome holoenzyme possesses a fragmentation function that disassembles large fibrils into smaller and more cytotoxic species.

SUBMITTER: Cliffe R

PROVIDER: S-EPMC6381791 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Filamentous Aggregates Are Fragmented by the Proteasome Holoenzyme.

Cliffe Rachel R Sang Jason C JC Kundel Franziska F Finley Daniel D Klenerman David D Ye Yu Y

Cell reports 20190201 8

Filamentous aggregates (fibrils) are regarded as the final stage in the assembly of amyloidogenic proteins and are formed in many neurodegenerative diseases. Accumulation of aggregates occurs as a result of an imbalance between their formation and removal. Here we use single-aggregate imaging to show that large fibrils assembled from full-length tau are substrates of the 26S proteasome holoenzyme, which fragments them into small aggregates. Interestingly, although degradation of monomeric tau is ...[more]

PMID: 30784595

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Project description:BackgroundProtein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.Principal findingsTo better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.ConclusionsOur results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity.

Project description:Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2-6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ₁₋₄₂ expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72-86% (P < 10(-6) ). In worms expressing Aβ₁₋₄₂, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10(-6)). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.

Dataset Information

Filamentous Aggregates Are Fragmented by the Proteasome Holoenzyme.

Publications

Filamentous Aggregates Are Fragmented by the Proteasome Holoenzyme.

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