FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022285303> ?p ?o ?g. }

• W2022285303 endingPage "9924" @default.
• W2022285303 startingPage "9919" @default.
• W2022285303 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. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (181) Google Scholar, 9Rodriguez M.S. Desterro J.M.P. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Crossref PubMed Scopus (557) Google Scholar), it is assumed that SUMO E3 ligases catalyze sumoylation at non-consensus sites, increase the rate of modification, or ensure substrate specificity (10Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 11Pichler A. Gast A. Seeler J.-S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 12Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 13Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Sumoylation is a highly dynamic and reversible process as specific proteases can rapidly remove SUMO from their substrates (for review, see Ref. 14Melchior F. Schergaut M. Pichler A. Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In contrast to ubiquitin, which mainly tags proteins for proteasome-mediated degradation, covalent modification by SUMO can have a number of functional consequences for the target proteins. For example, sumoylation modulates protein-protein interactions, affects subcellular localization and, in some cases, antagonizes the proteasome pathway by competing with ubiquitin (for review, see Ref. 1Dohmen R.J. Biochim. Biophys. Acta. 2004; 1695: 113-131Crossref PubMed Scopus (200) Google Scholar). Despite the rapidly growing number of SUMO substrates identified, in most cases, the physiological function and regulation of sumoylation remain elusive and may vary according to the nature of the target. Tau and α-synuclein belong to the family of natively unfolded proteins as they display an extended conformation in vitro with little ordered secondary structure (15Schweers O. Schönbrunn-Hanebeck E. Marx A. Mandelkow E. J. Biol. Chem. 1994; 269: 24290-24297Abstract Full Text PDF PubMed Google Scholar, 16Weinreb P.H. Zhen W. Poon A.W. Conway K.A. Lansbury Jr., P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1283) Google Scholar). Both proteins are highly soluble and heat-resistant. They are highly expressed in the brain and are associated with several neurodegenerative disorders including Alzheimer and Parkinson disease (reviewed in Ref. 17Goedert M. Curr. Opin. Genet. Dev. 2001; 11: 343-351Crossref PubMed Scopus (80) Google Scholar). As with other amyloidogenic proteins, tau and α-synuclein undergo a pathological transition from random coil to a β-pleated sheet conformation that is accompanied by extensive aggregation and fibril formation (18Barghorn S. Davies P. Mandelkow E. Biochemistry. 2004; 43: 1694-1703Crossref PubMed Scopus (178) Google Scholar, 19Conway K.A. Harper J.D. Lansbury Jr., P.T. Biochemistry. 2000; 39: 2552-2563Crossref PubMed Scopus (678) Google Scholar, 20Serpell L. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Crossref PubMed Scopus (636) Google Scholar). Post-translational modifications affect both protein structure and function and may also contribute to protein dysfunction. Tau is a phosphoprotein with up to 30 tightly regulated phosphorylation sites, and hyperphosphorylation is a common feature of paired helical filaments in Alzheimer disease (21Grundke-Iqbal I. Iqbal K. Tung Y.-C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2786) Google Scholar). Similarly, α-synuclein inclusions in the form of Lewy bodies are a pathological hallmark of Parkinson disease and other α-synucleinopathies (22Spillantini M.G. Goedert M. Ann. N. Y. Acad. Sci. 2000; 920: 16-27Crossref PubMed Scopus (374) Google Scholar). In addition to phosphorylation (23Okochi M. Walter J. Akihiko K. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P.J. Haass C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), α-synuclein is also subject to nitration (24Giasson B.I. Duda J.E. Murray I.V. Chen Q. Souza J.M. Hurtig H.I. Ischiropoulos H. Trojanowski J.Q. Lee V.M.-Y. Science. 