The proteasome, located in the cytosol and nucleus of eukaryotic cells, plays a central role in cellular regulation by controlling the concentrations of hundreds of regulatory proteins, removing misfolded proteins, and producing some peptides displayed in antigen presentation. It is an approx. 2MDa cylindrical particle with its active sites of proteolysis buried deep inside the structure where they are accessible only through a narrow channel. Substrate proteins must be unfolded to fit the channel and to be degraded. The long-term goals of the research proposed here are to understand protein unfolding by the proteasome using a combination of modern biochemical, computational, genetic, and cell biological methods. These experiments will provide an in-depth understanding of proteasome function and may reveal unexpected regulatory mechanisms. The immediate goal is to understand how the unfolding step affects the specificity of the proteasome for its substrates and how it affects the nature of its degradation products. We will first define how degradation is initiated after substrate binding by the proteasome. Proteins are targeted to the proteasome by the covalent attachment of a ubiquitin tag but degradation initiates at a different site in the substrate. The initiation step contributes to the specificity of degradation and the sequence and spatial requirements for efficient initiation will be determined in vitro. The effect of these rules on the specificity of degradation of natural proteins will be tested in vivo. Second, we will investigate how the proteasome unfolds and degrades only part of a protein in a process called processing. The proteasome degrades most proteins completely to small peptides but a novel type of signal can lead to partial protein degradation. The processing signal and mechanism will be characterized and physiological examples of processing will be detected by a bioinformatics search and a genetic screen. This work is relevant to human diseases;many of the proteins degraded by the proteasome regulate cell growth and division. Since proteins must be unfolded before they are cleaved by the proteasome, understanding unfolding during degradation may open new avenues to fight cancer, for example by stabilizing specific proteins against unfolding and degradation. Some neurodegenerative diseases are associated with the accumulation of protein aggregates, which the proteasome fails to remove for unknown reasons. The investigation of partial protein degradation may lend insight into this question.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM063004-09S1
Application #
8460607
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Wehrle, Janna P
Project Start
2001-04-01
Project End
2013-04-30
Budget Start
2010-05-01
Budget End
2013-04-30
Support Year
9
Fiscal Year
2012
Total Cost
$108,536
Indirect Cost
$37,012
Name
Northwestern University at Chicago
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
160079455
City
Evanston
State
IL
Country
United States
Zip Code
60201
Bhattacharyya, Sucharita; Renn, Jonathan P; Yu, Houqing et al. (2016) An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates. Anal Biochem 509:50-9
Wilmington, Shameika R; Matouschek, Andreas (2016) An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors. PLoS One 11:e0152679
Martinez-Fonts, Kirby; Matouschek, Andreas (2016) A Rapid and Versatile Method for Generating Proteins with Defined Ubiquitin Chains. Biochemistry 55:1898-908
Yu, Houqing; Singh Gautam, Amit K; Wilmington, Shameika R et al. (2016) Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome. J Biol Chem 291:14526-39
Yu, Houqing; Kago, Grace; Yellman, Christopher M et al. (2016) Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region. EMBO J 35:1522-36
Cannon, Joe R; Martinez-Fonts, Kirby; Robotham, Scott A et al. (2015) Top-down 193-nm ultraviolet photodissociation mass spectrometry for simultaneous determination of polyubiquitin chain length and topology. Anal Chem 87:1812-20
Fishbain, Susan; Inobe, Tomonao; Israeli, Eitan et al. (2015) Sequence composition of disordered regions fine-tunes protein half-life. Nat Struct Mol Biol 22:214-21
Takahashi, Kazunobu; Matouschek, Andreas; Inobe, Tomonao (2015) Regulation of Proteasomal Degradation by Modulating Proteasomal Initiation Regions. ACS Chem Biol 10:2537-43
Bhattacharyya, Sucharita; Yu, Houqing; Mim, Carsten et al. (2014) Regulated protein turnover: snapshots of the proteasome in action. Nat Rev Mol Cell Biol 15:122-33
van der Lee, Robin; Lang, Benjamin; Kruse, Kai et al. (2014) Intrinsically disordered segments affect protein half-life in the cell and during evolution. Cell Rep 8:1832-44

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