Protein Degradation Biochem 4511 Spring 2014 Figures Essentials of Biochemistry 3rd Ed OSU Custom Edition Principles of Biochemistry 5th Ed Moran et al Pubmed World Wide Web Protein turnover selective degradation Proteins are constantly degraded and synthesized Individual cellular proteins turn over degraded and re synthesized at different rates Half lives of selected enzymes of rat liver cells range from 0 2 to 150 hours Improperly folded proteins are recognized and targeted for degradation Protein aggregates are also targeted for degradation N end rule On average a protein s half life correlates with its N terminal residue Proteins with N terminal Met Ser Ala Thr Val or Gly have half lives greater than 30 hours Proteins with N terminal Phe Leu Asp Lys or Arg charged or large hydrophobic have half lives of 3 min or less Protein Degradation Ubiquitin dependent Proteasome Pathway 80 90 Most intracellular proteins Lysosomal processes via autophagy 10 20 Extracellular proteins Cell organelles Some intracellular proteins Protein aggregates Lysosomal degradation Digestive System of the Cell Digests ingested materials obsolete cell components Degrades macromolecules of all types Proteins Nucleic acids Carbohydrates Lipids Lysosomal Enzymes 50 different degradative enzymes Acid hydrolases Active at pH 5 inside lysosome Inactive if released into cytosol pH 7 2 Acidic pH of lysosomes maintained by a proton pump in the lysosomal membrane Requires ATP Different pathways lead to the lysosome 1 Phagocytosis Cell eating of material 250nm 2 Pinocytosis Cell drinking 150nm 3 Receptor Mediated Endocytosis clathrin coated pits 4 Autophagy self eat of old worn out organelles important in cell degradation during apoptosis Important under starvation and to recycle entire organelles aggregates Proteasomal degradation UBIQUITIN Small protein 76 amino acids ubiquitin PDB 1TBE G H K C terminal glycine isopeptide bond with the amino group of lysine residues on the substrate Attached as monoubiquitin or polyubiquitin chains Protein destined for degradation Polyubiquitination Proteins destined to be degraded are poly ubiquitinated K G All ubiquitins are attached to each other via iso peptide bonds Polyubiquitinated proteins are recognized and degraded by 26S proteasome Protein Degradation by the Proteasome Full size 26S proteasome cleaves only poly ubiquitinated or very small proteins in ATP dependent manner ATP is a source of energy Energy of ATP hydrolysis is used to unfold poly U targeted proteins 2014 John Wiley Sons Inc All rights reserved ATP hydrolysis provides energy for thermodynamically unfavorable reactions Products are more stable than the substrates i e G Gprod Gsub 0 The proteasome the proteasome is a cylindrical complex consisting of four stacked seven membered rings the two outer rings are alpha subunits inactive the two inner rings are beta subunits these are proteolytically active eukaryotic proteasome seven types of alpha subunits seven types of beta subunits regulatory cap at least 17 subunits core particle 20S regulatory particle 19S 19 2 20S 26S ATP dependent protease mechanisms 19S 26S 20S 8 9 a a long peptides Proteolytic activities of proteasome ACTIVE SITES SHOWN WITH CIRCLES ARE ON THREE SEPARATE BETA SUBUNITS THREONINE RESIDUES ARE CRITICAL DURING CATALYSIS hydrophobic a a CHYMOTRYPSIN LIKE 5 After Tyr Trp Phe acidic a a CASPASE LIKE 1 After Glu 20S basic a a TRYPSIN LIKE 2 after Arg Lys 26S vs 20S proteasome 26S Recognises poly U De ubiquitylation Unfolding Required ATP Translocation Degradation 20S Only good for extreme conditions Recognition of unfolded sequences Degradation Does not require ATP energy Under stressed conditions there are more unfolded proteins such as high temperature There are naturally disordered proteins that will be destroyed by Stress regulated 20S 26S equilibrium Stress Name of chaperone Heat shock proteins e g HSP70 HSP70 Norm Low level of unfolded proteins Only poly U proteins are degraded ATP is required to unfold poly U proteins Stress High level of unfolded proteins All unfolded disordered proteins are degraded ATP is not required Regulation of 20S by NQO1 Partially disordered Folded Under normal conditions Reducing potential is high FAD content is high NQO1 is stabilized 20S is inhibited by NQO1 disordered proteins e g p53 are not cleaved by 20S Under oxidative stress Unfolded protein content high Reducing potenital is low FAD content is low NQO1 is not stabilized by FAD NQO1 is degraded 20S is active unfolded and disordered proteins are degraded Summary Proteins are constantly degraded and synthesized Individual cellular proteins turn over degraded and resynthesized at different rates N rule Improperly folded proteins are recognized and targeted for degradation in proteasome Large protein aggregates are targeted for degradation in lysosomes Autophagy Proteasome switches from specific S26 ATP dependent to non specific 20S ATP independent modes of degradation Shock oxidative metabolic heat promotes non specific degradation of unfolded proteins This saves energy and promotes cell survival under critical conditions but also destines many usefull IDP e g p53 to be degraded