2000; 290: 985-989Crossref PubMed Scopus (1354) Google Scholar). Besides chemical modifications, proteins can also be modified by the conjugation of other polypeptides such as ubiquitin. It has been shown that both tau and α-synuclein are degraded by the proteasome in a ubiquitin-independent (25David D.C. Layfield R. Serpell L. Narain Y. Goedert M. Spillantini M.G. J. Neurochem. 2002; 83: 185Crossref Scopus (272) Google Scholar, 26Tofaris G.K. Layfield R. Spillantini M.G. FEBS Lett. 2001; 509: 22-26Crossref PubMed Scopus (324) Google Scholar) and -dependent manner (27Shimura H. Schwartz D. Gygi S.P. Kosik K.S. J. Biol. Chem. 2004; 279: 4869-4876Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 28Petrucelli L. Dickson D. Kehoe K. Taylor J. Snyder H. Grover A. De Lucia M. McGowan E. Lewis J. Prihar G. Kim J. Dillmann W.H. Browne S.E. Hall A. Voellmy R. Tsuboi Y. Dawson T.M. Wolozin B. Hardy J. Hutton M. Hum. Mol. Genet. 2004; 13: 703-714Crossref PubMed Scopus (565) Google Scholar, 29Babu J.R. Geetha T. Wooten M.W. J. Neurochem. 2005; 94: 192-203Crossref PubMed Scopus (242) Google Scholar, 30Bennett M.C. Bishop J.F. Leng Y. Chock P.B. Chase T.N. Mouradian M.M. J. Biol. Chem. 1999; 274: 33855-33858Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Alzheimer neurofibrillary tangles are strongly immunoreactive for ubiquitin (31Mori H. Kondo J. Ihara Y. Science. 1987; 235: 1641-1644Crossref PubMed Scopus (714) Google Scholar, 32Perry G. Friedman R. Shaw G. Chau V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3033-3036Crossref PubMed Scopus (533) Google Scholar), and tau is also a substrate for the CHIP-Hsc70 complex (27Shimura H. Schwartz D. Gygi S.P. Kosik K.S. J. Biol. Chem. 2004; 279: 4869-4876Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 28Petrucelli L. Dickson D. Kehoe K. Taylor J. Snyder H. Grover A. De Lucia M. McGowan E. Lewis J. Prihar G. Kim J. Dillmann W.H. Browne S.E. Hall A. Voellmy R. Tsuboi Y. Dawson T.M. Wolozin B. Hardy J. Hutton M. Hum. Mol. Genet. 2004; 13: 703-714Crossref PubMed Scopus (565) Google Scholar) as well as the ubiquitin E3 ligase TRAF6 (29Babu J.R. Geetha T. Wooten M.W. J. Neurochem. 2005; 94: 192-203Crossref PubMed Scopus (242) Google Scholar). Recently, ubiquitination of α-synuclein as well as the sites of the modification within both the soluble and the filamentous forms of the protein have been reported (33Nonaka T. Iwatsubo T. Hasegawa M. Biochemistry. 2005; 44: 361-368Crossref PubMed Scopus (104) Google Scholar). In the present study, we examined the sumoylation of these two native unfolded proteins, tau and α-synuclein, and showed that tau undergoes SUMO modification at a defined consensus motif. Additional evidence suggests that there is a dynamic interplay between tau sumoylation and proteasome inhibition. Functional studies involving phosphorylation and microtubule stability indicated that free soluble tau is targeted for covalent SUMO modification. These findings indicated a novel pathway for tau and α-synuclein regulation that may have unique consequences for the cellular regulation of these proteins and possibly their disease-related processes. Plasmids—The plasmids encoding N-terminally HA-tagged (peptide YPYDVPDYA) full-length SUMO1, SUMO2, and SUMO3 were generously provided by Dr R. T. Hay (University of St. Andrews, UK). These correspond to the human protein sequences as described previously (34Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M.P. