NMR assay targeting the ubiquitin-proteasome system to modulate protein folding and degradation
The ubiquitin-proteasome system has won the reputation of the cellular recycling center that degraded aggregated or misfolded proteins in the cell (ref.  and references therein). Although the ubiquitin system is now known to control many other physiological processes through regulating the turnover and activity of normal cellular proteins, the recycling or disposal function is still one of the more ..
BioActive Compounds: 7
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: XO1 MH078942
Assay Provider: Dr. Maurizio Pellecchia, Sanford-Burnham Medical Research Institute
The ubiquitin-proteasome system has won the reputation of the cellular recycling center that degraded aggregated or misfolded proteins in the cell (ref.  and references therein). Although the ubiquitin system is now known to control many other physiological processes through regulating the turnover and activity of normal cellular proteins, the recycling or disposal function is still one of the most vital activities of the ubiquitin system. For example, the failure to degrade hill-folded or aggregated proteins in neuronal cells has been linked to several neurodegenerative diseases, such as Alzheimer's and Parkinson's. The degradation of unfolded proteins by the ubiquitin system is coordinated with the other major protein folding control system in the cell, the Hsp chaperones, such as Hsp70 and Hsp90. It is believed that exposed hydrophobic regions in unfolded proteins are recognized by Hsp proteins via their beta-domains and eventually refolded. However, if refolding fails, misfolded proteins are thought to be degraded by the proteasome. Because of the multitude of binding events and associated cellular activities of Ubc13, we propose to use unbiased NMR-based screens to identify high-affinity ligands to further probe the role of this central ubiquitin conjugating enzyme in protein degradation, DNA repair and IKB alpha I kinase activation.
1. Wilkinson, K.D., The discovery of ubiquitin-dependent proteolysis. PNAS, 2005. 102(43): p. 15280-15282.
1. Protein expression and purification
Ubc13 protein expression and purification for NMR studies. We expressed and purified Ubc13 protein to provide the necessary protein reagents for 1D 1H NMR and 2D 15N 1H HSQC NMR studies. Ubc13 was previously cloned into pETShin-(His)9 vector at BamH1 and Xho1 sites and transformed into BL21/DE3 E.coli cells. Proteins in either labeled (15N form for 15N 1H HSQC studies) or unlabeled (for 1D NMR studies) forms were expressed as required using either minimal media supplemented with 15NH4Cl or LB media, respectively. Cells were grown at 37 oC to exponential phase (O.D ~ 0.6) and induced with 0.4 mM IPTG (Bio Vectra). The general protocol for labeled and unlabeled proteins expressed from pET vector remained the same. Briefly, cells were resuspended in a lysis buffer consisting of 1 mM Tris pH 7.0, 1 mM DTT, 0.2 mg/mL lysozyme and a mixture of protease inhibitors in the form of Complete Mini-EDTA free tablets (Roche). Lysates were incubated with Ni+-NTA agarose (Qiagen) previously washed with washing buffer consisting of 1 mM Tris, pH 7.0 and 1 mM DTT. Proteins were eluted with imidazole (Sigma) under linear gradient conditions followed by FPLC purification (Pharmacia BioTech) on a MonoQ column using gradient conditions (0 to 1 M NaCl). This protocol generally yielded proteins of > 90% purity. SDS-PAGE gel analysis of labeled and unlabeled versions of Ubc13 protein purified by FPLC yielded coomasie-stained bands that corresponded to MW of ~ 17 kDa. Gelcode blue stain reagent (Pierce) and DI water were used for staining and destaining, respectively. Unlabeled UBC13 was supplied in 50 mM HEPES @ 7.5 buffer and labeled UBC13 was supplied in 50 mM HEPES @ 7.5 buffer containing 20% D20.
2. Compound Library Screened by NMR
We have assembled a scaffold library composed of ~ 4,000 compounds. The compounds have been selected based on their anticipated use as building blocks or scaffolds components of further optimized molecules. The scaffold library has been acquired from three different sources and the chemical structures of the library have been deposited into PubChem. In line with the general NIH Molecular Libraries Screening Centers Network (MLSCN) library, we have also included a collection of 602 Natural Products (MicroSource) that could be screened by NMR
3. Screening description
Ligand binding was monitored by comparing the aliphatic region of 1D 1H NMR spectra of a 20 uM Ubc13 solution (100 mM sodium phosphate buffer at pH 7.5 containing 100% D2O; T= 300 K) in the presence and in absence of compounds tested at a final concentration of 100 uM. Compounds were initially tested at mixtures of 20, and then individual compounds for those mixtures that caused significant perturbations in the spectrum were further deconvoluted. No significant hits were reported by this screen. Only a few natural products were identified as weak binders (500 < Kd < 1000 uM) and their site of binding verified by 2D [15N, 1H] HSQC. These are Pubchem Substance IDs: 11537556; 11537407; 11537396; 11537489; 11537422; 11537399; 11537676.
The score is an estimate that reflects the binding affinity of the compound for the target and the accuracy of the method used to measure the dissociation constants. Compounds with an estimated Kd > 4000 uM are assigned a Kd of 4001 uM. Compounds for which we do not have a complete titration curve are scored on a scale from 0 to 40 according to the following scale:
Kd > 4000 uM is assigned score 0
100 < Kd < 500 uM is assigned a score of 30 and a Kd of 101 uM
Data Table (Concise)