Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein

Part of the Methods in Molecular Biology book series (MIMB, volume 1345)

Abstract

Posttranslational modifications (PTMs) serve as molecular switches for regulating protein folding, function, and interactome and have been implicated in the misfolding and amyloid formation by several proteins linked to neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Understanding the role of individual PTMs in protein misfolding and aggregation requires the preparation of site-specifically modified proteins, as well as the identification of the enzymes involved in regulating these PTMs. Recently, our group has pioneered the development of enzymatic, synthetic, and semisynthetic strategies that allow site-specific introduction of PTMs at single or multiple sites and generation of modified proteins in milligram quantities. In this chapter, we provide detailed description of enzymatic and semisynthetic strategies for the generation of the phosphorylated α-Synuclein (α-Syn) at S129, (pS129), which has been identified as a pathological hallmark of Parkinson’s disease. The semisynthetic method described for generation of α-Syn-pS129 requires expertise with protein chemical ligation, but can be used to incorporate other PTMs (single or multiple) within the α-Syn C-terminus if desired. On the other hand, the in vitro kinase-mediated phosphorylation strategy does not require any special setup and is rather easy to apply, but its application is restricted to the generation of α-Syn_pS129. These methods have the potential to increase the availability of pure and homogenous modified α-Syn reagents, which may be used as standards in numerous applications, including the search for potential biomarkers of synucleinopathies.

Key words

Parkinson’s disease Posttranslation modification Alpha-synuclein Amyloid Phosphorylation Semisynthesis Native chemical ligation Desulfurization 