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). PCR was used to generate His epitope tag (peptide AHHHHHHV), using the corresponding HA-tagged plasmids as templates. All SUMO constructs were cloned into pcDNA3 vectors and confirmed by DNA sequencing. Human wild-type tau 4R2N cDNA in bacterial expression vector pBluescript II was subcloned into pcDNA3, and α-synuclein was cloned into pcDNA6. Single mutants Tau (K340R, K385R), α-synuclein (K96R, K102R), and the conjugation-deficient double mutant SUMO1 (G96A,G97A) were generated by site-directed mutagenesis according to the manufacturer's instructions (Stratagene) and confirmed by DNA sequencing. Cell Culture and Transfection—Human embryonic kidney 293 cells (HEK293) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected in reduced serum medium at ∼50% confluence using Lipofectamine (Invitrogen), according to the manufacturer's instructions. For each transfection, cells were incubated in the presence of 5 μg of plasmid DNA encoding tau or α-synuclein, in the presence or absence of 5 μg of His-SUMO isoforms as indicated. Where necessary, pcDNA3 empty vector was used to bring the total amount of DNA to 10 μg. After 20 h of transfection, medium was replaced, and cells were incubated for an additional 24 h. Cells were treated for the final 16 h with 5 μm MG132 (Me2SO), 20 nm okadaic acid (ethanol), or 1 μm colchicine (H2O). Treated cells were harvested and washed in phosphate-buffered saline. Purification of His-tagged SUMO Conjugates—Transfected and treated cells were lysed in buffer (8 m urea, 100 mm NaH2PO4, pH 8.0, 10 mm β-mercaptoethanol, 1% Triton X-100, 10 mm iodoacetamide, 5 mm imidazole) containing complete protease inhibitor mixture (Roche Applied Science). Lysates were briefly sonicated to reduce viscosity and cleared by centrifugation. The protein content was determined using the Bradford assay. Clarified lysates were mixed with 35 μl of Ni2+-nitrilotriacetic acid-agarose prewashed with lysis buffer containing 20 mm imidazole and incubated for 2 h at 4°C. The beads were washed by centrifugation once with lysis buffer (pH 8.0) containing 10 mm imidazole and twice with lysis buffer (pH 6.4) containing 10 mm imidazole. His-tagged SUMO conjugates were eluted (lysis buffer, pH 6.4, containing 300 mm imidazole), diluted in Laemmli sample buffer, and analyzed by Western blotting. Western Blotting—Proteins were separated by electrophoresis on precast 4–20% polyacrylamide gels (Invitrogen) and transferred onto nitrocellulose (Amersham Biosciences). SUMO1 and SUMO2/3 antibodies were purchased from Zymed Laboratories Inc.. Anti-tau antibody CP27 was generously provided by Dr Peter Davies (Albert Einstein College of Medicine, New York, NY). Anti-α-synuclein antibody (Syn1, clone 42) was purchased from Pharmingen. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were used as secondary antibodies (Jackson ImmunoResearch). Immunoreactive bands were visualized by enhanced chemiluminescence using ECL detection kit (Amersham Biosciences), according to the manufacturer's instructions. Western blots presented were representative of 3–5 experiments, which displayed comparable results. Sumoylation of Tau and α-Synuclein—Tau and α-synuclein are two natively unfolded proteins frequently found in intracellular filamentous inclusions that define several neurodegenerative diseases. Both proteins are regulated through various post-translational modifications such as phosphorylation (21Grundke-Iqbal I. Iqbal K. Tung Y.-C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2786) Google Scholar, 23Okochi M. Walter J. Akihiko K. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P.J. Haass C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar) and ubiquitination (27Shimura H. Schwartz D. Gygi S.P. Kosik K.S. J. Biol. Chem. 2004; 279: 4869-4876Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 28Petrucelli L. Dickson D. Kehoe K. Taylor J. Snyder H. Grover A. De Lucia M. McGowan E. Lewis J. Prihar G. Kim J. Dillmann W.H. Browne S.E. Hall A. Voellmy R. Tsuboi Y. Dawson T.M. Wolozin B. Hardy J. Hutton M. Hum. Mol. Genet. 2004; 13: 703-714Crossref PubMed Scopus (565) Google Scholar, 29Babu J.R. Geetha T. Wooten M.W. J. Neurochem. 2005; 94: 192-203Crossref PubMed Scopus (242) Google Scholar, 33Nonaka T. Iwatsubo T. Hasegawa M. Biochemistry. 2005; 44: 361-368Crossref PubMed Scopus (104) Google Scholar). Sumoylation plays an important role in many cellular processes. Recent reports have also implicated sumoylation in neurodegeneration (35Ueda H. Goto J. Hashida H. Lin X. Oyanagi K. Kawano H. Zoghbi H.Y. Kanazawa I. Okazawa H. Biochem. Biophys. Res. Commun. 