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Ubiquitin Protein Ligase E3

Ubiquitin Protein Ligase E3. Regulation of Protein Expression Widespread Cancer Implications. John Prodromo. Regulatory Proteins. Protein expression is regulated on many levels Transcription operons, histones Translation Modification of mRNAs, excision of introns Post translation

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Ubiquitin Protein Ligase E3

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  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

  24. Questions?/Comments

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