References

  1. 1.
    Lashuel HA, Overk CR, Oueslati A, Masliah E (2013) The many faces of alpha-synuclein: from structure and toxicity to therapeutic target. Nat Rev Neurosci 14:38–48PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Oueslati A, Fournier M, Lashuel HA (2010) Role of post-translational modifications in modulating the structure, function and toxicity of alpha-synuclein implications for Parkinson’s disease pathogenesis and therapies. Prog Brain Res 183C:115–145CrossRefGoogle Scholar
  3. 3.
    Oueslati A, Schneider BL, Aebischer P, Lashuel HA (2013) Polo-like kinase 2 regulates selective autophagic alpha-synuclein clearance and suppresses its toxicity in vivo. Proc Natl Acad Sci U S A 110(41):E3945–E3954PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Anderson JP, Walker DE, Goldstein JM, de Laat R, Banducci K, Caccavello RJ, Barbour R, Huang J, Kling K, Lee M, Diep L, Keim PS, Shen X, Chataway T, Schlossmacher MG, Seubert P, Schenk D, Sinha S, Gai WP, Chilcote TJ (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J Biol Chem 281:29739–29752CrossRefPubMedGoogle Scholar
  5. 5.
    Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, Goldberg MS, Shen J, Takio K, Iwatsubo T (2002) alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nat Cell Biol 4:160–164CrossRefPubMedGoogle Scholar
  6. 6.
    Wang Y, Shi M, Chung KA, Zabetian CP, Leverenz JB, Berg D, Srulijes K, Trojanowski JQ, Lee VM-Y, Siderowf AD, Hurtig H, Litvan I, Schiess MC, Peskind ER, Masuda M, Hasegawa M, Lin X, Pan C, Galasko D, Goldstein DS, Jensen PH, Yang H, Cain KC, Zhang J (2012) Phosphorylated α-Synuclein in Parkinson’s disease. Sci Transl Med 4:121ra120Google Scholar
  7. 7.
    Foulds PG, Mitchell JD, Parker A, Turner R, Green G, Diggle P, Hasegawa M, Taylor M, Mann D, Allsop D (2011) Phosphorylated alpha-synuclein can be detected in blood plasma and is potentially a useful biomarker for Parkinson’s disease. FASEB J 25:4127–4137CrossRefPubMedGoogle Scholar
  8. 8.
    Schmid AW, Fauvet B, Moniatte M, Lashuel HA (2013) Alpha-synuclein post-translational modifications as potential biomarkers for Parkinson disease and other synucleinopathies. Mol Cell Proteomics 12:3543–3558PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Paleologou KE, Schmid AW, Rospigliosi CC, Kim HY, Lamberto GR, Fredenburg RA, Lansbury PT Jr, Fernandez CO, Eliezer D, Zweckstetter M, Lashuel HA (2008) Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of alpha-synuclein. J Biol Chem 283:16895–16905PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Mbefo MK, Paleologou KE, Boucharaba A, Oueslati A, Schell H, Fournier M, Olschewski D, Yin G, Zweckstetter M, Masliah E, Kahle PJ, Hirling H, Lashuel HA (2010) Phosphorylation of synucleins by members of the Polo-like kinase family. J Biol Chem 285:2807–2822PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Burre J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, Sudhof TC (2010) Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329:1663–1667PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Mahul-Mellier AL, Fauvet B, Gysbers A, Dikiy I, Oueslati A, Georgeon S, Lamontanara AJ, Bisquertt A, Eliezer D, Masliah E, Halliday G, Hantschel O, Lashuel HA (2014) c-Abl phosphorylates alpha-synuclein and regulates its degradation: implication for alpha-synuclein clearance and contribution to the pathogenesis of Parkinson’s disease. Hum Mol Genet 23(11):2858–2879PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Negro A, Brunati AM, Donella-Deana A, Massimino ML, Pinna LA (2002) Multiple phosphorylation of alpha-synuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation. FASEB J 16:210–212PubMedGoogle Scholar
  14. 14.
    Ellis CE, Schwartzberg PL, Grider TL, Fink DW, Nussbaum RL (2001) alpha-synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases. J Biol Chem 276:3879–3884CrossRefPubMedGoogle Scholar
  15. 15.
    Hodara R, Norris EH, Giasson BI, Mishizen-Eberz AJ, Lynch DR, Lee VM, Ischiropoulos H (2004) Functional consequences of alpha-synuclein tyrosine nitration: diminished binding to lipid vesicles and increased fibril formation. J Biol Chem 279:47746–47753CrossRefPubMedGoogle Scholar
  16. 16.
    Uversky VN, Yamin G, Munishkina LA, Karymov MA, Millett IS, Doniach S, Lyubchenko YL, Fink AL (2005) Effects of nitration on the structure and aggregation of alpha-synuclein. Brain Res Mol Brain Res 134:84–102CrossRefPubMedGoogle Scholar
  17. 17.
    Muir TW, Sondhi D, Cole PA (1998) Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci U S A 95:6705–6710PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Evans TC Jr, Benner J, Xu MQ (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci 7:2256–2264PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Fauvet B, Fares MB, Samuel F, Dikiy I, Tandon A, Eliezer D, Lashuel HA (2012) Characterization of semisynthetic and naturally Nalpha-acetylated alpha-synuclein in vitro and in intact cells: implications for aggregation and cellular properties of alpha-synuclein. J Biol Chem 287:28243–28262PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Hejjaoui M, Haj-Yahya M, Kumar KS, Brik A, Lashuel HA (2011) Towards elucidation of the role of ubiquitination in the pathogenesis of Parkinson’s disease with semisynthetic ubiquitinated alpha-synuclein. Angew Chem Int Ed Engl 50:405–409CrossRefPubMedGoogle Scholar
  21. 21.
    Shabek N, Herman-Bachinsky Y, Buchsbaum S, Lewinson O, Haj-Yahya M, Hejjaoui M, Lashuel HA, Sommer T, Brik A, Ciechanover A (2012) The size of the proteasomal substrate determines whether its degradation will be mediated by mono- or polyubiquitylation. Mol Cell 48:87–97CrossRefPubMedGoogle Scholar
  22. 22.
    Haj-Yahya M, Fauvet B, Herman-Bachinsky Y, Hejjaoui M, Bavikar SN, Karthikeyan SV, Ciechanover A, Lashuel HA, Brik A (2013) Synthetic polyubiquitinated alpha-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology. Proc Natl Acad Sci U S A 110:17726–17731PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Hejjaoui M, Butterfield SM, Fauvet B, Vercruysse F, Cui J, Dikiy I, Prudent M, Olschewski D, Zhang Y, Eliezer D, Lashuel HA (2012) Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: alpha-synuclein phosphorylation at tyrosine 125. J Am Chem Soc 134(11):5196–5210PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Wissner RF, Wagner AM, Warner JB, Petersson EJ (2013) Efficient, traceless semi-synthesis of alpha-synuclein labeled with a fluorophore/thioamide FRET pair. Synlett 24:2454–2458CrossRefGoogle Scholar
  25. 25.
    Fauvet B, Butterfield SM, Fuks J, Brik A, Lashuel HA (2013) One-pot total chemical synthesis of human alpha-synuclein. Chem Commun (Camb) 49:9254–9256CrossRefGoogle Scholar
  26. 26.
    Chen L, Periquet M, Wang X, Negro A, McLean PJ, Hyman BT, Feany MB (2009) Tyrosine and serine phosphorylation of alpha-synuclein have opposing effects on neurotoxicity and soluble oligomer formation. J Clin Invest 119:3257–3265PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Hejjaoui M (2012) Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis - phosphorylation at Tyrosine 125 and monoubiquitination at Lysine 6, EPFLGoogle Scholar
  28. 28.
    Khalaf O, Fauvet B, Oueslati A, Dikiy I, Mahul-Mellier AL, Ruggeri FS, Mbefo MK, Vercruysse F, Dietler G, Lee SJ, Eliezer D, Lashuel HA (2014) The H50Q mutation enhances alpha-synuclein aggregation, secretion, and toxicity. J Biol Chem 289:21856–21876PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Wan Q, Danishefsky SJ (2007) Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew Chem Int Ed Engl 46:9248–9252CrossRefPubMedGoogle Scholar
  30. 30.
  31. 31.
    Hackenberger CP, Schwarzer D (2008) Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed Engl 47:10030–10074CrossRefPubMedGoogle Scholar
  32. 32.
    Han JC, Han GY (1994) A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal Biochem 220:5–10CrossRefPubMedGoogle Scholar
  33. 33.
    Stevens R, Stevens L, Price NC (1983) The stabilities of various thiol compounds used in protein purifications. Biochem Educ 11:70CrossRefGoogle Scholar
  34. 34.
    Singletary AM (1997) Hazardous laboratory chemicals disposal guide margaret-Ann Armour, CRC Press, Inc., Boca Raton, FL, (1996), 546 Pages, [ISBN No.: 1–56670–108–2] U.S. List Price: 7$79.95. Environ Prog 16:S5CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind InstituteEcole Polytechnique Fédérale de LausanneLausanneSwitzerland
  2. 2.Qatar Biomedical Research InstituteHamad Bin Khalifa UniversityDohaQatar

Personalised recommendations