Discussion RPE cells and retina from a-crystallin KO mice are highly susceptible to oxidant injury

ags to full-length Parkin have been reported to increase auto-ubiquitination. Previous HTS screens for small molecules binding to E3 ligases have utilized enzymatic activity assays or biochemical assay approaches, such as in vitro ubiquitination assay. Our previous Parkin HTS was based on such an ubiquitination assay and resulted in the discovery of a variety of chemical scaffolds, which formed the basis of SAR efforts. However, hit expansion efforts did not markedly improve potency or increase residency times. Previously, a successful direct binding assay screen has not been reported in the literature. Herein, SPR was used to characterize the E3 ligase Parkin, to perform assay development for optimization of screening conditions, and to identify new scaffolds that bind to Parkin from a fragment screening campaign. This is, to our knowledge, the first fragment screen performed on an E3 ligase using SPR as the primary screening technology. Constructs and protein expression To generate the FLAG-Parkin expression construct, the entire 12419798 coding region from NM_004562.1 was cloned into pFastBac with an N-terminal 3xFLAG and tobacco etch virus protease cleavage site. Recombinant Bacmid was generated and P1 virus was produced and amplified according to the Halofuginone manufacturer’s protocol. For protein production, the amplified virus was used to infect either Sf9 or Sf21 cells; pellets were harvested 23 days post infection by centrifugation and snap frozen in liquid nitrogen prior to purification. To generate the R0-RBR expression construct, DNA encoding residues 141465 of Parkin were cloned into the Champion pET SUMO vector according to the manufacturer’s protocol. To generate the Ubch7 expression construct, DNA encoding residues 1154 of UBE2L3 was cloned into the pET30 vector with an N terminal 8xHis/TEV site. To generate the Ubl expression construct, 19187978 DNA encoding residues 1 76 of Parkin was cloned into the pET30 vector incorporating a C-terminal 6xHis tag followed by two stop codons. Bacterial expression of R0-RBR, Ubch7, and Ubl was as follows: plasmids were transformed into BL21 DE3 E.Coli. Overnight cultures inoculated from fresh colonies were grown in Terrific broth media containing 2% glucose and 50 mg/ml kanamycin at 37uC. The following morning overnight cultures were diluted to OD600 0.1 and shaking was continued at 37uC until OD600 reached 0.4, at which time the flasks were transferred to 16u. When the OD600 reached 0.8 to 0.9, cultures were induced with 0.1 mM IPTG supplemented with 50 uM zinc chloride and expression was allowed to proceed for 1820 hours at 16uC. Cells were then harvested by centrifugation and frozen at 280uC. Materials and Methods CM5 chips, amine coupling kit, HBS-P Buffer, Tween-20 solution were purchased from GE Healthcare, Glutathione-S Transferase and Carbonic Anhydrase II was obtained from R&D Systems. S5a, Ubiquitin and UbcH7 were purchased from Boston Biochem and dimethyl sulfoxide, pluronic F127 detergent, Anti-FLAG antibody M2 from Sigma Aldrich. Streptavidin XL665 conjugate and Ub-Eu K were obtained from Cisbio. Filter plates and vacuum device were obtained from Millipore Corporation and 96- or 384 well plates from Greiner Bio-One Inc.. High performance Ni sepharose, the Mono Q HR 10/10 anion exchange column, and the HiLoad 26/60 Superdex 200 column were from GE Life Sciences. The Anti-FLAG M2 agarose resin and the 3x-FLAG peptide were obtained from Sigma Aldrich. 10% Bis-Tris Nu PAGE gels and MES running Buffer wer