Mechanisms of Regulation of the Proteasome Activities

Project: Research project

Project Details

Description

DESCRIPTION (provided by applicant): The proteasome is a proteolytic assembly, which plays a central role in controlled nonlysosomal proteolysis in eukaryotic cells and therefore it emerged as an attractive anti-cancer drug target. The long-term goal of our laboratory is to understand the functioning of the proteasome. Activity of this giant multicatalytic complex is precisely regulated on many levels. Allosteric signaling constitutes the acknowledged but least known level of its regulation. The gate leading to the catalytic chamber is one of the most intriguing parts of proteasome, functioning of which depends on allostery. However, the experimental proof of this postulate is scarce. Therefore, in this project, we plan to test the hypothesis that the gate is involved in regulation of the activities of the proteasome by allosteric mechanism. Our hypothesis is based on the gated channel model, however it adds a dynamic aspect to the already accepted functions of the gate. Specifically, we will elucidate which parts of proteasome form the dynamic gate and how they participate in regulation of proteasome activities. Next, we will decipher how docking of the ligands to the face of a ring is sensed by proteasome and the role of the gate in the response of proteasome to such stimulus. For this purpose, we will employ two exceptional tools: protein PI31 and a family of Proline-Arginine rich peptides as ligands. The mechanism of PI31 is dearly missing, considering its diverse physiological roles as the modulator of proteasomal activity. The PR peptides are seriously considered as potential drugs; therefore, an elucidation of their mechanism of interactions with proteasome is invaluable. For our studies, we will use S. cerevisiae 20S particles, the best structurally and functionally described among eukaryotic proteasomes. To test our hypothesis, we will biochemically examine effects of deletion of the individual N-terminal sequences of all the subunits on functioning of the 20S particles. We will purify latent proteasomes from mutant yeast strains. Subsequently, we will determine their endoproteolytic efficiency, cooperatively between catalytic subunits, processivity, and products of degradation of model substrates. The conformation of the a ring will be assessed with the tapping mode atomic force microscopy in liquid and fluorescence spectroscopy of tryptophan residues. On this basis, we will attempt to create a model of the gate revealing possible roles of the gate elements in modulating enzymatic activities of proteasome. The proposed studies will provide a basis for the future targeting of the proteasomal activities with noncompetitive modulators interacting with the a rings and ultimately 19S caps.
StatusFinished
Effective start/end date2/1/041/31/10

Funding

  • National Institutes of Health: $209,510.00
  • National Institutes of Health: $243,217.00
  • National Institutes of Health: $198,654.00
  • National Institutes of Health: $230,010.00
  • National Institutes of Health: $13,086.00
  • National Institutes of Health: $198,654.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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