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Related Concept Videos

The Proteasome Structure01:17

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The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
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Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach
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Proteasomal conformation controls unfolding ability.

Julianna R Cresti1, Abramo J Manfredonia1, Christopher E Bragança1

  • 1Department of Chemistry, Villanova University, Villanova, PA 19085.

Proceedings of the National Academy of Sciences of the United States of America
|June 23, 2021
PubMed
Summary

The 26S proteasome undergoes conformational changes during protein degradation. New research identifies key interactions stabilizing these states, linking proteasome conformation to its protein unfolding and degradation efficiency.

Keywords:
ATP-dependent proteaseATPases associated with diverse cellular activities (AAA)proteasomeprotein degradationprotein unfolding

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Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • The 26S proteasome is crucial for protein degradation in eukaryotic cells.
  • Protein degradation involves conformational transitions from substrate-accepting to substrate-processing states.
  • Studying these dynamic conformational changes and stabilizing interactions is challenging.

Purpose of the Study:

  • To develop and utilize a conformationally sensitive assay to investigate the 26S proteasome.
  • To identify specific protein interactions critical for stabilizing substrate-processing conformations.
  • To link proteasome conformational dynamics to its protein unfolding and degradation capabilities.

Main Methods:

  • Development of a Förster resonance energy transfer (FRET) assay using fluorescently tagged Sem1 and Rpn6.
  • Measurement of proximity changes between Sem1 and Rpn6 to report on proteasome conformation.
  • Analysis of mutations affecting proposed interaction sites (Rpn5, Rpn2) and their impact on proteasome function.

Main Results:

  • The FRET assay successfully monitors conformational shifts in the 26S proteasome.
  • Interactions involving Rpn5 and Rpn2 are essential for stabilizing substrate-processing conformations.
  • Disruption of these interactions destabilizes processing states and impairs the degradation of difficult substrates.

Conclusions:

  • Specific interactions involving Rpn5 and Rpn2 play a vital role in stabilizing the substrate-processing states of the 26S proteasome.
  • Proteasome conformational stability is directly linked to its functional efficiency in unfolding and degrading ubiquitinated proteins.
  • The developed FRET assay provides a valuable tool for studying proteasome dynamics.