Cellular Protein Homeostasis: Folding, Interactions, Degradation, Slides of Biochemistry

Download Cellular Protein Homeostasis: Folding, Interactions, Degradation and more Slides Biochemistry in PDF only on Docsity! Cellular Protein Homeostasis regulated clding | degradation “~~ a —_—_> > SF c= transport Lf interactions = C \ » A’ docsity.com 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 docsity.com - confine proteolytic enzymes in a membrane compartment - Proteins to be degraded must be imported Lysosome docsity.com The lysosome hypothesis was challenged •  Lysosome inhibitors prevented degradation of extracellular proteins but has almost no effect on degradation of most intracellular proteins • Protein degradation requires ATP • Half-lives of different proteins vary by 103-fold and change with changing physiological conditions docsity.com 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)] docsity.com The 20S Core Proteolytic Particle docsity.com •  Archaea: two types of subunits α - catalytically inactive β - proteolytically active 14 copies of each subunits Composition of 20S Proteolytic Particle •  Eukaryotes: 2 copies each of 7 different α- and β-subunits docsity.com Eukaryotic proteasome subunits New systematic Traditional name H. sapiens name (S. cerevisiae) al_sc docsity.com Crude model from EM analysis Pühler et al, EMBO J. 11: 1607 (1992) docsity.com Crysal structure of Thermoplasma 20S proteasome particle • α-subunits form 7-membered outer rings • Barrel shaped structure • β-subunits form 7-membered inner rings • together forms 3 internal cavities ~ 50 Å in diameter Lowe et al, Science 268: 533 (1995) docsity.com General Fold of β-subunit β-sandwich structure typical of Ntn-hydrolases Lowe et al, Science 268: 533 (1995) docsity.com Yeast 20S particle has the same architecture Groll et al, Nature 386: 463 (2000) docsity.com A constricted gate into the Thermoplasma 20S Lowe et al, Science 268: 533 (1995) • Turn-forming loops with conserved Tyr126 (•) define port of entry (13 Å) docsity.com Wenzel & Baumeister, NSB 2: 199 (1995) Proteolysis by 20S requires completely unfolded polypeptides Oxidized somatostatin cannot be degraded by 20S docsity.com Proteolytic chamber of yeast 20S is occluded by the N-terminus of α1–α6 Groll et al, Nature 386: 463 (2000); Whitby et al, Nature 408: 115 (2000) Free 20S exists in an auto-inhibited state docsity.com α3 N-terminal deletion opens a gate into the 20S particle Groll et al, Nature 386: 463 (2000) WT α3NΔ docsity.com Activation of 20S particle by α3ΔN Groll et al, Nature 386: 463 (2000) α3ΔN mutation activates 20S for digestion of a variety of peptide substrates docsity.com Seem üller et al, Science 268: 579 (1995) N-terminal thr is the nucleophile? Deletion of N-terminal Thr yields inactive proteasome docsity.com A peptide aldehyde inhibitor (acLLnL) is bound to all 14 β-subunits of Thermoplasma 20S Lowe et al, Science 268: 533 (1995) docsity.com 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) docsity.com Which residue provides the general base? Model 1. Lys33 Groll et al, Nature 386: 463 (1997) Mutation of Lys33 disrupts proteolytic activity docsity.com Which residue provides the general base? Model 2. N-terminal -NH2 - Consistent with proteasome activation by autoprocessing - N-terminal amino group is a more effective base than Lys R N H R' O CH3 CH OH H2C CH NH2 R N H R' O CH3 CH OH H2C CH NH2 H O HOR docsity.com Groll et al, Nature 386: 463 (1997) Only three of the 7 β-subunits in eukaryotic proteasome are proteolytically active docsity.com Specificity of Cleavage: P1 Recognition or Not? docsity.com 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? docsity.com Hypothesis: Residue 45 determines the specificity of each active site β1: polyglutamyl-peptidase β2: Chymotrypsin-like? Groll et al, Nature 386: 463 (1997) docsity.com 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) docsity.com 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 docsity.com Size of Proteasome Digestion Products docsity.com 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) docsity.com Opening of Gate increases the mean size of peptide products from Proteasome Proteolysis continues until products are small enough to diffuse out of the proteasome WT α3NΔ docsity.com