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

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

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

Homology between Ub and Sumo Residues 22-97 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

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

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

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?

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

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

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

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

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

Crystal structure

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)

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

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?

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

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

Autophagy

Function of UBLs