Formation of ubiquitin dimers via azide-alkyne click reaction
2012, Eger, Silvia, Scheffner, Martin, Marx, Andreas, Rubini, Marina
The conjugation of poly-ubiquitin chains is a widespread post-translational modification of proteins that plays a role in many different cellular processes. Notably, the biological function of the attached ubiquitin chain depends on which lysine residue is used for chain formation. Here, we report a method for the modular synthesis of site-specifically linked ubiquitin dimers, which is based on click reaction between two artificial amino acids. In this way, it is possible to synthesize all seven naturally occurring ubiquitin connectivities, thus giving access to all ubiquitin dimers. Furthermore, this method can be generally applied to link ubiquitin to any substrate protein or even to link any two proteins site specifically.
Synthesis of Defined Ubiquitin Dimers
2010, Eger, Silvia, Scheffner, Martin, Marx, Andreas, Rubini, Marina
Many proteins are post-translationally modified by the attachment of poly-ubiquitin (Ub) chains. Notably, the biological function of the attached Ub chain depends on the specific lysine residue used for conjugate formation. Here, we report an easy and efficient method to synthesize site-specifically linked Ub dimers by click reaction between two artificial amino acids. In fact, we were able to synthesize all seven naturally occurring Ub connectivities, providing the first example of a method that gives access to all Ub dimers. Furthermore, these synthetic Ub dimers are recognized by the natural ubiquitination machinery and are proteolytically stable, making them optimal candidates to further investigate the function of differently linked Ub chains.
Generation of a mono-ubiquitinated PCNA mimic by click chemistry
2011-12-16, Eger, Silvia, Castrec, Benoît, Hübscher, Ulrich, Scheffner, Martin, Rubini, Marina, Marx, Andreas
Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive highfidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono-ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site-specifically mono-ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site-specifically by the CuI-catalyzed azide–alkyne cycloaddition. Finally, we show that the synthetic PCNA–Ub is able to stimulate DNA synthesis by DNA polymerase d, and that DNA polymerase h has a higher affinity for PCNA–Ub than to PCNA.
Titanocene Difluorides with Improved Cytotoxic Activity
2010, Eger, Silvia, Immel, Timo, Claffey, James, Müller-Bunz, Helge, Tacke, Matthias, Groth, Ulrich, Huhn, Thomas
Titanocene difluorides can be obtained by halide metathesis of the respective titanocene dichlorides with trimethyltin fluoride (Me3SnF), giving access to a new class of cytotoxic active substances. Furthermore, an improved method for the synthesis of diaryl-substituted titanocene dichlorides is presented.
Ubiquitination via Chemical Ligation between Artificial Amino Acids
2011, Eger, Silvia
Modification of proteins by the covalent attachment of ubiquitin (Ub) plays a fundamental role in the control of many biological processes including cell cycle regulation, transcription, DNA repair, and apoptosis. Substrate proteins are either mono-ubiquitinated or poly-ubiquitinated, i. e. several Ub monomers are attached to form poly-Ub chains. In these chains several Ub moieties are linked to each other via isopeptide bonds between a specific lysine residue of one Ub and the C-terminal glycine of the next Ub. Ub contains seven lysine residues and each of these lysines can be used for poly-Ub chain formation. Importantly, the actual lysine residue of Ub used for Ub-Ub conjugation seems to determine the biological function of the respective poly-Ub chain.
The aim of the present work was to develop a method to synthesize all naturally occurring Ub dimers and to mono-ubiquitinate substrate proteins in vitro. This will provide the basis to elucidate different functions of differently-linked poly-Ub chains and of mono-ubiquitination.
Cross-linking of Ub to other proteins (either a second Ub or a substrate protein) was achieved using the Cu(I)-catalyzed Huisgen cycloaddition, the so-called click reaction. The two orthogonal functional groups needed for click chemistry, an azide and an alkyne, had to be incorporated into the proteins via artificial amino acids. The azide function was introduced at the C-terminus of one Ub via the methionine analog azidohomoalanine (Aha) using selective pressure incorporation. The alkyne function was introduced via a pyrrolysine analog, the propargyl-protected lysine derivative Plk using amber suppression. It replaced the respective lysine residues naturally used for conjugation. Subsequent click reaction between the two modified proteins resulted in a hydrolytically stable triazole linkage. With this, the synthesis of all seven naturally occurring Ub dimers was possible, as well as the site-specific mono-ubiquitination of the two substrate proteins PCNA and DNA polymerase β.