Ubiquitin proteolytic system
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Presentation given by Dr. Karthikeyan at Department of Biochemistry, Maulana Azad Medical College. Addition: There are certain proteins which are degraded by proteasome without ubiquitin tag. one such example is ornithine decarboxylase - rate limiting enzyme of polyamine synthesis.
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Ubiquitin proteolytic system
- 1. Ubiquitin Moderator: Dr. Sarita Agarwal Presentor: Dr. Karthikeyan
- 2. Overview • Various ways of protein degradation • Ubiquitin in contrast to lysosomes • Discovery of Ubiquitin • Nature of Ubiquitin gene and protein • Signal sequences for ubiquitination • 3 steps of Ubiquitination • Proteasome assembly
- 3. contd., • Ubiquitin and Malignancies • Use of Proteasome inhibitors in Malignancies • Ubiquitin in cell cycle (cyclin) and gene expression (Histone) • Ubiquitin like proteins in intracellular trafficking (SUMOylation) • ERAD and Aggresomes
- 4. Topological diversity of proteolytic systems • Extracellular – Digestive enzymes – Blood Coagulation System • Intracellular – membrane secluded: Lysosomes – Free Floating: Ubiquitin System (Cytosolic, nuclear and ER)
- 5. Proteins have variable life-spans Enzyme Half-life Hours Ornithine decarboxylase 0.2 RNA polymerase I 1.3 Tyrosine aminotransferase 2.0 Serine dehydratase 4.0 PEPcarboxylase 5.0 Aldolase 118 GAPDH 130 cytochrome c 150
- 6. • Lysosomes – Receptor mediated endocytosis & phagocytosis in acidic pH • Proteasomes: for intracellular proteins – transcription factors – cyclins – virus coded proteins – improperly folded proteins – damaged proteins
- 7. • Ubiquitin mediates degradation of many but not all proteins.
- 8. Discovery
- 10. The Nobel Prize in Chemistry 2004 Aaron Ciechanover Avram Hershko Irwin Rose
- 12. Small protein with big function • small, heat-stable, compact globular protein (76 AA) • Found only in eukaryotic organisms • Highly conserved
- 13. Ubiquitin Genes • UBA52 • RPS27A • UBB • UBC
- 14. Ub genes typically exist in two states • The ubiquitin gene (red) can be fused to a ribosomal protein gene (blue) giving rise to a translation product that is a Ub- ribosomal fusion protein. Ub-C-term hydrolase
- 15. Ub genes can exist as a linear repeat • Translation product is comprised of a linear chain of Ub-molecules fused together (a polyubiquitin molecule). • Ub-C-term hydrolase cleaves the fusion proteins to yield Ub monomers Ub-C-term hydrolase
- 18. Signals for Degradation • N – Degron • PEST Sequence • Signals in Hydrophobic core • Masking and unmasking
- 21. Signals in Hydrophobic core
- 22. 3 steps of ubiquitination • Activation • conjugation • ligase action
- 26. Why couldn’t be done in a single step ? • E1 (1) • E2 (12-30) • E3 (>200) – HECT-type – RING-type – PHD-type – U-box containing
- 28. Proteasome
- 29. 26S proteasome
- 30. α subunit β subunits α subunit 20S core protein
- 31. Top view
- 33. Oncogenesis • ↑ ubiquitin mediated destruction of Tumor suppressor gene products. (HPV – p53) • ↓ ubiquitin mediated destruction of oncoproteins. (beta catenin)
- 34. ↓ ubiquitin mediated destruction of β-catenin P P
- 35. AUXIN – an eye opener
- 38. Death of Proteins is Required for Life P P
- 39. Ubiquitin in cell cycle and gene expression
- 40. Cyclin ubiquitination • key regulators of the cell cycle. • Cyclins themselves have no enzymatic activity but bind and activates Cdk. • G1-S-G2-M cycle follows successive oscillations in the levels of cyclins, D, E, A & B. • Precise control is achieved by ubiquitination
- 41. • Ubiquitination of protein does not always imply degradation of proteins only.
- 42. Histone Ubiquitination and Gene Expression • Histones are rich in lysine – potential site for ubiquitination. • H2A mono-ubiquitination is a repressive mark. • H2B mono-ubiquitination is an activation mark.
- 43. H2A mono-ubiquitination is a repressive mark.
