Dataset Information

Pull-down of nuclear proteins from HGC-27 cell line

ABSTRACT: To identify nuclear proteins that interact with KIT2KIT* G-quadruplex DNA, pull-down assays were performed with nuclear extracts from the KIT-positive HGC-27 cell line. Streptavidin-coated paramagnetic beads were derivatized with the biotinylated oligonucleotide and subsequently incubated with nuclear extracts. Bound proteins were eluted with a KCl gradient. A last fraction was obtained by boiling beads in denaturing Laemmli sample loading buffer. When solved by SDS-PAGE, this last fraction exhibited three main bands that were cut and subjected to in-gel trypsin digestion and LC-MSE analyses for protein identification. Two of them (bands S1 and S2 at ~15 kDa and ~28 kDa, respectively) corresponded to Streptavidin monomer and dimer that detached from the beads along boiling procedure. That aside, the band at ~50 kDa (band V) was associated to the intermediate filament protein Vimentin.

INSTRUMENT(S): Xevo G2-S Tof

ORGANISM(S): Homo Sapiens (human) Streptomyces Avidinii

TISSUE(S): Epithelial Cell

DISEASE(S): Stomach Carcinoma

SUBMITTER: Silvia Ceschi

LAB HEAD: Claudia Sissi

PROVIDER: PXD026505 | Pride | 2022-04-04

REPOSITORIES: Pride

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Publications

Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters.

Ceschi Silvia S Berselli Michele M Cozzaglio Marta M Giantin Mery M Toppo Stefano S Spolaore Barbara B Sissi Claudia C

Nucleic acids research 20220201 3

G-quadruplex (G4) structures that can form at guanine-rich genomic sites, including telomeres and gene promoters, are actively involved in genome maintenance, replication, and transcription, through finely tuned interactions with protein networks. In the present study, we identified the intermediate filament protein Vimentin as a binder with nanomolar affinity for those G-rich sequences that give rise to at least two adjacent G4 units, named G4 repeats. This interaction is supported by the N-ter ...[more]

PMID: 35100428

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Project description:Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences now serving and promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences, and allows the characterization of TF binding determinants within and outside of core binding sites. Binding experiments were carried out with one of two input libraries; Lib1 (also referred to as “main lib”) includes designed sequence variants of length 150bp, and Lib2 (also referred to as “constant flanks lib”) includes designed sequence variants of length 200bp. The sequences of the variants of interest can be found in the second column of the processed data file (BunDLE-seq_data, see 3 excel sheets). EXP1, EXP2 and EXP3 were carried out using Lib1, and EXP4 was carried out using Lib2. Binding experiments were carried out with different proteins (in different concentrations) – DNA was extracted from bound bands that were formed on the gel and amplified with a primer with a unique 5bp upstream tail specifying the identity of the originating band (also referred to as “band barcode”). Data from the following bands was used in the analysis: EXP1_band1, no-protein band – with band barcode “AACGA”, EXP1_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “TCGTA” , EXP1_band3, histones bound band – with band barcode “AGATA”, EXP2_band1, no-protein band – with band barcode “AACGA”, EXP2_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “AGATA”, EXP2_band2, single Gcn4 (conc 1:7) bound band – with band barcode “CACTT”, EXP2_band2, single Gcn4 (conc 1:6) bound band – with band barcode “TCGTA”, EXP2_band2, single Gcn4 (conc 1:5) bound band – with band barcode “CATCA”, EXP2_band2, single Gcn4 (conc 1:4) bound band – with band barcode “TATTA”, EXP2_band2, single Gcn4 (conc 1:3) bound band – with band barcode “AGTCA”, EXP2_band2, single Gcn4 (conc 1:2) bound band – with band barcode “ACGAA”, EXP2_band2, single Gcn4 (conc 1:1) bound band – with band barcode “ACAGA”, EXP2_band2, two Gcn4 (conc 1:1) bound band – barcoded with “GTAGA”, EXP3_band1, no-protein band – with band barcode “GCATA”, EXP3_band2, single Gal4 (conc 1:1) bound band – with band barcode “GATGT”, EXP3_band3, single Gal4 (conc 3:1) bound band – with band barcode “GTTCA”, EXP3_band4, two Gal4 (conc 3:1) bound band – with band barcode “CTCAA”, EXP4_band1, no-protein band – with band barcode “GAGTA”, EXP4_band2, single Gcn4 (conc 1:3) bound band – “TACCA”, EXP4_band3, no-protein band – with band barcode “TCGTT”, EXP4_band3, single Gal4 (conc 3:1) bound band – with band barcode “ACATC”, *biological replicates

Project description:In the present study, we report for the first time a proteomic profile of Buthotus saulcyi, Odontobuthus doriae and Androctonus crassicauda scorpion venom with the aim of looking ahead and determining the structural and functional characteristics of these compounds for use as medical tools. Molecular weight determination of isolated proteins was performed in order to better describe the B. saulcyi, O. doriae and A. crassicauda raw toxin proteins by both polyacrylamide electrophoresis and two-dimensional electrophoresis. 2D-PAGE data from the B. saulcyi, O. doriae and A. crassicauda raw toxin showed a molecular weight between 3.6 and 205 kDa (for B. saulcyi), 6.6 to 205 kDa (for O. doriae) and 6.6-109 kDa (for A. crassicauda). Then 14, 14 and 21 fractions of crude toxins were isolated using HPLC and their protein content was estimated for B. saulcyi, O. doriae and A. crassicauda, respectively. SDS-PAGE analysis of selected fractions of crude toxin showed 9 protein bands with a molecular weight between 13 and 217 kDa for B. saulcyi, 10 protein bands with a molecular weight between 3.8 to182 kDa for O. doriae and 5 protein bands with a molecular weight between 5.99 and 41.65 kDa for A. crassicauda. In case of B. saulcyi, the fraction 7 (F7) showed more cytotoxicity than other isolated fractions. Subsequently, the amino acid sequencing of fraction 7 led us to two protein bands designated as p3 and p4 peptide. For O. doriae, the peptide fraction of F17, obtained from the crude venoms of O. doriae scorpion, was found to be more cytotoxic than the crude venoms and other isolated fractions. Furthermore, F5 demonstrated significant anti-proliferative and apoptotic activity. Therefore, we performed PAGE on fraction F5 and found 5 protein bands. The two protein bands, each from fraction F5 that marked as P1 and P2 were selected for amino acid sequencing. The three peptide fractions F17, obtained from the crude venoms of A. crassicauda scorpion, was found to be more cytotoxic than the crude venoms and other isolated fractions. Furthermore, F17 demonstrated significant anti-proliferative and apoptotic activity. This makes this fraction better candidate for searching the peptide that might be used for selective killing of cancerous cells. Therefore, we performed PAGE on fractionsF17, and found 2 protein bands. One protein band from fraction F17 that marked as P5 was selected for amino acid sequencing. Finally, these protein bands were removed and molecular mass and amino acid sequence analysis was performed using Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). In-silico studies of P1, P2, P3, P4 and P5 for protein sequence alignment showed the most similarity with Hemoglobin beta-2 chain protein, Chaperonin HSP60, Chrysophsin2, pheromone-bound protein 2 and Trypsin- like serine proteinase, respectively

Dataset Information

Pull-down of nuclear proteins from HGC-27 cell line

Publications

Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters.

Similar Datasets

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