2002; 293: 307-313Crossref PubMed Scopus (74) Google Scholar, 36Terashima T. Kawai H. Fujitani M. Maeda K. Yasuda H. Neuroreport. 2002; 13: 2359-2364Crossref PubMed Scopus (71) Google Scholar, 37Chan H.Y. Warrick J.M. Andriola I. Merry D. Bonini N.M. Hum. Mol. Genet. 2002; 11: 2895-2904Crossref PubMed Google Scholar, 38Pountney D.L. Huang Y. Burns R.J. Haan E. Thompson P.D. Blumbergs P.C. Gai W.P. Exp. Neurol. 2003; 184: 436-446Crossref PubMed Scopus (88) Google Scholar, 39Pountney D.L. Chegini F. Shen X. Blumbergs P.C. Gai W.P. Neurosci. Lett. 2005; 381: 74-79Crossref PubMed Scopus (52) Google Scholar), and many proteins involved in these pathologies were found to be SUMO targets (40Riley B.E. Zoghbi H.Y. Orr H.T. J. Biol. Chem. 2005; 280: 21942-21948Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 41Steffan J.S. Agrawal N. Pallos J. Rockabrand E. Trotman L.C. Slepko N. Illes K. Lukacsovich T. Zhu Y.-Z. Cattaneo E. Pandolfi P.P. Thompson L.M. Marsh J.L. Science. 2004; 304: 100-104Crossref PubMed Scopus (546) Google Scholar, 42Shinbo Y. Niki T. Taira T. Ooe H. Takahashi-Niki K. Maita C. Seino C. Iguchi-Ariga S.M.M. Ariga H. Cell Death Differ. 2006; 13: 96-108Crossref PubMed Scopus (144) Google Scholar). We therefore evaluated whether the two brain proteins, tau and α-synuclein, were sumoylated. HEK293 cells were transfected with plasmids expressing tau or α-synuclein along with the different His-tagged SUMO isoforms. Cells were lysed under denaturing conditions, and total His-tagged SUMO substrates were isolated by nickel affinity chromatography. In the presence of SUMO1 and, to a lesser extent, SUMO2 and SUMO3, higher molecular weight tau species were observed (Fig. 1A, arrow). Unmodified tau appeared as a single band at ∼64 kDa. The most intense tau immunoreactive band at ∼98 kDa is compatible with the conjugation of a single SUMO protein (∼20 kDa). An additional higher molecular weight SUMO1- and tau-positive band was also observed (Fig. 1A, arrowhead). This could correspond to conjugation of more than one SUMO molecule to different tau target lysines (multisumoylation) or the extension of a SUMO chain on a single lysine (polysumoylation). Sumoylation of tau was not detected in cells lacking SUMO expression. In addition, co-transfection of tau and the conjugation-deficient SUMO1 GG-AA mutant abolished the higher molecular weight bands, consistent with the loss of a covalent modification (Fig. 1A). For α-synuclein and, in contrast to tau, a single primary sumoylated species at ∼36 kDa was observed when it was co-expressed with the His-tagged SUMO isoforms. Human α-synuclein was modified primarily by SUMO1, and to a lesser extent, by SUMO2 and SUMO3 (Fig. 1B, arrow). The SUMO/α-synuclein derivatives were also specific to the presence of SUMO expression as they were absent from cells transfected with empty vector. No polysumoylation or multisumoylation of α-synuclein was detected, and no bands were observed with the conjugation-deficient SUMO1 GG-AA mutant, suggesting a specific modification. Total SUMO conjugates were visualized using SUMO1- and SUMO2/3-specific antibodies (Fig. 1, C and D). The overall sumoylation by these proteins was comparable, which demonstrates that the observed bands were not the result of differences in expression levels or activity of the transfected SUMO proteins. This indicates a specific SUMO1 conjugation to tau and α-synuclein. Probing total cell extracts for the conjugation-deficient SUMO1 GG-AA mutant revealed that it existed exclusively as a monomeric species, consistent with the loss of tau and α-synuclein modification (Fig. 1C, asterisk). Cumulatively, these results demonstrate that both natively unfolded proteins tau and α-synuclein can be preferentially sumoylated by SUMO1. Mapping SUMO Modification Sites of Tau and α-Synuclein—A majority of SUMO-accepting lysines are defined by the consensus motif ΨKX(E/D), where Ψ corresponds to a hydrophobic residue, K is the target lysine for covalent conjugation, X is any amino acid, and the final amino acid is a glutamate or aspartate (E/D) residue. Tau (VK340SE, AK385TD) and α-synuclein (VK96KD, GK102NE) have two putative SUMO consensus motifs (Figs. 2A and 3A). Mutagenesis was used to examine sumoylation at these sites, and individual lysine-to-arginine mutants were generated. The tau mutants (K340R and K385R) were expressed at similar levels as the wild-type protein (data not shown). The single mutation K385R had no effect on SUMO1 modification of tau (Fig. 2B). This tau mutant was sumoylated to the same extent as the wild-type substrate, suggesting that the consensus motif containing Lys385 is not a target for SUMO1 conjugation. In contrast, sumoylation of the tau K340R mutant was significantly reduced, and virtually no SUMO1 conjugates were observed (Fig. 2B). These findings indicate that Lys340 represents one of the major SUMO1 acceptor sites for both mono- and poly/multisumoylation.FIGURE 3Identification of the SUMO modification sites in α-synuclein. A, the position of the two putative SUMO consensus sites within the C-terminal domain of human α-synuclein. The seven core repeats are shown as black boxes. B, modification by SUMO1 of α-synuclein (α-syn) K102R as compared with wild type (WT). Plasmids encoding α-synuclein wild type or K102R were co-expressed with His-SUMO1 and SUMO substrates were purified by nickel chromatography and analyzed by immunoblotting. WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether sumoylation of α-synuclein was also the result of a specific consensus motif, similar mutagenesis of the two potential target lysine residues (Lys96 and Lys102) was performed. Co-expression of the K102R mutant α-synuclein resulted in a slight decrease in the level of sumoylation as compared with wild type (Fig. 3B). This suggested that Lys102 may be one site of SUMO1 conjugation, but it is unlikely to be a primary target residue. Despite repeated attempts, the other putative SUMO site, Lys96, could not be directly investigated due to complications with antibody recognition. Lys96 lies within the sequence recognized by the Syn1 antibody, and the K96R substitution disrupted the epitope, which rendered the mutant protein undetectable (data not shown). To resolve this problem, other anti-α-synuclein antibodies such as the monoclonal Ab211 (Zymed Laboratories Inc.) and a polyclonal antibody raised against human α-synuclein (peptide epitope107APQEGILEDMPVDPDNEAY125) 3V. Dorval and P. E. Fraser, unpublished data. were examined. However, these were not suitable for our particular investigation since both antisera exhibited nonspecific bands in the molecular weight range expected for sumoylated α-synuclein (data not shown). However, the fact that the Syn1 antibody recognizes sumoylated α-synuclein strongly suggests that Lys96 is not a major SUMO target site (Fig. 1). It would be predicted that steric hindrance caused by SUMO1 conjugation at Lys96 would also result in epitope disruption and failure of antigen-antibody recognition caused by the K96R substitution as described above. In addition to Lys96 and Lys102, α-synuclein contains 13 other lysine residues that are localized mainly within the core repeats. It has also been shown that sumoylation is not exclusively restricted to consensus motifs (13Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 43Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchior F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 44Zhou W. Ryan J.J. Zhou H. J. Biol. Chem. 2004; 279: 32262-32268Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), and therefore, SUMO1 modification may occur at one or more of these other sites. This is the case for α-synuclein ubiquitination, which is confined to lysine residues within the N-terminal half of the protein and does not involve either Lys96 or Lys102 (33Nonaka T. Iwatsubo T. Hasegawa M. Biochemistry. 