Dataset Information

Site-selective incorporation of phosphorylated tyrosine into the p50 subunit of NF-κB and activation of its downstream gene CD40.

ABSTRACT: The NF-κB family of transcriptional activators is responsible for the expression of numerous genes that control key functions such as cell development and survival. Subunit p50 has been studied extensively and is known to include 13 tyrosines, but the extent and pattern of tyrosine phosphorylation that accompanies p50 function has not been defined in the literature, especially at the level of selectivity of gene expression. In this study, phosphorylated tyrosine (pTyr) was site-selectively incorporated into the p50 subunit using an E. coli in vitro expression system containing a modified ribosome. In human T cells, the NF-κBs containing a pTyr at position 60 or 82 of p50 strongly increased the expression of CD40, which is a potential target for cancer or viral immunotherapy. Promoter DNA binding was studied for CD40 promoters, and verified two pTyr residues in NF-κB p50/p65 heterodimers that facilitated this process, and that support the possible importance of phosphorylation stoichiometry. This study defines a new approach for studying tyrosine residues whose phosphorylation alters protein binding to DNA promoters, and contributes to the facility of DNA expression.

SUBMITTER: Chen S

PROVIDER: S-EPMC8843104 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Site-selective incorporation of phosphorylated tyrosine into the p50 subunit of NF-κB and activation of its downstream gene CD40.

Chen Shengxi S Ji Xun X Dedkova Larisa M LM Hecht Sidney M SM

Chemical communications (Cambridge, England) 20211125 94

The NF-κB family of transcriptional activators is responsible for the expression of numerous genes that control key functions such as cell development and survival. Subunit p50 has been studied extensively and is known to include 13 tyrosines, but the extent and pattern of tyrosine phosphorylation that accompanies p50 function has not been defined in the literature, especially at the level of selectivity of gene expression. In this study, phosphorylated tyrosine (pTyr) was site-selectively incor ...[more]

PMID: 34766616

Similar Datasets

Project description:The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.

Project description:The unregulated activation of nuclear factor-κB (NF-κB) is a critical event in the progression of various inflammatory diseases such as ulcerative colitis, asthma, rheumatoid arthritis, bacterial induced gastritis, etc. Hence, blocking the transcriptional activity of NF-κB is a promising strategy towards the development of an anti-inflammatory agent. In this study, an integrated molecular and quantum mechanical approach was carried out to find a new potent andrographolide (AGP)-based analog that can inhibit DNA binding to NF-κB p50 and manifest anti-inflammatory activity. Our approach includes multiple sequence alignment, virtual screening, molecular docking (protein-ligand and protein-DNA), in silico site-directed mutagenesis, ADMET prediction, DFT (HOMO, LUMO, HLG, and EPM energy) analysis, MD simulation, and MM/GBSA rescoring. The virtual screening analysis of 237 AGP analogs yielded the five lead compounds based on the binding affinity. Further, molecular interactive docking and ADMET prediction of hit analogs revealed that Ana2 ((3Z,4S)-3-[2-[(4aR,6aS,7R,10aS,10bR)-3,3,6a,10b-tetramethyl-8-methylidene-1,4a,5,6,7,9,10,10a-octahydronaphtho[2,1-d][1,3]dioxin-7-yl]ethylidene]-4-hydroxyoxolan-2-one) is the most potent moiety as it displays the strongest binding affinity and better molecular/pharmacokinetic features. Moreover, DFT, MD simulation, and MM/GBSA studies corroborated the docking results and demonstrated better chemical and dynamic stability with the least binding free energy (- 29.99 kcal/mol) for the Ana2. Site-directed mutagenesis investigation (Cys62Ala) establishes the importance of the Cys62 amino acid residue towards the binding interaction and stability of Ana2 with NF-κB p50. Overall, the identified NF-κB p50 inhibitor opens up a new research horizon towards the development of plant-based anti-inflammatory drugs to combat progressive inflammatory diseases.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-022-03431-9.

Project description:Phosphorylated proteins play important roles in the regulation of many different cell networks. However, unlike the preparation of proteins containing unmodified proteinogenic amino acids, which can be altered readily by site-directed mutagenesis and expressed in vitro and in vivo, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for in vitro studies, we have explored the use of phosphorylated amino acids in which the phosphate moiety bears a chemical protecting group, thus eliminating the negative charges that have been shown to have a negative effect on protein translation. Bis-o-nitrobenzyl protection of tyrosine phosphate enabled its incorporation into DHFR and I?B-? using wild-type ribosomes, and the elaborated proteins could subsequently be deprotected by photolysis. Also investigated in parallel was the re-engineering of the 23S rRNA of Escherichia coli, guided by the use of a phosphorylated puromycin, to identify modified ribosomes capable of incorporating unprotected phosphotyrosine into proteins from a phosphotyrosyl-tRNACUA by UAG codon suppression during in vitro translation. Selection of a library of modified ribosomal clones with phosphorylated puromycin identified six modified ribosome variants having mutations in nucleotides 2600-2605 of 23S rRNA; these had enhanced sensitivity to the phosphorylated puromycin. The six clones demonstrated some sequence homology in the region 2600-2605 and incorporated unprotected phosphotyrosine into I?B-? using a modified gene having a TAG codon in the position corresponding to amino acid 42 of the protein. The purified phosphorylated protein bound to a phosphotyrosine specific antibody and permitted NF-?B binding to a DNA duplex sequence corresponding to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated I?B-? also mediated the exchange of exogenous DNA into an NF-?B-cellular DNA complex isolated from the nucleus of activated Jurkat cells.

Dataset Information

Site-selective incorporation of phosphorylated tyrosine into the p50 subunit of NF-κB and activation of its downstream gene CD40.

Publications

Site-selective incorporation of phosphorylated tyrosine into the p50 subunit of NF-κB and activation of its downstream gene CD40.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets