In eukaryotes the ATP dependent protein degradation by the ubiquitin-proteasome pathway removes short lived signaling protein that is critical in regulation of cellular process, degrades misfolded and damaged proteins whose accumulation is toxic to the cell and breaks down foreign proteins to generate antigenic peptides for presenting to the immune system. It is fundamental in understanding the mechanism of many human diseases, especially cancer and neurodegenerative diseases, e.g. Huntington disease. The eukaryotic 26S proteasome is formed by a 20S proteasome with the proteolytic active sites sequestered inside it and two 19S regulatory particles each contain six ATPases in contact with the 20S. A key role of the ATPases is to open the gated channel in the 20S to facilitate substrates enter for destruction. An important question in proteasome biology is that how short peptides of proteolytic products are released efficiently from CP to ensure a continuous substrate entering and products release required for the degradation of large protein substrates. A widely accepted yet untested paradigm is that the 26S proteasome functions unidirectional in which unfolded substrates enter the CP from one end and the proteolytic products exit from the opposite end. Another important question is what is the role of ATP hydrolysis by Rpt subunits during the Rpt ring assembly, and if the assembly requires CP as a template? In this application, we aim to address these questions. We will use near atomic resolution single particle cryoEM as our main structural analysis tool, together with other methods in molecular biology, biochemistry and biophysical tool, to elucidate the mechanisms that regulates the asymmetrical functionality of the symmetrical protein degradation machinery.
The specific aims are (1) determine the mechanism of proteolytic products releasing from the 20S degradation chamber, (2) determine mechanism that coordinates the functions of proteasomal activators bound to the opposite ends of 20S core particle, and (3) determine the role of ATP hydrolysis in the assembly pathway of eukaryotic proteasomal ATPases. Substantial completion of these aims will advance our knowledge about the proteasome-mediated protein degradation that plays a key role in the pathogenesis of many human diseases.

Public Health Relevance

In eukaryotic cells most unwanted proteins are degraded by a large molecular machine named proteasome. The protein degradation process is tightly regulated and plays a key role in the pathogenesis of many human diseases, especially cancer and neurodegenerative diseases, e.g. Huntington's disease. This application studies the mechanism by which the proteasomal ATPases regulate the proteolytic activities of the proteasome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM082893-06
Application #
8629478
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Gindhart, Joseph G
Project Start
2008-01-01
Project End
2018-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
6
Fiscal Year
2014
Total Cost
$392,973
Indirect Cost
$134,262
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Wu, Shenping; Armache, Jean-Paul; Cheng, Yifan (2016) Single-particle cryo-EM data acquisition by using direct electron detection camera. Microscopy (Oxf) 65:35-41
Zhao, Minglei; Wu, Shenping; Zhou, Qiangjun et al. (2015) Mechanistic insights into the recycling machine of the SNARE complex. Nature 518:61-7
Barad, Benjamin A; Echols, Nathaniel; Wang, Ray Yu-Ruei et al. (2015) EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat Methods 12:943-6
DiMaio, Frank; Song, Yifan; Li, Xueming et al. (2015) Atomic-accuracy models from 4.5-Ã… cryo-electron microscopy data with density-guided iterative local refinement. Nat Methods 12:361-5
Chiu, Po-Lin; Li, Xueming; Li, Zongli et al. (2015) Evaluation of super-resolution performance of the K2 electron-counting camera using 2D crystals of aquaporin-0. J Struct Biol 192:163-73
Cheng, Yifan; Grigorieff, Nikolaus; Penczek, Pawel A et al. (2015) A primer to single-particle cryo-electron microscopy. Cell 161:438-49
Cheng, Yifan (2015) Single-Particle Cryo-EM at Crystallographic Resolution. Cell 161:450-7
Li, Xueming; Zheng, Shawn; Agard, David A et al. (2015) Asynchronous data acquisition and on-the-fly analysis of dose fractionated cryoEM images by UCSFImage. J Struct Biol 192:174-8
Li, Xueming; Mooney, Paul; Zheng, Shawn et al. (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584-90
Li, Xueming; Zheng, Shawn Q; Egami, Kiyoshi et al. (2013) Influence of electron dose rate on electron counting images recorded with the K2 camera. J Struct Biol 184:251-60

Showing the most recent 10 out of 20 publications