The strains were harvested and stored at 280uC before use

The ubiquitin-proteasome system is central to a variety of different cellular events, and its dysregulation could be a contributing factor to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Amyotrophic Lateral Sclerosis. The inhibition or activation of targets involved in the UPS MedChemExpress ATL-962 pathway has proved to be a difficult area for drug discovery and drug development partially due to a lack of good functional assays for screening Currently, only one drug targeting the UPS pathway has been approved by the Food and Drug Administration, an inhibitor of the 26Sproteasome subunit with an anticancer indication. UPS signaling is mediated by the covalent linkage of multiple units of ubiquitin to lysine residues of proteins. Ubiquitin is attached to proteins by the interplay of three enzymes, an E1 activating enzyme, an E2 conjugating enzyme and an E3 ubiquitin ligase. As E3 ubiquitin ligases are the last step in the regulatory enzyme cascade, they are an attractive drug target. However, the lack of robust and well defined high-throughput screening assays for E3 ubiquitin ligases has been a significant barrier to the discovery of agonists/antagonists. Currently, no direct binding screens or fragment screens for E3 ubiquitin ligases have been reported in the literature. Parkin is linked to Familial Parkinson’s Disease by mutations in PARK2 on chromosome 6q25.227 and is abundantly expressed in brain and a variety of tissues. Parkin is an E3 ubiquitin ligase, and its function involves the transfer of ubiquitin from Conjugating Enzyme E2 to substrates through attachment to e-amino atoms of lysines or nitrogen atoms at the N-terminus. Several mechanisms of ubiquitin transfer have been proposed, and a large variety of substrates has been identified. Ubiquitination of substrates leads to their proteosomal degradation, signaling events or formation of inclusions. Loss of Parkin’s E3 ligase activity, and therefore loss of ubiquitination of proteins, has been linked to neurodegeneration. In patients harboring Parkin mutations, a selective loss of dopaminergic neurons in the substantia nigra has been observed. Therefore, it is highly desirable to identify a small molecule agonist that is capable of stabilizing functionally active Parkin. Parkin consists of 465 amino acids and has a molecular mass of 51.65 kDa. Parkin is a difficult protein to screen; it is known to easily aggregate and contains 35 cysteines and 8 Zinc atoms, which are required for structural stability and enzymatic activity. Several models have been proposed for the coordination 26506265 of the 1 Parkin SPR Fragment Screening zinc atoms. Parkin comprises several domains: the Ubiquitinlike domain, a linker domain, three Ring domains R0, R1 and R2, and an IBR domain between R1 and R2. All three Ring domains coordinate two zinc atoms each, and an additional two zinc atoms are found in the IBR domain. The UblD structure is very similar to ubiquitin and differs only in the C-terminal region when both 15595852 structures are superimposed. It has been proposed that Parkin can exist in an auto-inhibited state in which the UblD domain is folded back and binds to a Parkin UblD-Ubq binding motif between the IBR domain and Ring2 domain. In this state Parkin cannot be auto-ubiquitinated. Binding of activators to Parkin may result in conformational changes that reveal motifs involved in substrate binding and charged E2-interaction. Consistent with this possibility, Nterminal t