Introduction Session 1 and 2 (Ubiquitin, proteasome and human disease) WHAT S“ UBIQUITIN”? Main role: become a " label to target a substrate protein Protein Substrate Source: "Peptide models for protein beta-sheets PhD thesis, University of Nottingham, 2001 Figure by MIT OCW. Appendage of ubiquitin monomers to a fter Goodsell, D.S. The Oncologist 8, 293-294 protein substrate Ubiquitin is a small, 76 aa protein which gets appended to another proteins, as a label". The protein substrate has amino groups in the side chains of its Lys residues Ubiquitin has a C-terminal Gly G The carboxyl group of this Gly forms an isopeptide bond with the amino group of Hn the Lys in the protein substrate(see Inure Ubiquitin also has several lys that can Protein substrate act as internal acceptors for binding to the C-t Gly of new ubiquitin molecules, allowing the formation of a chain
Introduction Session 1 and 2 (Ubiquitin, proteasome and human disease) Appendage of ub prote Figure by MIT OCW. After Goodsell, D.S. The Oncologist 8, 293-294. Courtesy of Sam Griffiths-Jones. Used with permission. Source: "Peptide models for protein beta-sheets." PhD thesis, University of Nottingham, 2001. iquitin monomers to a in substrate. Ubiquitin is a small, 76 aa protein which gets appended to another proteins, as a “label”. The protein substrate has amino groups in the side chains of its Lys aa residues. Ubiquitin has a C-terminal Gly. The carboxyl group of this Gly forms an isopeptide bond with the amino group of the Lys in the protein substrate (see figure). Ubiquitin also has several Lys that can act as internal acceptors for binding to the C-t Gly of new ubiquitin molecules, allowing the formation of a chain
DISCOVERy OF THE ROLE OF UBIQUITIN IN PROTEIN DEGRADATION HISTORICAL FACTS Courtesy of Sam Griffiths-Jones. Used with permission Source: "Peptide models for protein beta-sheets hD thesis, University of Nottingham, 2001 8(1975)Ubiquitin was first isolated by Gideon Goldstein and colleagues from the thymus(reason why it was originally thought to be a thymic hormone) .o But because it was later found in all tissues and eukaryotic organisms it received the name of UBIQUITIN (for ' ubiquitous protein) .( 1977) Harris Goldknopf and Ira Busch found a DNA-associated protein that had one C-t but two N-t The short arm of this y shaped unusual protein was joined through its c-terminal to the 8- amino group of an internal lys of the histone h2A g Margaret Dayhoff soon identified it as Ubiquitin (a protein initially described as free by Goldstein) 3(1969-1971)Avram Hershko studies regulation of tyrosine aminotransferase by its degradation > he found that degradation of the enzyme was arrested by inhibitors of cellular energy production (fluoride, azide) That was the first indication that an as-yet-unknown energy dependent proteolytic system must exist 8(1971-1980sHershko decided to identify this energy-dependent system responsible for the degradation of proteins, by means of classi cal biochemistry. His aims were to reproduce the ATP-dependent protein breakdown in a cell-free system to fractionate such system to find the mode of action of its components
DISCOVERY OF THE ROLE OF UBIQUITIN IN PROTEIN DEGRADATION. HISTORICAL FACTS. Courtesy of Sam Griffiths-Jones. Used with permission. Source: "Peptide models for protein beta-sheets." PhD thesis, University of Nottingham, 2001. (1975) Ubiquitin was first isolated by Gideon Goldstein and colleagues from the thymus (reason why it was originally thought to be a thymic hormone). But because it was later found in all tissues and eukaryotic organisms it received the name of UBIQUITIN (for ‘ubiquitous’ protein). (1977) Harris Goldknopf and Ira Busch found a DNA-associated protein that had one C-t but two N-t! The short arm of this Yshaped unusual protein was joined through its C-terminal to the ε- amino group of an internal Lys of the histone H2A. Margaret Dayhoff soon identified it as Ubiquitin (a protein initially described as free by Goldstein). (1969-1971) Avram Hershko studies regulation of tyrosine aminotransferase by its degradation Æ he found that degradation of the enzyme was arrested by inhibitors of cellular energy production (fluoride, azide) That was the first indication that an as-yet-unknown energydependent proteolytic system must exist. (1971-1980’s) Hershko decided to identify this energy-dependent system responsible for the degradation of proteins, by means of classi cal biochemistry. His aims were: - to reproduce the ATP-dependent protein breakdown in a cell-free system. - to fractionate such system to find the mode of action of its components
.( 1977) Etlinger and Goldberg discovered an ATP-dependent proteolytic system occurring in reticulocytes(red blood cells) Therefore, Hershko, helped by his grad student Aron ciechanover and Rose( Fox Chase cancer Center, Philadelphia) decided to isolate the ATP dependent proteolytic system from these cells Reticulocyte lysates were fractionated on DEAE-cellulose anionIC exchange chromatography) into two crude fractions Fxni(not adsorbed), with most of the hemoglobin Fxn2, proteins adsorbed to the resin and eluted with high salt Fxn2 had lost most of the A TP-dependent proteolytic activity and only was restored when combining back Fxnl with 2. The active component in Fxn1 was isolated in basis of its high stability upon heat treatment: APF-1, for ATP-dependent proteolysis factor 1 possible activator or regulatory subunit of other component/s of the system?? .Radiolabeling APF-1 they saw substantial ATP-dependent binding to high-molecular weight proteins (gel filtration chromatography) Interaction extremely stable in the presence of conditions that disrupt non-covalent interactions: interaction should be covalent (peptidic or amidic linkage) APF-1 interacting not with an active component of the proteolytic system but with protein substrates??
