The proteasome is a complex molecular machine and an essential mediator of ubiquitin-dependent protein degradation. Polyubiquitination of target proteins has been long considered the major regulator of proper protein degradation. However, little is known about a new and potent regulator of cellular protein degradation ? mechanisms that control the rate and quality of proteasome assembly. It is well established that cells require multiple evolutionarily conserved chaperones to regulate proteasome assembly. A catalyzed assembly mechanism serves as an active regulator of proper proteasome assembly, and therefore of proteolysis itself. Under some pathological situations, cells are known to exploit these chaperones to alter the rate of proteasome- mediated proteolysis. Moreover, chaperone-mediated assembly of proteasomes has also been linked to major pathways for growth control. Therefore, understanding how chaperones control proteasome assembly is of wide- ranging biological and biomedical significance. The objective of this proposal is to elucidate how these chaperones function to regulate the rate and the quality of proteasome formation. Our central hypothesis is that the chaperones determine correct versus incorrect assembly of the proteasome by regulating both the ATP hydrolysis and the ubiquitination of the ATPase subunits en route to proteasome formation.
Aim 1 will determine how chaperone-mediated inhibition of ATP hydrolysis by the proteasome subunits controls proteasome assembly events. Both in vitro and in vivo proteasome assembly assays will be used while the ATPase rates are directly modulated by specific yeast mutants.
Aim 2 will determine how ubiquitinations of the proteasome subunits provide quality control during chaperone-mediated proteasome assembly. Biochemical approaches similar to Aim 1 will be used in combination with yeast genetics. The goal is to identify how the chaperones regulate ubiquitination of the proteasome subunits, and how ubiquitination affects assembly intermediates and the assembled proteasome holoenzyme. The expected outcome is the identification of the mechanisms by which the chaperones determine incorrect versus correct assembly events, and provide quality control, thereby enabling only the correct assembly events to proceed in forming the proteasome holoenzyme. The proposed research is innovative because it aims to identify mechanisms that regulate the number of functional proteasomes in the cell, which have been missing from the current paradigm of proteasome-mediated proteolysis. This contribution is significant for human health to help understand how this critical pathway is exploited to alter protein degradation in pathological conditions, including cancer, neurodegenerative disease and aging. These conditions are known to be impacted by the ubiquitin-proteasome system, a proven therapeutic target for altering protein degradation in disease. We anticipate that understanding chaperone-mediated regulation of the proteasome subunits will contribute to the development of new targeted drugs.

Public Health Relevance

The proteasome is a central mediator of protein degradation, and has been connected to cancers and neurodegenerative syndromes. The proposed research aims to unravel how cells generate the proteasome at the proper rate and the quality to control the number of functional proteasomes for protein degradation. This study contributes to public health by providing the fundamental basis of exploitation of the proteasome in human diseases, and for strategies to combat them.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM127688-02
Application #
9750305
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Phillips, Andre W
Project Start
2018-08-01
Project End
2023-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80303