Many complex molecular transactions in the cell are catalyzed by large multisubunit protein machines. Often, these machines are organized into ring-shaped structures, creating a central channel or chamber, and macromolecules are moved into or out of these chambers, usually by integral ATP-hydrolyzing (ATPase) domains or by noncovalently associated ATPase complexes, which may themselves have a toroidal organization. The 26S proteasome, the central intracellular protease of eukaryotic cells, is among the most intricate of such ring-based ATP- driven machines. It consists of a cylindrical core particle, the 20S proteasome, which houses a central proteolytic chamber, and a 19S regulatory particle (RP) on each end. The RP includes six different ATPase subunits, also likely to be in a ring-shaped subcomplex, which binds and unfolds protein substrates and drives them into the 20S proteasome proteolytic chamber. How such complicated ring-shaped complexes are assembled in vivo is poorly understood, and this is certainly true for the assembly of the 20S proteasome, which has four heteroheptameric rings. Even less clear is the assembly mechanism of the ~20-subunit RP. The long-range goal of this application is to delineate the pathway(s) of proteasome biogenesis in vivo. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly could provide a useful new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, and biophysical methods and are centered on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. A major focus of the project will be on deciphering the pathway(s) by which the 20S proteasome assembles in vivo, including the identification of potential assembly factors (Specific Aim 1). Steps in 20S proteasome assembly will be reconstituted in vitro or in a bacterial co-expression system and the mechanisms of putative assembly factors will be tested (Specific Aim 2). A final set of experiments addresses the question of how the RP assembles, an issue for which there is only minimal information at present (Specific Aim 3). A potential RP assembly factor recently discovered in the PI's laboratory provides the starting point for these studies.
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| 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 |
| Hochstrasser, Mark (2016) Gyre and gimble in the proteasome. Proc Natl Acad Sci U S A 113:12896-12898 |
| 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 |
| 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 |
| 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 |
| 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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