Ubiquitin Protein Ligase E3

1. Ubiquitin Protein Ligase E3 Regulation of Protein Expression Widespread Cancer Implications John Prodromo

2. Regulatory Proteins • Protein expression is regulated on many levels • Transcription • operons, histones • Translation • Modification of mRNAs, excision of introns • Post translation • Turnover time of proteins • Degradative pathways

3. Ubiquitination and the Proteasome • Ubiquitination is a common method of marking a protein for degradation. • Ubiquitous in nature (hence the name) • Small polypeptide of 76 a.a. attaches to amino terminal lysine residue • Targets to proteasome • Can mono or poly-ubiquinate • Lys-48 linked poly-ubiquitin chains are common, others are also known. • Lys6, Lys11, Lys29, Lys63 Ubiquitin with purple Lysine Residues http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Proteasome.html

4. The E1, E2, E3 system • Ubiquitin-activating enzyme (E1) activates Ub by adenylating C-terminus, binding at its own Cys. • Ub transferred to an E2 • With help of E3, transferred to a lysine residue of substrate protein. • E3 helps in the transfer, exact mechanism is varied, and often confers specificity • E3 is not ALWAYS used

5. Target; Proteasome • Addition of Ubiquitin -> regulatory particle binds to the complex-> hydrolyzes ATP to unfold protein-> moves protein to proteasome-> cuts protein into smaller units • These units are either completely broken down to amino acids • Or presented to the immune system as antigens • Immune system activity releases cytokine interferon gamma, which modifies proteasome and makes it release antigenic determinants • Transporter associated with antigen processing (TAP) proteins move the antigens to ER, they pair to class I MHC and then are taken to the cell membrane for recognition by T cells • Ubiquitin is released and reused. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Proteasome.html

6. Signals for Degradation • Not completely known • Some motifs recur • N-terminal residue- the N degron • BUT- Also applies to bacteria (no ubiquitin) • PEST (proline-glutamic acid-serine-threonine) • 5 minute-50 minute half life • Misfolded proteins • Exposing core sequences http://homepages.bw.edu/~mbumbuli/cell/ublec

7. E3 ubiquitin protein ligase structure • Split into two distinct groups • Based on the presence of a HECT domain or a RING finger domain • HECT- catalytic contribution • RING finger- mostly acts as a scaffolding • Other Domains variable • May contain a cullin domain • In vitro activation of E2s Passmore, Lori A. and Barford, David. Biochem J. “Getting Into position; the catalytic mechanisms of protein ubiquitylation”

8. HECT • 350 aa sequence • Two lobes form an L shape and transfer Ub using a conserved Cysteine and transfers to a lysine via an undetermined general base deprotonation Passmore, Lori A. and Barford, David. Biochem J. “Getting Into position; the catalytic mechanisms of protein ubiquitylation”

9. RING Finger • Really Interesting New Gene • Also structurally related to U Box domain • 15th most common domain in human genome • Uncertain how many RING fingers act as E3s • Conserved Zn++ with cross brace arrangement • Divided into RING-HC or RING-H2 • based on Cys or His • All have 2 Zn sites http://pawsonlab.mshri.on.ca/index.php?option=com_content&task=view&Itemid=64&id=176

10. The RING-finger Domain • Binding of Zinc atoms- cross bridge x x x x x x x x x x x x x x x x x C C C C x \ / x x \ / x x Zn x x Zn x C / \ C H / \ C x x x x x x x x x x x x x x x x x Zn: Zinc C: Cysteine H: Histidine X: unspecified amino acid http://pfam.sanger.ac.uk/family?acc=PF00097 Ubox domain replaces Zn with H bonds

11. RING Finger • May allosterically activate, or stabilize transfer • Example: APC is a multisubunit E3 with 13 individual subunits. • Scaffolding; • Bring donor Ub closer to Lys residues Passmore, Lori A. and Barford, David. Biochem J. “Getting Into position; the catalytic mechanisms of protein ubiquitylation”

12. C-CBL/UBCH7 Complex: RING domain function in Ub-protein ligases • UBCH7- activity with steroid hormone receptors • ZAP70-zeta chain associated protein kinase • Role in T-cell signaling C-CBL ZAP70 Ubiquitin conjugating enzyme

13. Common Domains/patterns • N- terminal 4 helix bundle • EF Hand calcium binding domain • Src homology-2 (SH2)-like domain • The key is that the E3 has either the RING Finger domain or HECT domain, and aids an E2 in binding ubiquitin to a target protein http://www.rcsb.org/pdb/explore/explore.do?structureId=1FBV

