---
# System prepended metadata

title: p100 tuning of the NF-kB
tags: [science, NF-kB]

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---
title: p100 tuning of the NF-kB
description: View the slide with "Slide Mode".
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###### tags: `science` `NF-kB`

## The Canonical pathway



###### Typically, cell surface receptors of the canonical pathway bind to their ligands and mediate cell signalling via the activation of the IKK complex comprising IKKα, IKKβ and IKKγ (NEMO). The activated IKK complex phosphorylates IkBs leading to its subsequent ubiquitin-tagged proteosomal degradation. The NF-kB dimers (most commonly the *$p50–RelA$* dimers) release and translocate to the nucleus, bind DNA and activate the down-stream gene transcription.

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## The Non-Canonical Pathway

###### The target for IKKα homodimers in this pathway, is instead $NF-kB2/p100$, which is phosphorylated at two C-terminal sites. However, unlike the IKBs, the inhibitory C-terminal half parts of p100 are removed. The N-terminal portion of NF-kB (the p52 polypeptide that contains the RHD) is released. As the RHD of p100 is most commonly associated with RelB, activation of this ‘alternative’ pathway results in nuclear translocation of $p52–RelB$ dimers. ######

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## Figure 1: The NF-kB System

![](https://i.imgur.com/Y56zncN.png)
* ###### In the canonical pathway, NEMO-IKK2 phosphorylates the IKBs and it falls off, releasing $RelA: NF-kB(1)$ complex, resulting in it's translocation to the nucleus.
* ###### In the non-canonical pathway, NIK-IKK1 phosphorylates p100 (there is no IKBs), causing the translocation of some RelB as $RelB:p52$ complex into the nucleus.


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## Role of p100? #1
![](https://i.imgur.com/Vj0Vzkg.png) ![](https://i.imgur.com/3wIyBkd.png)
* ###### Simulating mutant cell systems devoid of one each NF-κB regulators (IKBs), and a $Nfkb2-/-$ system, as shown in the image above.
* ###### In the absence of p100, RelB appears in the nucleus as a minor $RelB:p50$ NF-kB activity, which visibly partially compensated for the absense of immune-organogenic $RelB:p52$ functions in $Nfkb2-/-$ mice. ######


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#### Role of p100? #2

![](https://i.imgur.com/vJIbYK2.png) 
* ###### Dynamic TNF stimulation: By TNFc or TNFp, in WT as well as mutants: $Nfkb2-/-$ and p52-null systems.
* ###### Biphasic in $Nfkb2-/-$, late-phase lasting >8h.


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#### Role of p100? #3
![](https://i.imgur.com/hagiboY.png)
* ###### Description of 4 NF-κB heterodimers: $RelA:p50$, $RelA:p52$, $RelB:p50$, $RelB:p52$.

* ###### Experimental data: NF-κBn activity consisted of mostly $RelA:p50$ in WT, while, $RelA:p52$ and $RelB:p50$ were negligible. In $Nfkb2-/-, however, $RelB:p50$ was predominant in the later phase.



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## Verification-EMSA

![](https://i.imgur.com/RFPfIEh.png)
* ###### Treated MEFs, immortalized using NIH 3T3 protocol, with TNFc/p gave similar results.



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## Verification-EMSA
![](https://i.imgur.com/hunMS40.png)

* ###### Dimer composition?: Shiftablation assay proved the predominance of $RelA:p50$ in WT, and a late-phase predominance of $RelB:p50$ in $Nfkb2-/-$.



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### Verification-EMSA


###### *"Our studies suggested that p100 imparted dynamical NF-κB control by preventing late-acting RelB:p50 response to shortlived IKK2 signals generated by pro-inflammatory cytokines."*

###### *"However, deficiency of p100 and that of the well-articulated negative feedback regulator, IκBα caused distinct aberrations with respect to the temporal profile and the composition of the  signal-induced nuclear NF-κB activity."*


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## Bio-chemical mechanism for late acting RelB:p50 in Nfkb2-/-
![](https://i.imgur.com/FoBgzU2.png)

###### * Model parameters, divided into _48_ distinct groups
###### * Monte Carlo simulation to explore parameter space surrounding the nominal values simultaneously among the different parameter groups.
###### * Local sensitivity analysis of Group 5 parameters 



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## Group 5 parameters?!