(1977) Etlinger and Goldberg discovered an ATP-dependent proteolytic system occurring in reticulocytes (red blood cells). Therefore, Hershko, helped by his grad student Aron ciechanover and Irwin Rose (Fox Chase cancer Center, Philadelphia) decided to isolate the ATPdependent proteolytic system from these cells. • Reticulocyte lysates were fractionated on DEAE-cellulose (anionic exchange chromatography) into two crude fractions: Æ Fxn1(not adsorbed), with most of the hemoglobin . Æ Fxn2, proteins adsorbed to the resin and eluted with high salt. • Fxn2 had lost most of the ATP-dependent proteolytic activity and only was restored when combining back Fxn1 with 2. •The active component in Fxn1 was isolated in basis of its high stability upon heat treatment: APF-1, for ATP-dependent proteolysis factor 1. …possible activator or regulatory subunit of other component/s of the system?? •Radiolabeling APF-1 they saw substantial ATP-dependent binding to high-molecular weight proteins (gel filtration chromatography). • Interaction extremely stable in the presence of conditions that disrupt non- covalent interactions: interaction should be covalent (peptidic or amidic linkage). …APF-1 interacting not with an active component of the proteolytic system but with protein substrates??
By using a good substrate for ATP-dependent proteolysis, lysozyme, they found 1) that similar high-molecular weight derivatives were for med when I-labeled APF-1 was incubated with unlabeled lysozyme than whenI-labeled lysozyme was incubated with unlabeled APF-1. 2)analysis of the ratio of radiactivity in APF-1 and lysozyme dicated that the various derivatives consisted of increasing numbers f APF-1 molecules linked to one molecule of lysozyme 1234567 (Hershko et al, 1980 C C Courtesy of A Hershko. Used with permission of ne author Source: Figure 1 in Hershko et al. " Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis. PNAS 1980 April 77(4):1783-1786
• By using a good substrate for ATP-dependent proteolysis, lysozyme, they found: 1) that similar high-molecular weight derivatives were for med when 125 I-labeled APF-1 was incubated with unlabeled lysozyme than when 125 I-labeled lysozyme was incubated with unlabeled APF-1. 2) analysis of the ratio of radiactivity in APF-1 and lysozyme indicated that the various derivatives consisted of increasing numbers of APF-1 molecules linked to one molecule of lysozyme. (Hershko et al., 1980) Courtesy of A. Hershko. Used with permission of the author. Source: Figure 1 in Hershko et al. "Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis." PNAS 1980 April; 77(4): 1783–1786
(Model of action proposed by Hershkoet al, 1980) 2 Protein K (APF)n Protein n APF n ATP n APF.x Amino acids Courtesy of A Hershko. Used with permission of the author. Source: Figure 6 in Hershko et al. "Proposed role of ATP in protein breakdown conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis PNAS1980Apml:77(4):1783-1786 Several molecules of APF-1 linked to E-amino groups of the protein substrate by an APF-1-protein amide synthetase (step1) Proteins ligated to several APF-1 are broken down by a specific protease that recognizes such conjugates(step 3). The protein is broken down to free amino acids and to APF-1 still linked by isopeptide linkage to a lysine or a small peptide, APF-1-X. Finally, free APF-1 would be released for re-use by specific amidase-isopeptidase (step 4). a hypothetical correcting isopeptidase would release free APF-1 and substrate protein from erroneous ligations(editing activity: step 2) g Short after Hershko's model proposal, Keith Wilkinson and Arthur Haas(pot-doctoral fellows in Irwin Rose's lab)indicated that APF-1 was indeed UBIQUITIN
(Model of action proposed by Hershkoet al., 1980) Courtesy of A. Hershko. Used with permission of the author. Source: Figure 6 in Hershko et al. "Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis." PNAS 1980 April; 77(4): 1783–1786. Several molecules of APF-1 linked to ε-amino groups of the protein substrate by an APF-1-protein amide synthetase (step1). Proteins ligated to several APF-1 are broken down by a specific protease that recognizes such conjugates (step 3). The protein is broken down to free amino acids and to APF-1 still linked by isopeptide linkage to a lysine or a small peptide, APF-1-X. Finally, free APF-1 would be released for re-use by specific amidase-isopeptidase (step 4). A hypothetical ‘correcting’ isopeptidase would release free APF-1 and substrate protein from erroneous ligations (editing activity; step 2). Short after Hershko’s model proposal, Keith Wilkinson and Arthur Haas (pot-doctoral fellows in Irwin Rose’s lab) indicated that APF-1 was indeed UBIQUITIN