- 45. SUMOylation • Post translational modification involved in nuclear-cytosolic transport • Small Ubiquitin-like Modifier (or SUMO) proteins • similar to ubiquitin but SUMO is not used to tag proteins for degradation
- 47. Endoplasmic-reticulum-associated protein degradation (ERAD) • cellular pathway which targets misfolded proteins of ER for ubiquitination and subsequent degradation by proteasome. • Molecular chaperones like calnexin/calreticulin try in correct folding of misfolded proteins. Terminally misfolded proteins are processed by mannosidase. • translocated into cytosol for destruction
- 48. Aggresomes: cellular response to misfolded proteins • occurs when the capacity of the proteasome is exceeded by the production of aggregation- prone misfolded proteins Neurofibrillary tangles - Alzheimer's disease Lewy bodies - Parkinson's diseas Pick bodies - Pick's disease Role of ubiquitin antibodies
- 49. Proteasome inhibitors • Bortezomib • Disulfiram • Epigallocatechin-3-gallate • Salinosporamide • carfilzomib
- 50. Bortezomib • Bortezomib - first therapeutic proteasome inhibitor • Approved by FDA for treating relapsed multiple myeloma and mantle cell lymphoma. • The boron atom in bortezomib binds the catalytic site of the 26S proteasome[4] with high affinity and specificity. • may prevent degradation of pro-apoptotic factors, permitting apoptosis
Editor's Notes
- Most of the cathepsins are found inside lysosomes except cathepsin K, which is secreted extracellularly after secretion by osteoclasts in bone resorption. Extracellular long lived proteins Ubiq – intracellular short lived regulatory proteins
- There are only 3 differences in the sequence when Ub from yeast is compared to human Ub. This strong sequence conservation suggests that the vast majority of amino acids that make up Ub are essential as apparently any mutations that have occurred over evolutionary history have been removed by natural selection. Titin , largest protein - connectin, molecular spring >30,000 AA
- 7 lysines – epsilon amino group participates in isopeptide bond C-terminal glycine binds to epsilon amino group of lysine.
- A degron is a specific sequence of amino acids in a protein that directs the starting place of degradation. The N-degron. Alexander Varshavsky in1986carried out an elegant set of experiments (Science 234, 179-186) that showed a correlation between the half-life of a protein and its N-terminal residue. For example, proteins that have Ser as the N-terminal amino acid were long-lived with a half-life of more than 20 hours. In contrast, proteins with Asp as the N-terminal amino have a half-life of only 3 minutes. The mechanism that couples recognition of the N-terminal amino acid and the protein's half-life is still unknown. In is interesting to note, however, that the N-end rule applies to bacteria even though they do not contain ubiquitin. 2. Certain amino acid sequences appear to be signals for degradation. One such sequence is known as the PEST sequence because short stretch of about eight amino acids is enriched with proline, glutamic acid, serine, and threonine. An example is the transcription factor Gcn4p. This protein is 281 amino acids in length and the PEST sequence is found at positions 91-106. The normal half-life of this protein is about 5 minutes. But if the PEST sequence (and only the PEST sequence) is removed, the half-life increases to 50 minutes. Some signals may also be subject to masking. A signal could be hidden if it is part of a protein-protein interaction. Or it may be masked by covalently attaching phosphate groups to the side chains of certain amino acids. Both of these mechanisms would thus allow for better control, as a protein degradation signal need only be unmasked to target it for degradation. Such reversible masking appears to be involved in the regulation of both transcription factor and cyclin concentrations. 3. Signals may also be buried in the hydrophobic core. This is why partially folded or abnormal, mutant proteins may be prone to degradation. When such proteins exist in their native state, the signals are hidden and the protein is thus long-lived. But in a partially unfolded state, the signals may be seen by the Ub machinery caused the protein to become tagged by Ub. This reaction appears to be hindered by chaperone activity.
- Activation: Ubiquitin-activating enzyme (E1) binds ATP-Mg2+ and ubiquitin. In the next step a catalytic cysteine on the E1 enzyme attacks the ubiquitin-AMP complex through acyl substitution, simultaneously creating a thioester bond and an AMP leaving group. Finally, the E1~ubiquitin complex transfers ubiquitin to an E2 enzyme through a transthioesterification reaction, in which an E2 catalytic cysteine attacks the backside of the E1~ubiquitin complex.
- Conjugation: Ubiquitin-conjugating enzyme Activated ubiquitin is then transferred to an E2 cysteine by transthioesterification. Once conjugated to ubiquitin, the E2 molecule binds one of several ubiquitin ligases
- Ubiquitin ligase:
- Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function.
- Lid – substrate recognition Base – substrate unfolding Core - Proteolysis
- β-catenin a subunit of cadherin complex is an oncoprotein. GSK-3 (a kinase) constitutively phosphorylates the β-catenin. Phosphorylated beta catenin is ubiquitinated and degraded.
- similar to ubiquitin - formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein, around 100 amino acids, very little sequence identity with ubiquitin at the amino acid level, it has a nearly identical structural fold