2005; 44: 361-368Crossref PubMed Scopus (104) Google Scholar). These findings suggest that sumoylation of α-synuclein may be dispersed within the protein sequence, and further investigations are required to map the precise modification sites. However, our findings indicated that Lys102 may be one minor sumoylation target that contributes to this process. Dynamic Interplay between Sumoylation and Proteasome Inhibition—Lysine residues are common targets for several post-translational modifications, including acetylation, methylation, ubiquitination, and sumoylation. Therefore, it is possible that SUMO conjugation to a target lysine may prevent and/or regulate other modifications at the same site. This paradigm has been reported for the substrates NF-κB essential modulator (NEMO) (45Huang T.T. Wuerzberger-Davis S.M. Wu Z.-H. Miyamoto S. Cell. 2003; 115: 565-576Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), proliferating cell nuclear antigen (PCNA) (46Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1716) Google Scholar), Smad4 (43Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchior F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 47Lee P.S.W. Chang C. Liu D. Derynck R. J. Biol. Chem. 2003; 278: 27853-27863Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and Huntingtin (41Steffan J.S. Agrawal N. Pallos J. Rockabrand E. Trotman L.C. Slepko N. Illes K. Lukacsovich T. Zhu Y.-Z. Cattaneo E. Pandolfi P.P. Thompson L.M. Marsh J.L. Science. 2004; 304: 100-104Crossref PubMed Scopus (546) Google Scholar), which are either sumoylated or ubiquitinated at the same lysine in response to different signaling events. One of the most extensively characterized proteins is IκBα (48Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar), in which conjugation of SUMO directly antagonizes the ubiquitin-proteasome pathway by competing with ubiquitin for a single target lysine. It has been proposed that this provides a new mechanism to regulate protein stability. Tau and α-synuclein can be degraded by the proteasome through ubiquitin-dependent (27Shimura H. Schwartz D. Gygi S.P. Kosik K.S. J. Biol. Chem. 2004; 279: 4869-4876Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 28Petrucelli L. Dickson D. Kehoe K. Taylor J. Snyder H. Grover A. De Lucia M. McGowan E. Lewis J. Prihar G. Kim J. Dillmann W.H. Browne S.E. Hall A. Voellmy R. Tsuboi Y. Dawson T.M. Wolozin B. Hardy J. Hutton M. Hum. Mol. Genet. 2004; 13: 703-714Crossref PubMed Scopus (565) Google Scholar, 29Babu J.R. Geetha T. Wooten M.W. J. Neurochem. 2005; 94: 192-203Crossref PubMed Scopus (242) Google Scholar, 30Bennett M.C. Bishop J.F. Leng Y. Chock P.B. Chase T.N. Mouradian M.M. J. Biol. Chem. 1999; 274: 33855-33858Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) as well as independent processes (25David D.C. Layfield R. Serpell L. Narain Y. Goedert M. Spillantini M.G. J. Neurochem. 2002; 83: 185Crossref Scopus (272) Google Scholar, 26Tofaris G.K. Layfield R. Spillantini M.G. FEBS Lett. 2001; 509: 22-26Crossref PubMed Scopus (324) Google Scholar). Given the specific conjugation of SUMO1 to both proteins, it is possible that there is similar competition between the two pathways that could be perturbed by proteasome inhibition. Cells co-expressing tau or α-synuclein and His-tagged SUMO1 were treated with MG132 prior to purification of SUMO1 conjugates. Proteasome inhibition significantly increased the levels of monomeric tau (2.7-fold ± 1.2; n = 3), consistent with a reduced catabolism of the free pool of protein (Fig. 4A). Under these conditions in wh" @default.
• W2022285303 created "2016-06-24" @default.
• W2022285303 creator A5007554690 @default.
• W2022285303 creator A5027242816 @default.
• W2022285303 date "2006-04-01" @default.
• W2022285303 modified "2023-10-11" @default.
• W2022285303 title "Small Ubiquitin-like Modifier (SUMO) Modification of Natively Unfolded Proteins Tau and α-Synuclein" @default.
• W2022285303 cites W1481130297 @default.
• W2022285303 cites W1525659758 @default.
• W2022285303 cites W1544371346 @default.
• W2022285303 cites W1708445842 @default.
• W2022285303 cites W1964162423 @default.
• W2022285303 cites W1964381171 @default.
• W2022285303 cites W1970061483 @default.
• W2022285303 cites W1970685852 @default.
• W2022285303 cites W1971611931 @default.
• W2022285303 cites W1972166544 @default.
