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Assays for the Degradation of Misfolded Proteins in Cells
Published on: August 28, 2016
Rpt5-Derived Analogs Stimulate Human Proteasome Activity in Cells and Degrade Proteins Forming Toxic Aggregates in
Katarzyna Cekała1, Karolina Trepczyk1, Julia Witkowska1
1Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.
Abstract:
Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.
Insights
Researchers developed peptide activators to boost 20S proteasome activity, crucial for clearing damaged proteins during aging. These peptides successfully entered cells and enhanced the degradation of disease-associated proteins like alpha-synuclein and Tau.
Area of Science:
- Biochemistry
- Molecular Biology
- Gerontology
Background:
- Aging leads to reduced protein turnover and accumulation of damaged proteins.
- The ubiquitin-independent proteasome system (UIPS), particularly the 20S proteasome, degrades misfolded and aggregated proteins.
- Declining proteasome activity contributes to age-related diseases.
Purpose of the Study:
- To develop peptide-based activators targeting the 20S proteasome.
- To investigate if these activators can counteract age-related decline in proteostasis.
- To assess the therapeutic potential of enhancing 20S proteasome function.
Main Methods:
- Synthesis of peptide and peptidomimetic activators based on the Rpt5 subunit C-terminus.
- Assay of 20S proteasome activity using synthesized compounds.
- Cellular studies using HEK293T cells and the cell-permeable substrate TAS3.
- Evaluation of degradation of aggregation-prone proteins (α-synuclein, Tau-441).
Main Results:
- Several synthesized peptides effectively stimulated human 20S proteasome activity.
- Cell-penetrating peptide TAT enabled cellular uptake and activation of proteasome activity in HEK293T cells.
- The most potent activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441.
Conclusions:
- Peptide-based activators can enhance 20S proteasome function.
- TAT-conjugated peptides demonstrate cell permeability and efficacy in stimulating proteasome activity.
- These compounds show potential for treating age-related diseases characterized by proteostasis imbalance.
