Large protein machines control almost all biological processes, including DNA replication, RNA processing, and both protein synthesis and degradation. The 26S proteasome is responsible for the great majority of protein degradation in eukaryotes and is essential for numerous cellular pathways. Aberrant proteasomal activity impacts many human diseases, such as neurodegenerative disorders, cancer, and diabetes. Like many multisubunit catalytic complexes, the proteasome forms ring-shaped structures. The proteasome consists of a cylindrical 20S proteasome core particle (CP) with a 19S regulatory particle (RP) on one or both ends. In the CP, two outer heteroheptameric ? rings sandwich a pair of ? rings; the latter creates the central proteolytic chamber. Each RP can be divided into ld and base complexes. The RP base includes six ATPases that form a heterohexameric ring; the RP binds and unfolds substrates and drives them into the CP interior. How these large, essential complexes are assembled in vivo remains poorly understood. Proteasome assembly appears to be an ordered process conserved across species. As proteasomes are highly abundant, assembly must occur with high fidelity to avoid excessive accumulation of nonproductive and potentially toxic off-pathway intermediates. Both CP and RP assembly depend on dedicated assembly chaperones. The long-range goal of this grant is to delineate the pathways of eukaryotic 26S proteasome biogenesis. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly will provide a new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, cell biological and biophysical methods and are focused primarily, but not entirely, on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. The first major goal will be to decipher the assembly-promoting mechanism of a putative novel CP assembly factor and how it interacts functionally with known CP assembly chaperones. A second goal is to determine how CPs with alternative ?-subunit arrangements assembles in both yeast and human cells and to evaluate their functional significance. In a third aim, the focus is on a set of RP assembly chaperones (RACs) to determine their contributions to proper assembly of the ATPase ring of the RP base; the mechanism of RP lid-base joining will also be probed. Lastly, experiments will address how proteasome assembly is linked to import into the nucleus and how proteasomes in quiescent cells rapidly relocalize from cytoplasmic foci to the nucleus in response to glucose addition.

Public Health Relevance

Many human diseases result from an abnormally high or low level of some crucial protein that controls cell or tissue function. Some cancers, for instance, ar caused by insufficient amounts of proteins called tumor suppressors. The proteasome is a huge enzyme complex that controls the levels of important proteins by degrading them; adjustment of proteasome assembly or activity can therefore have a profound impact on the course of many human disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083050-11
Application #
9439805
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Barski, Oleg
Project Start
2008-02-01
Project End
2020-02-29
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
11
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Hochstrasser, Mark (2016) Gyre and gimble in the proteasome. Proc Natl Acad Sci U S A 113:12896-12898
Huber, Eva M; Heinemeyer, Wolfgang; Li, Xia et al. (2016) A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat Commun 7:10900
Hu, Ronggui; Hochstrasser, Mark (2016) Recent progress in ubiquitin and ubiquitin-like protein (Ubl) signaling. Cell Res 26:389-90
Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark (2016) Substrate specificity of the ubiquitin and Ubl proteases. Cell Res 26:441-56
Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark (2016) Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells. Cell Rep 14:2962-74
Li, Xia; Li, Yanjie; Arendt, Cassandra S et al. (2016) Distinct Elements in the Proteasomal ?5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly. J Biol Chem 291:1991-2003
Tomko Jr, Robert J; Taylor, David W; Chen, Zhuo A et al. (2015) A Single ? Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly. Cell 163:432-44
Li, Yanjie; Tomko Jr, Robert J; Hochstrasser, Mark (2015) Proteasomes: Isolation and Activity Assays. Curr Protoc Cell Biol 67:3.43.1-20
Tomko Jr, Robert J; Hochstrasser, Mark (2014) The intrinsically disordered Sem1 protein functions as a molecular tether during proteasome lid biogenesis. Mol Cell 53:433-43

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