three-dimensional reconstruction of the pa200-20s proteasome complex by cryo-em
DESCRIPTION
Three-dimensional Reconstruction of the PA200-20S Proteasome Complex by Cryo-EM Joaquin Ortega. Ph.D. Biochemistry & Biomedical Sciences Mcmaster University. Hamilton, ON. Canada. Protein Quality Control System. From: Wickner et al. Science (1999). v. 286, 1888-1893. - PowerPoint PPT PresentationTRANSCRIPT
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Three-dimensional Reconstruction of the
PA200-20S Proteasome Complex by Cryo-EM
Joaquin Ortega. Ph.D.
Biochemistry & Biomedical SciencesMcmaster University. Hamilton, ON. Canada
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Protein Quality Control SystemProtein Quality Control System
From: Wickner et al. Science (1999). v. 286, 1888-1893
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*20S Proteasome Major cellular protease
*HtrA (Deg P)Periplasmic chaperone / protease
Current Projects in the lab
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Major Functions of the Major Functions of the ProteasomeProteasome
*Regulation of protein functions*Regulation of protein functionsTerminate lifespan of regulatory proteinsTerminate lifespan of regulatory proteins
*Protein quality control*Protein quality controlDegrade damaged/misfolded proteinsDegrade damaged/misfolded proteins
*Immunocompetent peptide generation*Immunocompetent peptide generationDegradation of foreign antigensDegradation of foreign antigens
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Structure of Structure of 20S Proteasome20S Proteasome
Top view Side viewFrom: Baumeister et al. (1998) Cell 92, 367-380
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Inside the BarrelInside the Barrel
Channel Gate
Catalytic Chamber
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Proteasome Regulatory Complexes
The 11S/PA28 activators: An ATP-independent activator
The 19S activator: An ATP-dependent activator
From Baumeister et al. (1998)Cell 92, 367-380
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PA200: Novel Proteasome PA200: Novel Proteasome Activator FamilyActivator Family
*200 kDa, 1843 aa.*200 kDa, 1843 aa.
*Subject to phosphorylation.*Subject to phosphorylation.
*Functionally more like PA28 than 19S.*Functionally more like PA28 than 19S.-ATP-independent-ATP-independent-Activate peptide hydrolysis only-Activate peptide hydrolysis only
*DNA Repair Involvement?*DNA Repair Involvement?-Localizes in nucleus-Localizes in nucleus-After -After -irradiation, PA200 forms intranuclear foci-irradiation, PA200 forms intranuclear foci
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Questions
*What is the structure of the PA200 activator?
*Where does PA200 bind to the 20S proteasome?
*Does PA200 binding induce structural changes in the 20S proteasome that correlate with its activation?
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Electron Microscopy of the PA200-20S Proteasome Complex
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Data collection and image processing for the cryo-images
*CM200 FEG 120 kV Magnification x 38000 21,091 particles
*Software: -Bsoft-EMAN
*Starting models: -Atomic structure of 20S -Featureless barrel
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3D Reconstruction of the Singly Bound PA200-20S Proteasome Complex.
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Visualization of the Axial Channel Gate Upon Binding of PA200
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Questions Revisited:Questions Revisited:*What is the structure of the PA200 activator?
PA200 is a highly asymmetric molecule organized in HEAT-like repeats, probably as -solenoid.
*Where does PA200 bind to the 20S proteasome?
A monomer of PA200 binds to one or both ends of 20S.
*Does PA200 binding induce structural changes in the 20S proteasome that correlate with its activation?
PA200 opens the axial channel in the proximal -ring of the 20S particle.
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Jack IwanczykFaisal UddinDaniela Damjanovic
LSBR. NIAMS. NIH, MD, USA Alasdair C. StevenBernard Heymann
University of Utah, UT, USAMartin Rechsteiner
McMaster University, Hamilton, ON, Canada
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Orientation of the 20S proteasome in the PA200-20S complex
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Side Views Fill 3D SpaceSide Views Fill 3D Space
Side & Top Views Side Views Only