Project description:His6-Cdc48a and associated proteins purified from H. volcanii. A) SDS- PAGE of proteins purified by Ni2+ -chromatography. H. volcanii carrying empty vector control (H1209-pJAM202c, lane 1) and ectopically expressing His6-Cdc48a (H1209- pJAM1409, lane 2). Red bars indicate regions of gel excised. B) Proteins identified in gel slices by LC-MS/MS analysis. Criteria for protein inclusion: FDR < 0.01%, normalized total spectra minus control > 50, protein threshold > 99% and 2 peptide minimum. Based on spectral counting, Cdc48a and DUF111 were most abundant in regions (a) and (b), respectively, as indicated. DHS1, deoxyhypusine synthase. DUF111, Cdc48a gene neighbor of unknown function. TnaA, tryptophanase; Rps3, ribosomal protein S3; n.d., not detected.
Project description:H. volcanii Cdc48a, RecJ3/4 and RNase J associate with Ubl-coated beads. A) Strategy to isolate H. volcanii proteins that bind agarose beads charged with monomeric Ubl (vs. BSA) as bait. Cell lysate was from triplicate cultures of H. volcanii NH02-pJAM957. B) Left, Non-reducing SDS-PAGE of H. volcanii proteins that bound BSA- (control, lane 1) vs. Ubl- (lane 2) decorated beads in the presence of ATP. Gels were stained with SYPRO Ruby. Red bar, HMW(250kDa) and LMW(50kDa) (high and low molecular weight) regions of gel excised from lanes 1 and 2 for LC- MS/MS analysis. Right, Proteins identified at > 99.9% probability and < 0.1 % false discovery rate (FDR) in the Ubl (not BSA) samples. Similar trend was observed in independent experiment. ATP was required for this association. Theor. Mr, theoretical molecular mass based on genome sequence. Normalized total spectra of sample minus control is indicated.
Project description:CLPB is a mitochondrial intermembrane space AAA+ domain–containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we define a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We show an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.
Project description:Cisplatin (CP) is a chemotherapeutic drug that is used to cure different types of cancer. CP induces DNA damage and leads to cell cycle arrest. The cyclin-dependent kinase inhibitor 1B (CDKN1B), also termed p27, plays an important role in the drug response ; and increased levels of p27 correlated with CP resistance. In HEK293 cells, we observed that p27 mRNAs levels increased whereas protein level drastically decreased in cells treated with CP; suggesting post-transcriptional regulatory events. To further understand the underlying mechanisms, we applied a biochemical approach combined with mass-spectrometry to systematically identify the RNA-binding proteins (RBPs) that are bound to the 3’UTR of p27 mRNAs in CP-treated versus non-treated cells in vivo. We found that 24 proteins, most of them known RBPs such HuR, hNRNPD, changed their association with p27 mRNA upon CP treatement. Furthermore, knock-down of a subset of the identified RBPs led to the inhibition of the CP-induced increase of p27 mRNA levels. In conclusion, these results highlight substantial rearrangement between RBPs and p27 mRNA upon CP treatment and corroborate the importance of post-transcriptional control in cellular drug response.
Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip ⢠The goal of the experiment Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae ⢠Experimental factor Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter) Cell condition 1: G1 arrest with alpha-factor Cell condition 2: HU treatment ⢠Experimental design ChIP analyses: ChIP using anti-c-Myc antibody. ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used. ⢠Quality control steps taken Confirmation of several loci by quantitative real time PCR. Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.
Project description:Proteomic analysis of murine stress granule and G3BP1 granulome in infected cells. BV2 GFP-G3BP1 cells were either treated with 0.1 mM arsenite for 1h to induce SG assembly or infected with MNV for 9h. G3BP1 granules were enriched by sequential centrifugation and purified by immunoprecipitation using antibodies to GFP (to trap GFP-G3BP1) or IgG (as a control) followed by pull down with Protein A-conjugated epoxy Dynabeads as previously described. To characterize the identity of G3BP1 partners within these, Mass Spectrometric analysis was performed.