Dataset Information

MudPIT analyses of the proteins associated with Kelch-like protein 6 (KLHL6) in a human multiple myeloma cell line, ARP-1, and in HEK293T cells

ABSTRACT: Kelch-like protein 6 (KLHL6) is a highly conserved and uncharacterized BTB-Kelch protein with a lymphoid tissue-restricted expression pattern. To gain insight into the molecular function of KLHL6 and to understand the impact of the cancer-associated mutations, we compared the protein interactome of KLHL6(WT) to that of the cancer mutant KLHL6(L65P), a BTB-domain mutant frequently mutated in B-cell cancers. FLAG-KLHL6(WT) or FLAG-KLHL6(L65P) complexes were biochemically immunopurified from two different cell lines (HEK293T and ARP-1).

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Laurence Florens

LAB HEAD: Laurence Florens

PROVIDER: PXD008963 | Pride | 2018-03-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Hs_FLAG-Emptyvector_293-scl_FLAG-IP_Ti_L2_1.mzid.gz	Mzid
	Hs_FLAG-Emptyvector_293-scl_FLAG-IP_Ti_L2_1.pride.mztab.gz	Mztab
	Hs_FLAG-Emptyvector_293-scl_FLAG-IP_Ti_L2_101.RAW	Raw
	Hs_FLAG-Emptyvector_293-scl_FLAG-IP_Ti_L2_101.ms2	Other
	Hs_FLAG-Emptyvector_293-scl_FLAG-IP_Ti_L2_101.pride.mgf.gz	Mgf

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Project description:Kelch-like protein 6 (KLHL6) is a highly conserved and uncharacterized BTB-Kelch protein with a lymphoid tissue-restricted expression pattern. Recently, whole-genome and exome sequencing have revealed cancer-associated mutations of the KLHL6 gene in mature B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). DLBCL is the most common lymphoid malignancy with two distinct molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB) lymphoma. How KLHL6 mutations contribute to the pathology of human DLBCL and whether they influence NF-kB activation is currently unknown. To gain insight into the molecular function of KLHL6 and to understand the impact of the cancer-associated mutations, we compared the protein interactome of KLHL6(WT) to that of the cancer mutant KLHL6(L65P), a BTB-domain mutant frequently mutated in B-cell cancers. FLAG-KLHL6(WT) or FLAG-KLHL6(L65P) complexes were biochemically immunopurified from two different cell lines (HEK293T and ARP-1). In brief, TCA-precipitated protein eluates were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. The peptide mixtures were loaded onto microcapillary fused silica columns (100um i.d.), packed with C18 reverse phase (Aqua; Phenomenx), SCX (Luna; Phenomenex) and C18-RP, placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on linear ion traps. MS/MS datasets were searched using SEQUEST (v. 27.9) against a non-redudant human protein database (NCBI, 2012-08-27) containing 160 usual contaminants. To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized. Peptide/spectrum matches were sorted and selected using DTASelect with the following criteria set: spectra/peptide matches were retained only if they had a DeltCn of at least 0.8, and minimum XCorr of 1.8 for singly, 2.0 for doubly, and 3.0 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic. In the two cell lines, high unique spectral counts corresponding to Cullin3 were identified in KLHL6(WT) immunoprecipitates as opposed to none in the KLHL6(L65P) purifications, indicating that Cullin3 is a novel KLHL6 binding partner and that a BTB-domain mutation (L65P) abolishes this interaction. Since KLHL6(L65P) is unable to promote ubiquitylation, we reasoned that it might trap (i.e.; interact with, but not ubiquitylate or degrade) natural KLHL6 substrates offering an opportunity to identify them. Thus, we ranked the identified proteins by the number of unique spectral counts associated to KLHL6(L65P) and compared them to KLHL6(WT). Roquin2 was enriched in the KLHL6(L65P) complex in both HEK293T cells and ARP-1 cells. Roquin2 is the first identified bona fide substrate of the CRL3KLHL6 E3 ubiquitin ligase complex.

Project description:Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. And quantitatively analyze the cell signaling pathways that regulated by the studied mutants. Methods: total mRNA was extracted from U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E.llumina compatible libraries were prepared by using the KAPA Stranded mRNA-Seq Library Preparation Kit for Illumina® platforms (KAPA Biosystems). In brief, 250 ng of total RNA was enriched for poly-A-tailed mRNA by using Kapa’s mRNA Capture beads. Poly-A-enriched RNA was fragmented to a median size of 150 bp by using chemical fragmentation and converted into double-stranded cDNA with dUTP incorporated into the second cDNA strand. The ends of the double-stranded cDNA were polished, 5’-phosphorylated, and 3’-A-tailed for the ligation of indexed adapters. Adapter-ligated DNA fragments were amplified by 8 cycles of PCR. The strand with incorporated dUTP was not amplified. The resulting libraries were quantified by qPCR and assessed for size distribution by using the 4200 TapeStation (Agilent Technologies), and then multiplexed, 4 per pool, and sequenced on the Illumina’s NextSeq500 by using the mid-output, 75-bp paired-end read format. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the human genome and quantified the expression of protein coding genes in the U251 cells of WT Gcn5 and Gcn5 Y645A mutant/WT DLST and DLST R224A/K226E mutant. Altered expression of 3 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that Gcn5 Y45A mutant and DLST R224A/K226E mutant, which reduce histone H3 K79 succinylation, can regulate several cell signaling pathways that important for cancer cell proliferation.

Dataset Information

MudPIT analyses of the proteins associated with Kelch-like protein 6 (KLHL6) in a human multiple myeloma cell line, ARP-1, and in HEK293T cells

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