Targeted Protein Degradation

The complexity of targeting substrates to the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) is based on several aspects, such as the 8 different linkage types possible in ubiquitin (Ub) chains, the chain length, the modification site(s) in the target proteins and, only recently discovered, PTMs of Ub. Several approaches to assemble homogeneous, mono- and polyubiquitinated peptides and proteins that cannot easily be isolated from natural sources have been developed over the past three decades. Among them, enzymatic approaches that have advanced to a stage where different linkages and modification sites can be addressed if E2 and E3 enzymes are available but control over chain length and overall homogeneity is still lagging behind.

Combining intein-based generation of activated Ub chains with solid phase peptide synthesis (SPPS) to obtain Ubs with a potentially large set of modifications has arguably been the most successful strategy to generate fully defined Ub chains and Ub-modified proteins. As defined Ub chains can now be site-selectively linked to target proteins, degradation signals can be deciphered at much higher molecular resolution as demonstrated for several target proteins. In combination with defined Ub chains and binding assays for key components of the UPS and ALP, we will investigate the role of site-specific attachment of differently linked chains of up to 4 Ubs within this SFB.