1. Post-translational Modification by UBLs
Kathleen M. Sakamoto, M.D., Ph.D.
Departments of Pediatrics, Pathology & Laboratory Medicine,
Molecular Biology Institute, UCLA
and Division of Biology, Caltech

2. Ubiquitin-like modifiers
Sumo-1*
Discovered in 1996
Nedd8*
Discovered in 1997
Apg8 and 12
Required for autophagy

3. SUMO (small ubiquitin-like modifier)
Also known as Sentrin-1, PIC-1, GMP-1, UBL-1, SMT3C
Sumo-1, Sumo-2, Sumo-3
101 amino acid protein
Contains unique ubiquitin-homology domains

4. Homology between Ub and Sumo
Residues of Sumo-1 is 18% identical and 48% homologous to human ubiquitin
Sumo-2 is a 95 aa protein 46% identical and 66% homologous to Sumo-1
Sumo-3 is a 103 aa protein which is 97% identical to Sumo-2 in the ubiquitin homology domain
Gly-Gly

5. Activation and conjugation of Sumo
Activating enzyme complex has two subunits
AOS1 is similar to the N-terminal half of ub E1
UBA2 is homologous to the C-terminal half of ub E1 and contains the cysteine necessary for linkage with Sumo
Conjugating enzyme is UBC9

8. Desumoylation Desumoylating enzymes
Cleave the isopeptide linkage between Sumo and target proteins
Ulp1
Smt3 from its conjugates
Cleaves Sumo-1 but not ubiquitin in vitro
Has both isopeptidase and C-terminal hydrolase activity
Required for G2/M transition
Sumo C-terminal hydrolases, e.g. SENP1

9. Remaining questions Function of sumoylation?
Sumo conjugation on proteins?
Specificity of Sumo-1, -2, and -3?
Regulation of sumoylation?
Other Sumo E2s?
Sumo E3 ligases?

10. Nedd8 (neural precursor cell-expressed developmentally downregulated)
Novel mRNA enriched in fetal mouse brain
Developmentally downregulated
Expression restricted to heart and skeletal muscle
Enriched in the nucleus

11. Nedd8 Rub 1 (Yeast and plant homologues)
Encodes a 81 amino acid polypeptide
60% identical and 80% homologous to ubiquitin
Structurally more closely related to ubiquitin

12. Activation of NEDD8 Activating enzyme complex (2 subunits)
APP-BP1 and UBA3
The APP-BP1 N-terminal half is homologous to the N-terminal half of ubiquitin E1
UBA3 is homologous to the C-terminal half of ubiquitin E1 and contains the cysteine required for thiol ester linkage with Nedd8
Specificity is due to preferential binding of UBA3 to NEDD8, but not ubiquitin or Sumo

13. Conjugation of NEDD8 Conjugating enzyme is UBC12 Necessary
supported by observation in Ubc12 null yeast mutants
Dominant-negative form of UBC12 abolishes NEDD8 conjugation in vivo and affects cell growth

14. Function of Neddylation
Modification of E3 Cullins
Cdc53 in yeast
In humans, NEDD8 modifies cullin family members
Modifies nuclear proteins
?involved in centrosome cycle

15. Crystal structure

17. COP9 Signalosome (CSN) 500 kDa protein complex 8 distinct subunits
CSN5 or Jab1 is catalytic component of isopeptidase
Homology to subunits of the proteasome (CSN5=RPN11)

18. NEDD8-specific hydrolases and isopeptidases
Cross-reactivity with UCHs and UBPs
UCH-L3 has NEDD8 C-terminal hydrolase activity
USP21 deconjugates ubiquitin and deneddylates from NEDD8 conjugates

19. Remaining questions
Is NEDD8 modification required for assembly or function of SCF?
Role of NEDD8 conjugation of cullins on Lys 689?
How does neddylation modulate ligase activity?
other substrates?
Role of NEDD8 or mixed ubiquitin/NEDD8 polymers?

20. Interferon-stimulated gene 15
Induced by IFN, lipopolysaccharides, or upon viral infections
E1 (ISG15 activating enzyme)
UBP43, ISG15-specific isopeptidase
Modified proteins: JAK-STAT proteins
Overexpression of ISG15 leads to neurologic disorders in mice; deletion leads to cancer

21. Autophagy
Aut7 is necessary for formation of membrane structures in autophagosomes
Target is a phospholipid
Primary amino group = e amino group of lysine of UBL proteins

22. Autophagy

23. Function of UBLs