BMB170c_2011_03_29_LECTURE

BMB170c_2011_03_29_LECTURE - BMB 170c Lecture 1 Regulated...

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Unformatted text preview: BMB 170c Lecture 1 Regulated Proteolysis 1 March 29, 2011 Welcome to BMB/Bi/Ch 170c Macromolecular Machines Tuesdays and Thursdays 10:30 am – 12:00 pm 151 Crellin Coordinator: Shu-ou Shan x3879 sshan@caltech.edu TAs: Fay Bi, Aileen Ariosa, David Akopian Course Philosophy • Discuss interesting molecular machines in biology • Reinforce the principles and techniques learned in BMB 170a & b • Develop critical thinking, reading and presentation skills Topics • Regulated Proteolysis – Shan • Chaperones – Clemons • Virus / HIV – Jensen • Nuclear Pore Complex – Hoelz • Channel – Rees For each topic there will be • One principal and invited guest lecturers • Lecture notes downloadable from the website • Assigned readings (3–5 research articles), links provided on the website • Problem sets based on the lectures and readings, downloadable from the website For each topic there will be • A discussion session (journal club format) during the last lecture on the topic - 2–3 student presentations of select research articles, 30 minutes each (20–25 min presentation + Q & A). - Student participation and discussion is critical - Students who did the oral presentations are exempt from the homework on the topic. The Final • An original, out of field research proposal on topics of your interest/choice. • Students will form ‘Review groups’ to evaluate and discuss each other’s proposal at study sessions. Two reviewers / proposal. • Pre-proposal due May 19. • Detailed guidelines on the proposal and evaluation will be posted as we get closer to the finals dates. Literature discussion signup • Regulated proteolysis • Chaperones • Virus / HIV • Nuclear Pore Complex • Channels Cellular Protein Homeostasis folding transport regulated degradation interactions ‘The dynamic state of body constituents’ The new results imply that not only the fuel, but the structural materials [of the cell] are in a steady state of flux. The classical picture [that body structural proteins are stable and static] must thus be replaced by one which takes into account of the dynamic state of body structure. Schoenheimer, Edward K. Durham lecture, 1941 Importance of Regulated Proteolysis • Maintaining protein homeostasis while cellular structures are continually rebuilt • Handle aggregation of misfolded proteins • Provide a means to terminate the lifespan of regulatory proteins at distinct times examples: cyclins, transcription factors, signal transduction pathways • Partial processing to generate biologically active protein fragments (e.g, Ci, NF-κB) • Facilitate maturation of immune system Compartmentalization is a basic strategy in controlling proteolysis • Prevent the destruction of proteins not destined to degradation • Confine proteolysis to sites that can only be accessed by proteins displaying degradation signal Lysosome - confine proteolytic enzymes in a membrane compartment - Proteins to be degraded must be imported The lysosome hypothesis was challenged • Half-lives of different proteins vary by 103-fold and change with changing physiological conditions • Lysosome inhibitors prevented degradation of extracellular proteins but has almost no effect on degradation of most intracellular proteins • Protein degradation requires ATP Discovery of a new proteolytic system • Reconstitution of a two-component, ATP-dependent proteolytic system from rabbit reticulocyte extract [Ciechanover, Yod, Hershko, BBRC 81: 1100 (1978)] • Ubiquitination shown to be a key step for targeting proteins for proteolytic degradation [Ciechanover et al, PNAS 77: 1365 (1980); Hershko et al, PNAS 77: 1783 (1980)] • Isolation