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Published on: November 24, 2015
Distinguishing aggregate formation and aggregate clearance using cell-based assays.
Evelien Eenjes1, Joanna M Dragich2, Harm H Kampinga3
1Columbia University, Department of Neurology, New York, NY 10032, USA University Medical Center Groningen, University of Groningen, Department of Cell Biology, Groningen 9713, The Netherlands.
Researchers developed a new assay to understand how protein aggregate burden is managed. This tool reveals that HspB7 and Alfy (WDFY3) impact distinct cellular processes, and that new protein aggregate formation can hinder the clearance of existing ones.
Area of Science:
- Cellular Biology
- Biochemistry
- Neuroscience
Background:
- Protein aggregate accumulation, characterized by ubiquitylated inclusions, results from an imbalance between protein aggregate formation and clearance.
- Strategies to reduce aggregate burden, such as decreasing formation or enhancing clearance, can have differential impacts on disease pathogenesis.
- Understanding the distinct mechanisms underlying aggregate formation and clearance is crucial for developing effective therapeutic interventions.
Purpose of the Study:
- To develop and utilize a novel cell-based assay capable of differentiating between newly formed and pre-existing protein aggregates.
- To elucidate the specific cellular processes influenced by HspB7 and Alfy (WDFY3) in the context of expanded polyglutamine aggregate clearance.
- To investigate the impact of constitutive expression of aggregation-prone proteins on the rate of protein aggregate elimination.
Main Methods:
- Development of a novel cell-based assay to distinguish newly formed from preformed proteinaceous inclusions.
- Application of the assay to study the roles of HspB7 and Alfy (WDFY3) in the autophagic clearance of expanded polyglutamine inclusions.
- Assessment of the effect of constitutive expression of aggregation-prone proteins on aggregate clearance rates.
Main Results:
- HspB7 and Alfy (WDFY3) were found to influence distinct cellular processes affecting aggregate burden, rather than acting solely on autophagic clearance.
- The cell-based assay demonstrated that constitutive expression of aggregation-prone proteins can impede the elimination of existing protein aggregates.
- Not all protein aggregates appear to be equally accessible for degradation, suggesting differential targeting mechanisms.
Conclusions:
- The novel cell-based assay provides a powerful tool to determine the specific step at which modifiers influence aggregate burden.
- This assay offers new insights into the mechanisms by which protein aggregates are targeted for degradation.
- The findings highlight the complexity of protein aggregate management, involving distinct roles for proteins like HspB7 and Alfy (WDFY3) and the influence of ongoing aggregate formation on clearance efficiency.
