MassIVE MSV000081926

Partial Public PXD008668

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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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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Kelch-like protein 6 (KLHL6) ; diffuse large B-cell lymphoma (DLBCL) ; Cullin-Ring ubiquitin ligase (CRL) ; mRNA decay factor Roquin2

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens
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