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
Ubiquitin proteolytic pathway ATP AMP+PPi 2 Ub-C-OH El-S-C-Ub Protein E1-SH E2-SH (Ub)n ADP+PI ATP 3VE n Ub Ub-peptides Protein(Ub)n 26S Proteasome Peptides 8 Amino acids Courtesy of Annual Reviews. Used with permission Source: Figure 1A in Hershko A, Ciechanover A. The ubiquitin system Annu Rev biochem. 1998: 67: 425-79 A: Conjugation of ubiquitin to the target substrate E1: Ub-activating enzyme E2: Ub-conjugating enzyme: Ubc's Image removed due to copyright considerations E3: Ub-ligase See Figure 1 in Ciechanover A, Orian A, Schwartz AL destruction. " Bioessays. 2000 May: 22(5): 442-51 B: Degradation of the polyubiquitinated substrate by the 26s proteasome complex and Ub recycling by deubiquitinating enzymes(Ub C-terminal hydrolases dUBs or UCHs)
Ubiquitin proteolytic pathway Courtesy of Annual Reviews. Used with permission. Source: Figure 1A in Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425-79. A: Conjugation of ubiquitin to the target substrate E1: Ub-activating enzyme E2: Ub-conjugating enzyme; Ubc’s Image removed due to copyright considerations. E3: Ub-ligase See Figure 1 in Ciechanover A, Orian A, Schwartz AL. "Ubiquitin-mediated proteolysis: biological regulation via destruction." Bioessays. 2000 May; 22 (5): 442-51. B: Degradation of the polyubiquitinated substrate by the 26S proteasome complex and Ub recycling by deubiquitinating enzymes (Ub C-terminal hydrolases, DUBs or UCH’s)
.(1980s)Hershko and Ciechanover purified the components of the first part of the reaction (APF-1-protein amide synthetase): E1, E2 and enzymes, by covalent'affinity chromatography over immobilized Ub s The protease step was further clarified with the characterization purification of the 26 proteasome complex, by martin Rechsteiner and colleagues(1986) .o It was first thought that multiple ubiquitin attached to different Lys sites in the substrate protein but Hershko( 1985)and then A xander Varshavsky and colleagues(1989)demonstrated that a single poly- ubiquitin chain could be form through attachment to a single internal Lys residue Attachment of subsequent Ub to the first one occurs through linkages of the Gly c-t of the next Ub to specific lysines present in the precedent Ub Poly-ubiquitin chains might have different roles dependin on the lys utilized K48> recognition by proteasome K63> signal for traffic and degradation in the lysosome, vacuole; subunit selectivity K29. K6. etc First evidence showing that ubiquitin constitutes a signal for protein degradation in vivo Hershko lab: 1982): immunochemical analysis of Ub adducts in cells> by using anti-Ub abs they saw that after incubation of cells in the presence of aa analogs the resulting abnormal proteins were short-lived and their rapid degradation was accompanied y a transient increase in Ub adducts But the strongest confirmation came with alexander varshavsky' s work
(1980’s) Hershko and Ciechanover purified the components of the first part of the reaction (APF-1-protein amide synthetase): E1, E2 and E3 enzymes, by ‘covalent’ affinity chromatography over immobilized Ub. The ‘protease’ step was further clarified with the characterization and purification of the 26 proteasome complex, by Martin Rechsteiner and colleagues (1986). It was first thought that multiple ubiquitin attached to different Lys sites in the substrate protein but Hershko (1985) and then Alexander Varshavsky and colleagues (1989) demonstrated that a single poly-- ubiquitin chain could be form through attachment to a single internal Lys residue. Attachment of subsequent Ub to the first one occurs through linkages of the Gly C-t of the next Ub to specific lysines present in the precedent Ub. Poly-ubiquitin chains might have different roles depending on the Lys utilized: K48 Æ recognition by proteasome K63 Æ signal for traffic and degradation in the lysosome, vacuole; subunit selectivity K29, K6, etc.. First evidence showing that ubiquitin constitutes a signal for protein degradation in vivo (Hershko lab; 1982): immunochemical analysis of Ub adducts in cells Æ by using anti-Ub Abs they saw that, after incubation of cells in the presence of aa analogs the resulting abnormal proteins were short-lived and their rapid degradation was accompanied by a transient increase in Ub adducts. But the strongest confirmation came with Alexander Varshavsky’s work
