Small Ubiquitin-like Modifier (SUMO) Modification of Natively Unfolded Proteins Tau and α-Synuclein

Abstract

Sumoylation is an important post-translational modification that provides a rapid and reversible means for controlling the activity, subcellular localization, and stability of target proteins. We have examined the covalent attachment of the small ubiquitin-like modifier (SUMO) proteins to tau and α-synuclein, two natively unfolded proteins that define several neurodegenerative diseases. Both brain proteins were preferentially modified by SUMO1, as compared with SUMO2 or SUMO3. Tau contains two SUMO consensus sequences, and mutational analyses identified Lys340 as the major sumoylation site. Although both tau and α-synuclein are targets for proteasomal degradation, only tau sumoylation was affected by inhibitors of the proteasome pathway. Tau is a microtubule-associated protein, whose ability to bind and stabilize microtubules is negatively regulated by phosphorylation. Treatment with the phosphatase inhibitor, okadaic acid, or the microtubule depolymerizing drug, colchicine, up-regulated tau sumoylation. This suggests that SUMO modification may preferentially target a free soluble pool of the substrate. These findings revealed a new, possibly regulatory, modification of tau and α-synuclein that may also have implications for their pathogenic roles in neurodegenerative diseases. Sumoylation is an important post-translational modification that provides a rapid and reversible means for controlling the activity, subcellular localization, and stability of target proteins. We have examined the covalent attachment of the small ubiquitin-like modifier (SUMO) proteins to tau and α-synuclein, two natively unfolded proteins that define several neurodegenerative diseases. Both brain proteins were preferentially modified by SUMO1, as compared with SUMO2 or SUMO3. Tau contains two SUMO consensus sequences, and mutational analyses identified Lys340 as the major sumoylation site. Although both tau and α-synuclein are targets for proteasomal degradation, only tau sumoylation was affected by inhibitors of the proteasome pathway. Tau is a microtubule-associated protein, whose ability to bind and stabilize microtubules is negatively regulated by phosphorylation. Treatment with the phosphatase inhibitor, okadaic acid, or the microtubule depolymerizing drug, colchicine, up-regulated tau sumoylation. This suggests that SUMO modification may preferentially target a free soluble pool of the substrate. These findings revealed a new, possibly regulatory, modification of tau and α-synuclein that may also have implications for their pathogenic roles in neurodegenerative diseases. Small ubiquitin-like modifier proteins (SUMO) 2The abbreviations used are: SUMO, small ubiquitin-like modifier; E1, SUMO activating enzyme; E2, SUMO-conjugating enzyme; E3, SUMO ligase. 2The abbreviations used are: SUMO, small ubiquitin-like modifier; E1, SUMO activating enzyme; E2, SUMO-conjugating enzyme; E3, SUMO ligase. display similarities to ubiquitin in both the structure and the biochemistry of their conjugation (for review, see Ref. 1Dohmen R.J. Biochim. Biophys. Acta. 2004; 1695: 113-131Crossref PubMed Scopus (200) Google Scholar). SUMO isoforms are expressed in humans and display cell type-specific expression levels and distinct, although not exclusive, subcellular localizations (2Su H.-L. Li S. S.-L. Gene (Amst.). 2002; 296: 65-73Crossref PubMed Scopus (127) Google Scholar). Each SUMO paralog is expressed as a precursor protein that undergoes processing by a C-terminal hydrolase (3Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Once cleaved, the mature protein has a diglycine motif exposed at the C terminus and is ready to enter a multistep enzymatic pathway, which is similar but quite distinct from ubiquitination. Mature SUMO proteins are primed in an ATP-dependent manner by the SUMO-activating (E1) enzyme Sua1/hUba2 (4Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (439) Google Scholar, 5Desterro J.M.P. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Activated SUMO is then transferred, through a trans-esterification reaction, to a unique conjugating (E2) enzyme, Ubch9 (6Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 7Desterro J.M.P. Thomson J. Hay R.T. FEBS Lett. 1997; 47: 297-300Crossref Scopus (302) Google Scholar). The final step is the formation of an isopeptide bond between the C-terminal glycine of SUMO and the lysine ϵ-amino group of the target substrate. A majority of the acceptor lysine residues are found within a SUMO consensus motif ΨKX(E/D), in which Ψ corresponds to a hydrophobic residue. Although E1 and E2 are sufficient for SUMO conjugation to various substrates (8Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H.