NF-κB Rel subunit exchange on a physiological timescale.

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Signaling

Background:

• Nuclear Factor kappa B (NF-κB) transcription factors regulate gene expression and are involved in immune responses and cell survival.
• The NF-κB complex comprises various Rel subunits that form homo- and heterodimers, but their assembly kinetics and stability are poorly understood.
• Understanding NF-κB dynamics is crucial for deciphering cellular signaling pathways and developing targeted therapies.

Purpose of the Study:

• To investigate the dissociation and reassociation kinetics of NF-κB p50 and p65 Rel subunits.
• To determine the stability and formation preference of NF-κB homo- and heterodimers.
• To propose a new model for NF-κB activity modulation based on subunit dynamics.

Main Methods:

• Utilized a soluble expression system for efficient production of NF-κB Rel subunits.
• Employed biophysical techniques to analyze protein-protein interactions and complex formation.
• Verified canonical nucleic acid binding properties of the expressed NF-κB proteins.

Main Results:

• NF-κB homodimers (p50/p50 and p65/p65) exhibit rapid subunit exchange with half-lives under 10 minutes at physiological temperatures.
• The p50/p65 heterodimer demonstrates significantly greater kinetic stability, at least ten times that of homodimers.
• Formation of homodimers is transient, suggesting specific DNA targets are primarily bound by heterodimers or require exclusive subunit expression.

Conclusions:

• NF-κB subunit exchange dynamics favor the formation of a stable p50/p65 heterodimer.
• Cellular NF-κB activity can be modulated by controlling the relative abundance and expression timing of p50 and p65 subunits.
• This provides a quantitative framework for understanding Rel dimer distribution and modeling NF-κB pathway regulation.