SUMMARY Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and the polyUb chains to a range of cellular protein targets. Best documented Ub signaling pathway is the one that delivers polyUb-proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb-conjugates. Our work demonstrates the value of UbPT and we expect that its future uses will help define and investigate the ubiquitin interactome.

Tyrosinase belongs to the type 3 copper enzyme family, containing a dinuclear copper center, CuA and CuB. It is mainly responsible for melanin production in a wide range of organisms. Although copper ions are essential for the activity of tyrosinase, the mechanism of copper uptake is still unclear. We have recently determined the crystal structure of tyrosinase from Bacillus megaterium (TyrBm) and revealed that this enzyme has tighter binding of CuA in comparison with CuB. Investigating copper accumulation in TyrBm, we found that the presence of copper has a more significant effect on the diphenolase activity. By decreasing the concentration of copper, we increased the diphenolase to monophenolase activity ratio twofold. Using a rational design approach, we identified five variants having an impact on copper uptake. We have found that a major role of the highly conserved Asn205 residue is to stabilize the orientation of the His204 imidazole ring in the binding site, thereby promoting the correct coordination of CuB. Further investigation of these variants revealed that Phe197, Met61, and Met184, which are located at the entrance to the binding site, not only play a role in copper uptake, but are also important for enhancing the diphenolase activity. We propose a mechanism of copper accumulation by the enzyme as well as an approach to changing the selectivity of TyrBm towards L-dopa production.

Osteoarthritis is an incurable joint disease manifesting itself with gradually progressing degenerative changes, leading to premature motor disability. These changes mainly occur owing to an imbalance between the processes of degeneration and regeneration of articular cartilage structures. Until now many risk factors favoring the development of degenerative joint disease have been identified. These include age, weight, previously sustained traumas to joints, sports, sex and genetic predisposition. The latest scientific reports confirm that the pathogenesis of changes in osteoarthritic joints is complex and occurs on many levels. Enzymes belonging to the metalloproteinases family are mainly responsible for the degeneration of articular cartilage. Their activity is regulated by numerous pro-inflammatory cytokines, transcription factors and miRNA. A thorough analysis of all processes occurring in the afflicted joints needs to be carried out before effective therapeutic strategies can be developed.

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