Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di-or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications. eukaryotes is the covalent attachment of ubiquitin (Ub) to proteins. This reversible modification, which regulates a variety of biological processes, such as protein degradation, trafficking, and DNA damage response (1, 2), involves the attachment of the C terminus of Ub mainly to the side chain of a Lys residue in a protein substrate via an isopeptide linkage. The process is catalyzed by three enzymes that act in concert: the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2), and the Ub ligase (E3). The reaction is repeated, and a second Ub is attached to an internal Lys in the previously conjugated ubiquitin. Several repeats result in the synthesis of a poly-Ub chain that can be of varying lengths and internal linkages. The presence of seven Lys residues as possible anchoring sites within Ub in addition to the N-terminal amine results in a highly complex landscape of diverse Ub bioconjugates, which accounts for the diversity of the Ub signaling (3).Research in the Ub field, which aims at understanding the ubiquitination system at the molecular level, has been hampered by the difficulties of controlling ubiquitination in the cell and challenges associated with the preparation of specific Ub conjugates in vitro. These limitations have inspired the development of novel synthetic strategies to facilitate site-specific ubiquitination of proteins (4, 5). Recent advancements in the field have enabled the synthesis of relatively large amounts of highly complex Ub conjugates of defined covalent structure and provided novel insights into the structural, biochemical, and functional consequences of protein ubiquitination, along with unique opportunities to elucidate the molecular basis of Ub signaling. For example, monoubiquitinated α-Synuclein (α-Syn) and histone H2B bearing native isopeptide bonds were prepared and used to shed light on the role of monoubiquitination in regulating α-Syn aggregation a...

Posttranslational modifications (PTMs) play a vital role in regulating the structure and dynamics of chromatin as well as DNA-driven cellular processes by covalently modifying the primary structures of the histone proteins (H2B, H2A, H3, H4) with various chemical entities.[1] One such example is the modification of the histone H2B by ubiquitin (Ub) at multiple lysine residues. Of these, ubiquitination at Lys120 (H2K120Ub) is best studied. This PTM was suggested to modulate gene expression, trigger H3 methylation, and activate DNA damage response pathways.[2] In contrast, little is known about the effects and functions of the other sites of ubiquitination in H2B, K34, K46, K108, and K116. [3] Notably, the preparation of any of these ubiquitinated histone proteins for biochemical analyses has been a challenge in the field and has thus hampered a variety of studies aiming to understand the effect of ubiquitination on chromatin. Only recently, novel nonenzymatic approaches were introduced and enabled the preparation of homogeneously native H2K120Ub on a milligram scale for structural and functional analyses.[4] These approaches proved to be successful for the incorporation of this single modification or any other modification within the short C-terminal synthetic peptide.In order to investigate the role of ubiquitination at other sites and to study the interplay of multiple modifications, a new synthetic strategy is needed. For example, it has been reported recently that ubiquitination of H2B at Lys34 directly regulates H3K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells.[5] Hence, the preparation of such an analogue will enable studies aiming to understand how this modification regulates H3 methylation and potentially affects the structure of chromatin, as well as help to delineate mechanisms of K34 ubiquitination and deubiquitination. Here, we report our endeavors toward the total chemical synthesis of the H2B protein, and the successful sitespecific ubiquitination at Lys34 (H2K34Ub) for initial functional characterization.If one considers the preparation of H2K34Ub semisynthetically through the expression of a large C-terminal H2B fragment that bears an N-terminal Cys or through total chemical synthesis by applying a ligation approach with three fragments, the 57mer H2B(1-57), which bears the K34 modification, has to be prepared synthetically. Because H2B lacks any Cys residue, an Ala to Cys mutation for native chemical ligation (NCL) combined with desulfurization [6] should be performed at Ala58 (Scheme 1). Even if the semisynthetic method were applicable, it would not allow the insertion of different PTMs along the sequence but only at the N-terminal fragment. [7] Our initial studies with a threefragment approach revealed that the synthesis of the 57mer peptide via Fmoc-SPPS was very difficult. We thus concluded that a total chemical synthesis of the H2B protein from four fragments is inevitable. Hence, we divided the H2B sequence, taking into consideration the position of Ala residu...

The desulfurization reaction introduced by Yan and Dawson as a postnative chemical ligation step greatly expanded the scope of ligation chemistry beyond Xaa-Cys (Xaa is any amino acid) by making ligation at Xaa-Phe, Xaa-Val, Xaa-Lys, Xaa-Leu, Xaa-Thr, and Xaa-Pro junctions accessible in the synthesis of functional proteins. A new ligation site based on Xaa-Gln utilizing γ-mercaptoglutamine is reported, and several examples on the efficiency of ligation coupled with desulfurization are provided.

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