![](https://i.imgur.com/csIyWJn.png)
*  ###### Highest total effect index
*  ###### Dominant role in determining the late RelB:p50 response
*  ###### Rate parameters associated with NF-κB driven and constitutive syntheses of _RelB_ mRNA as well as translation of _Relb_ mRNA.


 

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## Results from simulation

* ###### _Computational simulations_: disruption of either RelA-mediated or RelB-driven transcription of RelB diminished the late RelB:p50 response in  p100-deficient cells.
* ###### TNFp included RelB activity only in engineered cells expressing RelB from the NF-kB promoter, but not in cells expressing RelB from the constitutive promoter.
* ###### Suggested that NF-kB induced synthesis of RelB was required for triggering late-acting RelB:p50 response to TNFp in absence of p100.



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## Microarray analysis
![](https://i.imgur.com/Nz1HdaZ.png)
![](https://i.imgur.com/X9wrAsl.png) 
* ###### 304 NF-kB-dependent genes were found.
* ###### Based on DE in Relb-/-Nfkb2-/- and Rela-/- Nfkb2-/- MEFs, they were grouped into 6 distinct clusters, which were further arranged into 4 gene-groups.
* ###### Following microarray analysis, these gene-groups were subjected to GO (BP) analysis which showed their roles in the canonical pathway: Group 1 and 2 were enriched in innate and adaptive immune responses. 
* ###### Group 3 and 4 were enriched in cellular differentiation, aging, cell death and metabolic processes. They scored poorly in immune response.

![](https://i.imgur.com/DQI9DtX.png)

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## ChIP-seq analysis and results

* ###### Chromatin level binding: Overlapping gene control of RelA and RelB
* ###### Nfkb2-/- MEFs to TNFc, 6 h and ChIP using anti-RelA and anti-RelB antibodies.
* ###### Top 2077 RelA binding peaks and top 2241 RelB binding peaks were measured, and indeed they bound to overlapping regions.
* ###### Browser track of selected genes were examined and it revealed distinct sets of RelA, RelB important genes


![](https://i.imgur.com/DaRrths.png)


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## Discussion

![](https://i.imgur.com/bcePZ81.png)

* ###### The canonical pathway involves RelA:p50 heterodimers, when stimulated by TNF. Brief and chronic stimulation induced transient/ long-lasting activites of the same, respectively.
    
* ###### p100, known for involving in the non-canonical pathway mediates activation of RelB heterodimers. Absence of p100 invokes a late-phase NF-kB activity, not by RelA:p50, but by RelB:p50. To study this, the gene Nfkb2 that encodes the p100 mRNA was knocked out, and it was seen that a subset of immune response genes were still being activated, along with additional RelB-related genes. 
    
* ###### In response to periodic TNFp, unnecessary RelA:p50 activity was controlled, however, the late-phase RelB:p50 activity strengthened in p100 deficient cells.
    
* ###### In the absence of p100, some RelB is sequestered in the cytoplasm by ikBa, while some migrated to the nucleus to produce a minor RelB:p50 activity. Chronic treatment, however degraded ikBa, which increases the otherwise weak RelB:p50 activity in the nucleus. This paralleled the RelA:p50 activity with a ~1h peak activity and a late ~8h activity.
    
* ###### ChIP studies revealed that RelA and RelB bind to the same chromatin loci associated with RelB-important genes.
    
* ###### It was suggested that the varied cellular abundance of p100 might provide a mechanism of tuning TNF responses involving RelB:p50. In addition, Nfkb2 was seen to be frequently mutated in cancer cells, with reported aberrant TNF signalling.
    
* ###### p100 depletion enabled late-acting RelB-dependent expression of pro-survivalgenes in myeloma cells. Furthermore, altered metabolism was also noticed.



