Project description:Serial Capture Affinity Purification (SCAP) samples analyzed using Multidimentional Protein identification Technology (MudPIT) on Orbitrap Velos Elite. Baits are SNAP-SPIN1 and Halo-WDR76. SNAP purification was performed first, then Halo purification was performed. SCAP was performed using whole cell extract from a stable cell line of 293FRT cells in WDR76 KO background. 3 biological replicates of E1, UB2 and E2 fractions.
Project description:SCAP-XL of Halo-WDR76 and SNAP-SPIN1 from stable cell line in WDR76 KO background. Serial Capture Affinity Purification (SCAP) was performed on whole cell extract of a stable cell line of 293FRT cells in WDR76 KO background. DSSO crosslinked in E2 sample. Data was acquired using a ms1/ms2/ms3 method. Data was searched using Proteomics Discoverer 2.4 with XlinkX.
Project description:Halo purification samples analyzed using Multidimentional Protein identification Technology (MudPIT) on Orbitrap Velos Elite. Baits are Halo-WDR76, Halo-SPIN1, Halo-CBX5. The bait for Control Purification is Halo tag only. Purification for each bait was performed using whole cell extract from a stable cell line in 293FRT cells. 3 biological replicates for each type of bait.
Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles
Project description:Profiling of bone marrow-derived erythroid progenitor cells at E1 (CFU-e), E2 (proerythroblasts), and E3 (maturing erythroblast) stages of development under stress erythropoiesis conditions and in response to EPO challenge. MACS-isolated E1, E2, and E3 stage EPCs were cultured in SP34ex media for 6 hrs in the absence of hematopoietic growth factors and the presence of insulin (to enforce survival and anti-apoptotic effects) and then exposed to rhEPO for 90 minutes. Gene expression analysis was performed using Affymetrix Mouse Genome 430 2.0 arrays; microarray data were analyzed using Bioconductor for R.
Project description:We have developed Halo-seq, an RNA proximity labeling method that allows the quantification of subcellular transcriptomes. We have demonstrated the efficacy of Halo-seq here by using it to quantify chromatin-proximal, nucleolar, and cytoplasmic transcriptomes. In Halo-seq, RNA molecules in close proximity to a spatially restricted protein are specifically marked and biotinylated, facilitating their separation from bulk cellular RNA and their quantification.
Project description:In eukaryotes, E1 initiates the ubiquitin cascade by adenylation and thioesterification of the ubiquitin C-terminus and subsequent transfer of ubiquitin to E2 enzymes. A clinical-grade small molecule that binds to the E1 ATP binding site and covalently derivatizes the ubiquitin C-terminus effectively shuts down E1 enzymatic activity. However, mutation at or near the ATP binding site of E1 causes resistance, mandating alternative approaches to blocking what is otherwise a promising cancer target. Here, we identified a helix-in-groove interaction between the N-terminal alpha-1 helix of E2 and a pocket within the ubiquitin fold domain of E1 as a druggable site of protein interaction. By generating and optimizing stapled alpha-helical peptides (SAHs) modeled after the E2 alpha-1 helix, we achieve site-specific engagement of E1, induce a consequential conformational change, and effectively block E1 enzymatic activity, resulting in a generalized disruption of E2 ubiquitin-charging that suppresses ubiquitination of cellular proteins. Thus, we provide a blueprint for an alternative E1-targeting strategy for the treatment of cancer. Hydrogen exchange mass spectrometry was used to characterize the predominant E1 enzyme in mammals (UBE1, a 118 kDa multi-domain enzyme that catalyzes both ubiquitin adenylation and thioesterification) in the unbound state. We then interrogated the structural impact of UBE1 interaction with the stapled peptide SAH-UBE2A and several mutants. The observed peptide-induced exposure of the ubiquitin-fold domain (UFD) linker hinge in UBE1 was consistent with an inhibitory mechanism whereby SAH-UBE2A locks UBE1 into its proximal UFD conformation.