of enzymes involved in the ubiquitin conjugation cascade [Ciechanover et al, JBC 257: 2537 (1982); Hershko et al, JBC 258: 8206 (1983)] • Discovery and reconstitution of 26S proteasome [ Hough et al, JBC 261: 2400 (1986); ibid 262: 8303 (1987); Waxman et al, JBC 262: 2451 (1987); Hoffman et al, JBC 267: 22362 (1992)] Self-Compartmentalizing Proteases Core particle: enclosed proteolytic compartment Regulatory particle - recognizes proteolytic tags - controls access to CP - unfolds target protein (requires ATP) - translocates protein to CP (requires ATP) Self-compartmentalizing Proteases are Evolutionarily conserved Core Particle Regulatory Particle Eukaryotes 20S 19S Archaea 20S PAN Prokaryotes HslU HslV ClpP ClpX & ClpA The 20S Core Proteolytic Particle Composition of 20S Proteolytic Particle • Archaea: two types of subunits α - catalytically inactive β - proteolytically active 14 copies of each subunits • Eukaryotes: 2 copies each of 7 different α- and β-subunits Eukaryotic proteasome subunits Architecture of 20S particle 110 Å - cylinder shaped barrel - two outer disks and two inner rings 150 Å - tripartite inner compartments - immuno-EM shows that α subunits constitute the outer rings and β-subunits constitute the inner rings Grziwa et al, FEBS lett 290: 186 (1991); Pühler et al, EMBO J. 11: 1607 (1992) Rings have seven-fold symmetry α and β rings are not completely in register Pühler et al, EMBO J. 11: 1607 (1992) Crude model from EM analysis Pühler et al, EMBO J. 11: 1607 (1992) Crysal structure of Thermoplasma 20S proteasome particle • Barrel shaped structure • α-subunits form 7-membered outer rings • β-subunits form 7-membered inner rings • together forms 3 internal cavities ~ 50 Å in diameter Lowe et al, Science 268: 533 (1995) General Fold of β-subunit β-sandwich structure typical of Ntn-hydrolases Lowe et al, Science 268: 533 (1995) α- and β-subunits have the same general fold N-terminal extention α-helices β-strands loops Lowe et al, Science 268: 533 (1995) Tight packing of α- and β-rings Particle is not penetrable through the walls Lowe et al, Science 268: 533 (1995) Yeast 20S particle has the same architecture Groll et al, Nature 386: 463 (2000) A constricted gate into the Thermoplasma 20S • Turn-forming loops with conserved Tyr126 (•) define port of entry (13 Å) Lowe et al, Science 268: 533 (1995) Proteolysis by 20S requires completely unfolded polypeptides Oxidized somatostatin cannot be degraded by 20S Wenzel & Baumeister, NSB 2: 199 (1995) Proteolysis by 20S requires completely unfolded polypeptides Reduced somatostatin is completely digested by 20S Wenzel & Baumeister, NSB 2: 199 (1995) ‘Peptide with a knot’ identifies the site of substrate entry nanogold-labeled insulin B-chain trapped during transit Wenzel & Baumeister, NSB 2: 199 (1995) Proteolytic chamber of yeast 20S is occluded by the N-terminus of α1–α6 Free 20S exists in an auto-inhibited state Groll et al, Nature 386: 463 (2000); Whitby et al, Nature 408: 115 (2000) α3 N-terminal deletion opens a gate into the 20S particle WT α3NΔ Groll et al, Nature 386: 463 (2000) Activation of 20S particle by α3ΔN α3ΔN mutation activates 20S for digestion of a variety of peptide substrates Groll et al, Nature 386: 463 (2000) Narrow constrictions and hydrophobic nature keep polypeptides in extended/unfolded form hydrophobic Peptidase Activity of the Core Particle Deletion of N-terminal Thr yields inactive proteasome N-terminal thr is the nucleophile? Seemüller et al, Science 268: 579 (1995) A peptide aldehyde inhibitor (acLLnL) is bound to all 14 β-subunits of Thermoplasma 20S Lowe et al, Science 268: 533 (1995) LLnL is bound close to Thr1 and may form a hemiacetal with Thr-OH Lowe et al, Science 268: 533 (1995) Groll et al, Nature 386: 463 (1997) Covalent Catalysis using Thr1 at proteasome active site O OH N H R H2O O CH2 Thr1 CH3 O- R OH CH CH CH3 H2N O R' R O R' CH CH2 Thr1 CH3 CH2 Thr1 Which residue provides the general base? O OH N H :B R O H2O OH +HB CH CH2 Thr1 O- R CH CH3 H2N O R' R O R' CH3 :B CH CH2 Thr1 CH3 CH2 Thr1 Which residue provides the general base? Model 1. Lys33 Mutation of Lys33 disrupts proteolytic activity Groll et al, Nature 386: 463 (1997) Which residue provides the general base? Model 2. N-terminal -NH2 O O R' R' R OH CH3 CH NH2 H2C R N H CH OH OR N H H O H CH3 CH NH2 H2C CH - Consistent with proteasome activation by autoprocessing - N-terminal amino group is a more effective base than Lys Only three of the 7 β-subunits in eukaryotic proteasome are proteolytically active Groll et al, Nature 386: 463 (1997) Only three of the 7 β-subunits in eukaryotic proteasome are proteolytically active • β-subunits are synthesized as inactive proforms • pro-sequences are processed between Gly(-1) and Thr1 during proteasome assembly and maturation Seemüller et al, Science 268: 579 (1995) Only three of the 7 β-subunits in eukaryotic proteasome are proteolytically active • Only β1, β2 and β5 have the full set of essential catalytic residues (Lys33, Asp17) and their pro-peptides are processed to liberate Thr1 • In contrast, β3 – β4 are not processed, and β6 – β7 are partially processed Seemüller et al, Science 268: 579 (1995) Specificity of Cleavage: P1 Recognition or Not? Eukaryotic proteasomes have three major types of activities • Chymotrypsin-like: Gly-Gly-Leu-pNA • Trypsin-like: Ala-Gly-Arg-pNA • Peptidyglutamyl peptidase: Leu-Leu-Glu-pNA Do these activities arise from one active site with broad specificity, or from several different active sites? Hypothesis: Residue 45 determines the specificity of each active site β1: polyglutamyl-peptidase β2: Chymotrypsin-like? Groll et al, Nature 386: 463 (1997) Hypothesis: Residue 45 determines the specificity of each active site β5: can be both chymotrypsin or trypsin-like Nevertheless, all sites are adaptable in size and polarity Groll et al, Nature 386: 463 (1997) Mutation of Thr1 at specific β subunits disrupt distinct proteolytic activities • PUP1 (β2) is primarily responsible for trypsin activity • PRE3 (β1) is primarily responsible for PGPH activity Arendt & Hochstrasser, PNAS 94: 7156 (1997) Analysis of cleavage patterns in Enolase Mutation of β5 did not abolish Chymotrypsin-like activity => Overlap in cleavage preference of different active sites Nussbaum et al, PNAS 95: 12504 (1998) Bottom line • The ancestral core particle is a non-specific protease, the active sites evolved to become more specific • Individual active sites have some degree of specificity but are also highly adaptable • Unfolded substrates and high concentration (50 mM) of active sites within a sequestered space ensure cleavage of all peptide bonds Size of Proteasome Digestion Products The proteasome generates peptide products ~ 7aa long Wenzel et al, FEBS lett. 349: 205 (1994) The Molecular Ruler Model Wenzel et al, FEBS lett. 349: 205 (1994) Lowe et al, Science 268: 533 (1995) However… • Eukaryotic and archaeal proteasomes generate products of similar size despite different number of active sites • Reducing the number of active sites did not change the size of proteolysis products Nussbaum et al, PNAS 95: 12504 (1998) Opening of Gate increases the mean size of peptide products from Proteasome WT α3NΔ Proteolysis continues until products are small enough to diffuse out of the proteasome ...
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This note was uploaded on 01/03/2012 for the course BI 170c taught by Professor List during the Fall '09 term at Caltech.

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