Objective/Rationale:
Despite increasing evidence linking specific modifications to alpha-synuclein (alpha-syn), alpha-synuclein aggregation, and Lewy body (LB) formation, little is still known concerning the effect of these modifications on the physiopathologic properties of alpha-syn and the relative contribution of each modification in the pathogenesis of Parkinson’s disease (PD). For many of these modifications, the enzymes involved remain unknown and/or achieving site specific modification in vitro or in vivo has not been possible. This knowledge gap has significantly limited our ability to quantitatively assess the levels of these modifications in vivo, and the relationship between these modifications and disease progression.
Project Description:
The primary objective of this project is to use and extend the recombinant and synthetic methods developed in our group to introduce single or multiple disease-associated site specific modifications in alpha-syn (e.g. pS129, pS87, pY125 alpha-syn) and produce the desired modified proteins in sufficient quantities to advance research and drug discovery efforts in Parkinson’s disease. Through this grant, we plan to further develop and simplify these methods and facilitate the transfer of these technologies to other research groups in the field. In addition, we will work closely with the foundation to make these reagents, and the wild-type (naturally formed and without mutation) alpha-syn protein, available to researchers in the field for the developments of assays, biomarker research, and as standards for cellular and in vivo studies.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The availability of these proteins is crucial for advancing current research programs aimed at 1) the characterization of modifications in humans and in pre-clinical models of PD; 2) biomarkers research, target identification and drug discovery research; 3) development approaches for detection and quantitative assessment of these modifications in vivo and in biological fluids (e.g. ELISA or LUminx-based). Indeed, the lack of such tools has been a major obstacle for advancing these efforts and enabling quantitative understanding of the relationship between specific modifications and PD progression and pathology. We are confident that we can produce sufficient amounts of these proteins as enabling tools to support many of the research programs in these areas.
Anticipated Outcome:
The availability of these reagents is crucial to unraveling the roles of modifications in regulating the structure, aggregation, and toxicity of alpha-syn in PD and related synucleinopathies. In addition, these research tools will enable us and others in the field to address key mechanistic questions that cannot be addressed using existing tools, including
- What percentage of alpha-syn is modified in vivo and is there a correlation between the level of modified alpha-syn and disease progression?
- What is the effect of each modification on the structure of monomeric alpha-syn and its binding to membranes, oligomerization and fibrillogenesis?
- Do alpha-syn modifications occur before and/or after alpha-syn aggregation and LB formation?
- Is there cross-talk between the different post-translational modifications in alpha-syn?
- What are the natural enzymes involved in regulating each of these modification?
- What is the effect of each modification on the stability, degradation and functional properties of alpha-syn?
Progress Report
The primary objective of this project is to use and extend the recombinant and synthetic methods developed in our group to introduce single or multiple disease-associated site specific modifications in α-syn (e.g. pS129, pS87, pY125 α-syn) and produce the desired modified proteins in sufficient quantities to advance research and drug discovery efforts in Parkinson’s disease. We are pleased to report that we will be able to meet all the milestones defined for the first year of the project. We successfully prepared the three proteins (wild-type, p129, and p87) requested by the foundation in larger quantities than requested. In addition, we have also generated, for the first time -syn phosphorylated specifically at Y125 (pY125) and Nterminally acetylated (N-Ac- -syn) form of α-syn. Recent studies have shown that -syn produced in pre-clinical cells is acetylated at the N-terminus. We have already shared these proteins with several groups, including some that are supported by the foundation. These achievements represent important advances toward unraveling the roles of PTMs in determining α-syn structure, aggregation and functions in health and disease.
Presentations & Publications
1. Hejjaoui M, Butterfield S, Fauvet B, Vercryusse F, Cun J, Dikiy I, Prudent M, Olschewski D, Zhang Y, Eliezer D., Lashuel HA* “Elucidating the role of C-terminal post-translational modifications using protein semisynthetic strategies: -synulcein phosphorylation at Tyrosine 125”, J. Amer. Chem. Soc, 2012 Feb 16. [Epub ahead of print]
2. Oueslati A, Paleologou KE, Schneider BL, Aebischer P., Lashuel HA* “Mimicking phosphorylation at Serin87 inhibits the aggregation of human alpha-synuclein and protects against its toxicity in a pre-clinical model of Parkinson’s disease, J. Neuroscience, 2012, 1;32(5):1536-44.
3. Fauvet B, Fares B, Samuel F, Tandon A, Lashuel HA* “Semisynthesis and characterization of N-terminally acetylated : -synulcein: Implications for : -syn aggregation and cellular properties”, PlosOne, 2012, Submitted
4. Hejjaoui H, Haj-Yahya M, Kumar KS, Brik A* and Lashuel HA* “Towards elucidating the role of ubiquitination in the pathogenesis of Parkinson’s disease using semisynthetic ubquitinated α-synuclein”. Angew Chem Int Ed, 2011, 10;50(2):405-9