14. The Immune System • Major Histocompatability complex (MHC) • Outside of cell membranes • Lymphocyte recognition and antigen presentation • Determine immune response • Self/nonself recognition • Class I • Marks cells as self to immune system • Presents to T cells • Endogenous antigen presentation to cytotoxic T cells- part of viral protein and tumor protein recognition • T cells • Lymphocytes that mature in the thymus • T cell receptors recognize antigens and can start immune response • Used in labeling, can terminate cells that are infected • B cells • Lymphocytes that mature in the spleen and produce antibodies • Positive recognition- destroyed if paired to self http://www.cehs.siu.edu/fix/medmicro/mhc.htm

15. http://youtube.com/watch?v=E2e5J1dfiE0

16. Viral Immunoevasion • Poxvirus and gamma-2 herpesvirus use E3s with RING domain • Downregulates their cell surface glycoproteins (MARCH proteins) • Catalyze ubiquitin conjugation to surface proteins in vitro • Ubiquitination leads to protein endocytosis • This makes the cells avoid recognition by MHC I • Avoids detection by the immune system http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=321412

17. Receptor Tyrosine Protein Kinases • E3 proteins help to recognize and degrade receptor protein tyrosine kinases (PTKs) • Receptor dimerizes when it binds to a growth factor ligand, then phosphorylation of a Tyrosine, and signal transduction. • PTKs are involved in: • Mitosis • Differentiation • Apoptosis • 70% of onco and proto-oncogenes are PTKs • Activated kinases are ubiquinated to terminate the signal • E3 protein recruits an E2, and the complex with the activated PTK transfers a ubiquitin to destroy the protein. • Deactivates PTK. http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Key_Resources/Protein_Kinase_Explorer/Tyrosine_Kinase_Overview.html

18. Cbl-c • TKB (tyrosine kinase binding) • RING finger domains • a short proline-rich region http://www.rcsb.org/pdb/explore.do?structureId=3BUO

19. Cell Cycle Regulation • Cyclin dependent kinases (Cdks) regulate transitions in the cell cycle • E1 uses ATP to bind and transfer ubiquitin to an E2, which can sometimes transfer directly, or often uses an E3 for specificity in ubiquitin transfer to the protein. • This pathway is necessary to • enter S phase • Destroy Sic1p- by CDC 4, CDC53, CDC34- ubiquitin conjugation • separate sister chromatids- destruction of kinetochores in Anaphase via Ub • end mitotic division. • Cyclins have a destruction box motif • Cyclin A and B have similar boxes, but A is degraded first http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195BWM-D&_coverDate=07%2F26%2F1996&_alid=471901450&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=7051&_sort=d&view=c&_acct=C000052510&_version=1&_urlVersion=0&_userid=1381001&md5=4ff0df5d107483b178ceb6b6e808ba4e

20. Cyclin levels • Cyclins are used to regulate and control the transition between checkpoints of the cycle. • Cyclin dependent kinases (Cdks) phosphorylate proteins of the cell cycle to change activity and aid in the progress of the cycle. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195BWM-D&_coverDate=07%2F26%2F1996&_alid=471901450&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=7051&_sort=d&view=c&_acct=C000052510&_version=1&_urlVersion=0&_userid=1381001&md5=4ff0df5d107483b178ceb6b6e808ba4e

21. The Cell Cycle • Rising levels of cyclin D in the G1 phase bind to their corresponding Cdks, which then begin the process of chromosomal replication • S phase- DNA duplication occurs as cyclin A bound to Cdk2 enters the nucleus. This leads to the destruction of cyclin E and the end of S phase. • Cyclins B and A rise. • As mitosis occurs, metaphase marks the central chromosomal alignment, which activates the anaphase promoting complex, and ubiquitination of cyclin B • the anaphase promoting complex (APC) degrades cohesins and causes separation of sister chromatids and degradation of cyclin B • This causes a rise in cyclin D to begin the cycle all over again. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195BWM-D&_coverDate=07%2F26%2F1996&_alid=471901450&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=7051&_sort=d&view=c&_acct=C000052510&_version=1&_urlVersion=0&_userid=1381001&md5=4ff0df5d107483b178ceb6b6e808ba4e

22. E3 activity and (more) Cancer • P53- the “guardian of the genome” • HDM2 is a ubiquitin E3 ligase that is a key negative regulator of the tumor suppressor p53 • Has 3(10) helix followed by four beta-strands • Zinc finger motif http://www.rcsb.org/pdb/explore/pubmed.do?structureId=2C6A

23. The key points • RING Finger is very common in the genome, and often acts as an E3 • Widespread cancer implications on many levels • Cell cycle regulation, tyrosine kinases, immune system • E3s help with specificity and transfer of Ub to target proteins to Proteasome • Ub targeting varies • Zn++ helps to stabilize the transfer • E3 may be a scaffold for E2 specificity • Exact mechanism is undetermined

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