• W2022285303 cites W1972939055 @default.
• W2022285303 cites W1983153401 @default.
• W2022285303 cites W1983882750 @default.
• W2022285303 cites W1987927755 @default.
• W2022285303 cites W1988369116 @default.
• W2022285303 cites W1995456120 @default.
• W2022285303 cites W1996811697 @default.
• W2022285303 cites W1997241945 @default.
• W2022285303 cites W1997920940 @default.
• W2022285303 cites W1998440070 @default.
• W2022285303 cites W2001029086 @default.
• W2022285303 cites W2012781495 @default.
• W2022285303 cites W2018633688 @default.
• W2022285303 cites W2024935012 @default.
• W2022285303 cites W2026263038 @default.
• W2022285303 cites W2027649342 @default.
• W2022285303 cites W2029189334 @default.
• W2022285303 cites W2031982078 @default.
• W2022285303 cites W2036755018 @default.
• W2022285303 cites W2037828638 @default.
• W2022285303 cites W2040825545 @default.
• W2022285303 cites W2045893413 @default.
• W2022285303 cites W2046014998 @default.
• W2022285303 cites W2046037983 @default.
• W2022285303 cites W2046720254 @default.
• W2022285303 cites W2047206971 @default.
• W2022285303 cites W2050895547 @default.
• W2022285303 cites W2050971426 @default.
• W2022285303 cites W2053060343 @default.
• W2022285303 cites W2061630866 @default.
• W2022285303 cites W2061995199 @default.
• W2022285303 cites W2067371211 @default.
• W2022285303 cites W2073148985 @default.
• W2022285303 cites W2074177924 @default.
• W2022285303 cites W2074649216 @default.
• W2022285303 cites W2080101736 @default.
• W2022285303 cites W2081345134 @default.
• W2022285303 cites W2083312621 @default.
• W2022285303 cites W2091829752 @default.
• W2022285303 cites W2094209629 @default.
• W2022285303 cites W2094423062 @default.
• W2022285303 cites W2100090579 @default.
• W2022285303 cites W2108228282 @default.
• W2022285303 cites W2118518385 @default.
• W2022285303 cites W2118895416 @default.
• W2022285303 cites W2119301405 @default.
• W2022285303 cites W2128932095 @default.
• W2022285303 cites W2131898220 @default.
• W2022285303 cites W2153398746 @default.
• W2022285303 cites W2154492443 @default.
• W2022285303 cites W2155438201 @default.
• W2022285303 cites W2168079270 @default.
• W2022285303 cites W2354791377 @default.
• W2022285303 doi "https://doi.org/10.1074/jbc.m510127200" @default.
• W2022285303 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16464864" @default.
• W2022285303 hasPublicationYear "2006" @default.
• W2022285303 type Work @default.
• W2022285303 sameAs 2022285303 @default.
• W2022285303 citedByCount "265" @default.
• W2022285303 countsByYear W20222853032012 @default.
• W2022285303 countsByYear W20222853032013 @default.
• W2022285303 countsByYear W20222853032014 @default.
• W2022285303 countsByYear W20222853032015 @default.
• W2022285303 countsByYear W20222853032016 @default.
• W2022285303 countsByYear W20222853032017 @default.
• W2022285303 countsByYear W20222853032018 @default.
• W2022285303 countsByYear W20222853032019 @default.
• W2022285303 countsByYear W20222853032020 @default.
• W2022285303 countsByYear W20222853032021 @default.
• W2022285303 countsByYear W20222853032022 @default.
• W2022285303 countsByYear W20222853032023 @default.
• W2022285303 crossrefType "journal-article" @default.
• W2022285303 hasAuthorship W2022285303A5007554690 @default.
• W2022285303 hasAuthorship W2022285303A5027242816 @default.
• W2022285303 hasConcept C100631289 @default.
• W2022285303 hasConcept C104317684 @default.
• W2022285303 hasConcept C139447449 @default.
• W2022285303 hasConcept C144519050 @default.
• W2022285303 hasConcept C158617107 @default.
• W2022285303 hasConcept C171350616 @default.
• W2022285303 hasConcept C181199279 @default.

• next