Towards elucidation of the role of ubiquitination in the pathogenesis of Parkinson's disease with semisynthetic ubiquitinated α-synuclein.

The ubiquitination of lysine residues has emerged as an important mechanism for the regulation of a variety of cellular processes, such as cell signaling, DNA repair, and protein degradation by the 26S proteasome. Until recently, studies aimed at deciphering the effect of ubiquitination on protein function have relied primarily on biochemical approaches to reconstitute the ubiquitinated protein in vitro. As a result, progress in the field has been very much dependent on the discovery of the E2–E3 enzymatic machinery that is highly specific to the protein of interest. The lack of an effective and general method for the site-specific incorporation of ubiquitin (Ub) or polyubiquitin chains has hampered efforts to elucidate the molecular and structural basis underlying the effects of ubiquitination on the regulation of protein function. Several research groups have recently reported elegant chemical methods that facilitate site-specific peptide and protein ubiquitination. Several of these methods are based on the attachment of ubiquitin through non-native isopeptide bonds to generate enzymatically stable Ub conjugates of peptides or recombinant proteins. The site-specific ubiquitination of peptides and proteins through the formation of a native isopeptide bond is now possible as a result of studies by Muir and co-workers, Liu and co-workers, and our research group. We recently developed a highly efficient and chemoselective method to facilitate the site-specific ubiquitination of peptides and proteins. In our approach, a d-mercaptolysine residue is used to mediate transthioesterification with an ubiquitin thioester, followed by S–N acyl transfer to form an isopeptide bond between ubiquitin and the lysine derivative. The thiol handle of d-mercaptolysine is then removed by desulfurization to furnish the desired native isopeptide linkage between ubiquitin and lysine without altering the native sequence of the target peptides/proteins. To facilitate the use of this unique residue in solid-phase peptide synthesis (SPPS) and sequential ligation, we also synthesized dmercaptolysine with different protecting groups. Herein, we describe an efficient strategy based on the use of these (advanced) synthetic tools for the semisynthesis and characterization of a-synuclein monoubiquitinated at Lys6 (T7-Uba-syn(K6)). a-Synuclein (a-syn) is a natively unfolded 140 amino acid presynaptic protein that is implicated in the pathogenesis of Parkinson s disease (PD) and related neurodegenerative diseases that are collectively termed “synucleinopathies”. The pathology of PD is characterized by the loss of dopaminergic neurons and the presence of intracellular inclusions, known as Lewy bodies (LBs), which are composed primarily of a-syn. Several posttranslational modifications, including phosphorylation, C-terminal truncations, and ubiquitination, have been shown to be closely associated with PD pathology and were identified in a-syn within LBs isolated from PD patients brains. Understanding of the role of these modifications in the regulation of a-syn aggregation, LB formation, and toxicity is crucial to the understanding of the biology of a-syn and the elucidation of its role in the pathogenesis of PD, and may lead to the identification of novel therapeutic targets for the treatment of the disease. The majority of a-syn species found in Lewy bodies are monoor diubiquitinated at multiple lysine residues. The directed site-specific ubiquitination of a-syn at a single or multiple lysine residues has not been possible. For example, the coexpression of a-syn with ubiquitin ligases results predominantly in monoand diubiquitination at multiple sites. Similarly, the ubiquitination of recombinant monomeric or fibrillar a-syn using rabbit reticulocytes fraction II or rat-brain extracts revealed that ubiquitination occurs at multiple but distinct lysine residues. Thus, it was not possible to investigate the effect of ubiquitination at specific lysine residues. To address this problem, we developed a semisynthetic strategy by combining cysteineand d-mercaptolysine-based methods for native chemical ligation (NCL) for the site-specific incorporation of ubiquitin and the preparation of highly homogenous monoubiquitinated forms of a-syn. [*] M. Hejjaoui, Prof. H. A. Lashuel Brain Mind Institute The Swiss Federal Institute of Technology EPFL Station 19, 1015 Lausanne (Switzerland) Fax: (+41)21-693-9665 E-mail: [email protected] Homepage: http://nmnf.epfl.ch M. Haj-Yahya, Dr. K. S. A. Kumar, Prof. A. Brik Department of Chemistry Ben-Gurion University of the Negev Beer Sheva 84105 (Israel) Fax: (+972)8-647-2943 E-mail: [email protected] Homepage: http://www.bgu.ac.il/~abrik [] These authors contributed equally.