2. Ubiquitin proteasome system (UPS)

The discovery of the ubiquitin proteasome system seems to be a never ending story of surprise, starting with the first observations of non-lysosomal, ATP-dependent protein degradation [4], recognizing ubiquitin as a barcode label for proteins dedicated for proteasomal degradation [5] and, finally, realizing that ubiquitin labeling is not an absolute requirement for proteasomal recognition, unfolding, and decay [6].

#### 2.1. Ubiquitin conjugation and barcode properties

The enzymatic machinery-conjugating ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to lysine is based on hundreds of enzymes, activators, inhibitors, and substrate adaptors, establishing an information-based system for multiple purposes in signal transduction. Fifteen human E1 enzymes (ubiquitin-activating enzymes (UBAs)) initiate the conjugation cascade either specifically using ubiquitin or one of the ten different UBLs as substrate. Thioesterlinked E1-Ub/UBL can be transferred to about 80 E2 enzymes (ubiquitin-conjugating UBC) via transesterification to the thiol group of an active site cysteine. Finally, more than 600 monomeric or multimeric E3 enzymes (ubiquitin protein ligases), either harboring RING or HECT domain motifs, constitute molecular scaffolds catalyzing the substrate-specific transfer of thioester-linked Ub/UBL mostly to lysine residues of substrate proteins. Since ubiquitin contains seven putative conjugation sites, linear or branched Ub conjugates can be formed by isopeptide bonds creating individual barcodes. Processing of conjugates, either as a requirement for substrate channeling into proteasomes or for fine tuning the barcode information, is performed by cysteine proteases of the deubiquitinase (DUB) protein family [7]. The resulting barcodes are guiding proteins into different fate decisions, namely, affecting subcellular locations (UBL:SUMO), enzymatic activity (UBL:NEDD8), or 26S/30S proteasomal turnover (UBL: ubiquitin) [3–5, 8].

Ubiquitin-K48 conjugates are mostly used as degradation signal [5] that can be recognized by 19S regulatory particles (PA700) attached to one or both ends of 20S proteasomes, either forming 26S or 30S proteasomes, respectively [9].
