NF- κB represents a protein family of transcription factors that control many physiological processes in eukaryotes. NF- κB is activated due to many different cellular responses such as immune responses, viral infections, radiation, oxidative stress, and more. NF- κB transcription factors play an essential role in nearly every cell’s processes. Regulation is key to its various purposes with plenty of PTMs to specify cellular responses. Its recognition motif allows for site-specific binding of DNA to initiate transcription.
Click for a full representation of the protein with DNA[1].
Structure
NF- κB is generally found bound to DNA as homodimers. Generally all NF- κB proteins have a Rel domain in their N-terminus, in which can be regulated via phosphorylation. Some proteins are made up of larger precursor proteins that are shortened via proteolysis into active DNA-binding proteins.
DNA Interactions
The N-terminal domain of NF- κB contains recognition loops that interact with DNA bases. The conserved, defined recognition motif for two sequential guanines is the Arg 57, Arg 59, and Glu 63 that with DNA bases. The arginine residues are coplanar to the guanines are able to donate hydrogen bonds to O6 and N7. The glutamic acid accept hydrogen bonding from the N4 of the paired cytosines.
Phosphate interactions anchor the dimer to the DNA through hydrogen bonding. The N-terminal domain donates hydrogen bonding with the main-chain -NH of Lys 147 and also with the side chains from Tyr 60 and His 144 [2]. The C-terminal domain has Lys 275, Gln 277, Arg 308, and Gln 309 that participate in hydrogen bonding with the backbone phosphates. In general, many polar and charged amino acid such as Lys, Tyr, His, Gln, or Arg residues all play into the hydrogen bonding between the protein and DNA. occur in both the C- and N-terminal domains and are generally conserved throughout the family.
Regulation
The activation of NF- κB occurs in a few different pathways; however, the most prominent are the canonical and the non-canonical pathways. In the canonical pathway, NF- κB is normally in an inactive dimer form in the cytosol, bound to an inhibitor kappa-B protein (IκB). The binding of IκB interferes with nuclear localization signal of NF- κB, which as in case of the p50 homodimer is found *** just beyond the last ordered residues***. According to Muller et al., I-κB may inhibit DNA binding of NF-κB through a few different ways. I-κB is a large enough protein that is could interfere with NF-κB-DNA contact directly by inserting itself in the groove between the two. On the other hand, it could just interact with the N-terminal domain to change the angle needed for DNA binding.
Once a ligand binds to a cellular receptor, the signal is relayed through adaptors such as TRAFs to an IκB kinase (IKK) complex. The canonical IKK complex is built up by alpha and beta catalytic subunits and two regulatory scaffold NF-κB essential modulator (NEMO) proteins. This IKK complex is activated by clusters of adaptors, and upon activation it will phosphorylate the IκB. Once phosphorylated, IκB is subsequently ubiquitinated and degraded by the proteasome. With its inhibitor degraded, NF- κB’s nuclear localization signal is freed, allowing it to move to the nucleus and bind to κB sites of DNA to prompt transcription. These are usually 9-10 base pairs that follow the general form 5'-GGGRNWYYCC-3' (R: A or G; N: any nucleotide; W: A or T; Y: C or T)[3]. In the scene, Gs are pink, As are blue, Ts are blue, and Cs are yellow, and we see that it follows the general form well.
Alternatively, NF- κB can be activated through the non-canonical pathway, however it affects mainly p100/RelB complexes. The non-canonical pathway is initiated upon binding of very specific ligands (B-cell activating factor, CD40, etc.). This cellular signal is then passed onto the NF-κB-inducing kinase (NIK), which in turn phosphorylates and activates an alpha IKK catalytic dimer. This activated catalytic dimer phosphorylates serine residues in the C-terminal domain of p100[4]. This phosphorylation prompts partial proteolysis, creating a p52/RelB complex which will go on to enter the nucleus and bind to DNA.
Active NF- κB promotes IκB expression, creating a negative feedback loop. Since NF- κB is essential to so many processes, its regulation includes post-translational modifications to differentiate between the processes. Moreover, NF- κB response can depend on its modifications, as the degradation of IκB does not guarantee maximal activity. NF- κB, IκB, and IKK can each be modified for different purposes with phosphorylation, ubiquitination, and acetylation.
Dysregulation and deficiencies of NF- κB lead to serious consequences such as immunodeficiency, autoimmunity, or cancer.
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