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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM069819-01
Application #
6712483
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
2004-02-01
Project End
2009-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
1
Fiscal Year
2004
Total Cost
$230,010
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Other Basic Sciences
Type
Other Domestic Higher Education
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
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Osmulski, Pawel A; Hochstrasser, Mark; Gaczynska, Maria (2009) A tetrahedral transition state at the active sites of the 20S proteasome is coupled to opening of the alpha-ring channel. Structure 17:1137-47
Rosenzweig, Rina; Osmulski, Pawel A; Gaczynska, Maria et al. (2008) The central unit within the 19S regulatory particle of the proteasome. Nat Struct Mol Biol 15:573-80
Gaczynska, Maria; Osmulski, Pawel A (2008) AFM of biological complexes: what can we learn? Curr Opin Colloid Interface Sci 13:351-367
Gaczynska, Maria; Rodriguez, Karl; Madabhushi, Srividya et al. (2006) Highbrow proteasome in high-throughput technology. Expert Rev Proteomics 3:115-27
Tan, Xiaolin; Osmulski, Pawel A; Gaczynska, Maria (2006) Allosteric regulators of the proteasome: potential drugs and a novel approach for drug design. Curr Med Chem 13:155-65
Gaczynska, Maria; Osmulski, Pawel A (2005) Characterization of noncompetitive regulators of proteasome activity. Methods Enzymol 398:425-38
Osmulski, Pawel A; Gaczynska, Maria (2005) Atomic force microscopy of the proteasome. Methods Enzymol 398:414-25
Gaczynska, Maria; Osmulski, Pawel A; Jiang, Yun et al. (2004) Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA. Proc Natl Acad Sci U S A 101:17952-7