Regulation of SIGNALLING PATHWAYS INVOLVING NUCLEAR FACTOR KAPPA B

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a p50 DNA binding protein and a p65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. However, this transcription factor plays a role in the induction of diverse sets of genes throughout the body in response to hundreds of different inducers. Overall, there are 5 Rel protein subunits, including p50 and p65, that form a variety of hetero- or homodimers that have been widely studied and have important roles in the immune system. Nevertheless, very little is known about the kinetics of NF-kB dimer formation or the stability or interconversion of dimerized Rel subunits. We investigated the biophysical properties that underlie the frequency of NF-kappaB dimer populations to gain a better understanding of the assembly dynamic and equilibrium of these vital proteins in their cellular context using a combination of techniques such as gel filtration, chemical cross-linking, and native mass spectrometry. Our results found that the core p50 and p65 homodimeric components of NF-kappaB routinely exchange subunits with a half-life of less than ten minutes at physiological temperature, highlighting a dramatic preference for the formation of the p50/p65 heterodimer. The heterodimer was more kinetically stable than either homodimer. Our data also showed the relative proportion of NF-kappaB Rel subunits themselves, as opposed to the abundance of inhibitory subunits like I-kappaB alpha, may produce an inherent degree of DNA binding-site selectivity. Despite the preference for formation of the p50/p65 heterodimer in vitro, additional experimentation is needed to confirm this phenomena in vivo. A better understanding of these biophysical properties is essential to further understanding and probing of NF-kappaB activity, which has important implications for our understanding of how this transcription factor system controls genes in the healthy and diseased immune system.