UBIQUTTIN CONJUGATION REQUIRED FOR PROTEIN DEGRADATION IN VIVO (first evidence through the studies carried out by Alexander varshavsky, Dan Finley and aaron Ciechanover) varshavsky studied chromosome structure and requlation of gene expression and came to boston in the fall of 1977 where a month later became a faculty member in the Biology Department here at MIt. .o He became interested in the role of protein degradation in gene regulation and expression at the light of hershko's and wilkinsons experiments (showing that the ATP-dependent proteolytic factor 1,was ubiquitin) g Considering that he already knew that Ub had been found attached to histones, he became very excited when in 1980 he read a paper, by yamada and coll. that described a conditionally lethal, temperature sensitive mouse cell line called ts85 (they had seen that in this cell line at restrictive temperature a nuclear protein disappeared and varshavsky confirmed it was UbH2A) ADan Finley, from his lab, and A. Ciechanover helped him to study ts85 and saw that cells were arrested in $/G2 phase of cell division cycle and had induced synthesis of heat shock proteins at non permi ssive temperature They found that ts85 mouse cells had a temperature-sensitive Ub-activating enzyme(E1)and that these cells stopped degrading the bulk of their normally short-lived proteins at the nonpermissive temperature g Later studies(by Hunt and coll. ) with rapidly dividing fertilized clam eggs, led to the discovery of cyCLINS and in 1984 Varshavsky proposed that cyclins were degraded at the exit from mitosis by the ubiquitin system
UBIQUITIN CONJUGATION REQUIRED FOR PROTEIN DEGRADATION IN VIVO (first evidence through the studies carried out by Alexander Varshavsky, Dan Finley and Aaron Ciechanover) Varshavsky studied chromosome structure and regulation of gene expression and came to Boston in the fall of 1977 where, a month later, became a faculty member in the Biology Department here at MIT. He became interested in the role of protein degradation in gene regulation and expression at the light of Hershko’s and Wilkinson’s experiments (showing that the ATP-dependent proteolytic factor 1, was ubiquitin). Considering that he already knew that Ub had been found attached to histones, he became very excited when in 1980 he read a paper, by Yamada and coll. that described a conditionally lethal, temperature sensitive mouse cell line called ts85 (they had seen that in this cell line at restrictive temperature a nuclear protein disappeared and Varshavsky confirmed it was UbH2A). ÆDan Finley, from his lab, and A. Ciechanover helped him to study ts85 and saw that cells were arrested in S/G2 phase of cell division cycle and had induced synthesis of heat shock proteins at non permi ssive temperature. They found that ts85 mouse cells had a temperature-sensitive Ub-activating enzyme (E1) and that these cells stopped degrading the bulk of their normally short-lived proteins at the nonpermissive temperature. Later studies (by Hunt and coll.), with rapidly dividing fertilized clam eggs, led to the discovery of CYCLINS and in 1984 Varshavsky proposed that cyclins were degraded at the exit from mitosis by the ubiquitin system
Ubiquitination is an essential regulatory process that occurs at multiple levels and in all mammalian cellular pathways .o selective protein degradation modulation of transcription, translation and protein localization For example in the regulation of apoptosis in the regulation of transcription Survival (Radiation) Glucocorticoids Receptor Receptor Ubiquitination Image removed for copyright considerations. Source: Figure 5 in Muratani M, W. P. Tansey How the Ubiquitin-proteasome System Controls Transcription. "Nat Rev Mol Cell Biol no.3(Mar2003):192-201 Ubiquitination Diablo Cytochrome c Ubiquitination biquitination Caspase-Dependent Apoptosis Pathway Figure by MIT OCW. After Yang and Xu, "Regulation of apoptosis the ubiquitous way. FASEB J17(2003)790-799 E3 ubiquitin ligases ( many) Proteasome El activating enzyme (one?) (several) Zyme Ub E1-Ub Ub thiol ester thiol esters Ubiquitinated Proteins Figure by MIT Oc
Stress Death Survival (Radiation) Glucocorticoids Receptor Receptor Ubiquitination Mdm2 Receptor Ubiquitination cFlip Ubiquitination Image removed for copyright considerations. p53 Source: Figure 5 in Muratani M, W. P. Tansey. "How the Ubiquitin-proteasome System Controls Transcription." Nat Rev Mol Cell Biol. 4, no. 3 (Mar 2003):192-201. Ubiquitination Bcl-2 Mitochondrion NFxB IxB Ubiquitination Smac/Diablo Cytochrome c Omi NFxB IxB ub ub Ubiquitination IAPs ub ub ub Caspase-Dependent Apoptosis Pathway Degradation Ubiquitination Figure by MIT OCW. After Yang and Xu, "Regulation of apoptosis: the ubiquitous way." FASEB J 17 (2003) 790-799. Figure by MIT OCW
How are the substrates recognized by the E3s? Image removed due to copyright considerations See Figure 2 in Ciechanover A. et al. "Ubiquitin-mediated proteolysis: biological regulation destruction. Bioessays. 2000 May: 22(5): 442-51
How are the substrates recognized by the E3s? Image removed due to copyright considerations. See Figure 2 in Ciechanover A. et al. "Ubiquitin-mediated proteolysis: biological regulation via destruction." Bioessays. 2000 May; 22(5): 442-51