This document discusses the role of NF-κB in regulating apoptosis. It describes how NF-κB is a transcription factor that regulates the expression of many anti-apoptotic genes. The regulation of NF-κB involves its interaction with the inhibitory protein IκB and transport between the cytoplasm and nucleus. Phosphorylation of IκB leads to its degradation and the release of NF-κB to enter the nucleus and activate gene transcription. This tight regulation of NF-κB activity and localization determines whether a cell survives or undergoes apoptosis.

1. “To be or not to be”
A structural approach to the regulation of NF-κB in Apoptosis
By
Rajendra K Kothinti

2. outlineoutline
 Introduction: ApoptosisIntroduction: Apoptosis
 NF-kB role in ApoptosisNF-kB role in Apoptosis
 Structure and Function: NF-kBStructure and Function: NF-kB
 Regulation of NF-kBRegulation of NF-kB
 ConclusionConclusion

3. CELL DEATH
Cell death:
 non programmed
(e.g., Necrosis)
 Programmed way
(e.g., Apoptosis)
Characteristics :
 DNA
 Membrane integrity
 Inflammation
 Cell volume
 Cell fragmentation

4. Why Apoptosis In cells ?
 To maintain cell homeostasis a balance between the increase
and decrease of cell population has to be efficiently balanced.
 Need to destroy cells that represent a threat to the integrity of
the organism.
 Formation of the proper connections (synapses) between
neurons in the brain during development.
 Formation of the fingers and toes of the fetus requires the
removal of the tissue between them.

5. Apoptosis
 This phenomenon recognized prior by Carl Vogt over a 150 years
ago in studying mid wife toad development.
 John Kerr et al. in 1972,
“The mechanism responsible for the physiological deletion of
cells and appears to be intrinsically programmed”.
 Some times used synonymous to programmed cell death (PCD).

6. Too much apoptosis:
- Degenerative diseases,
- Parkinson’s,
- Alzheimer’s,
- Spinal Muscular atrophy,
- AIDS, etc.
Too little apoptosis:
- Cancer (by virus infection or by DNA mutations)
- Autoimmune diseases like diabetes type-I.
Pathological significance

7. What is NF-kB?
“Nuclear Factor Kappa B”
A ~300 kDa protein which is a
latent, primary transcription
factor ubiquitously found in all
cell types.
-Sidney Harris

8. What is Transcription?
DNA sequence is enzymatically
copied by an RNA polymerase to
produce a complementary RNA.
In other words, the transfer of
genetic information from DNA into
RNA which subsequently
translates to protein in cytoplasm
www.scientificpsychic.com/.../aminoacids1.html

9. What is a Transcription factor?
 Protein that binds DNA at a
specific promoter or enhancer
region where it regulates
transcription.
 These are selectively activated
or deactivated by other proteins
 They regulate of many different
gene products. (proteins)
promoter
Genes for
different Protein.
ds-DNA
Transcription
Factor
m-RNA
Protein
RNA polymerase
Translation
n
Cytoplasm
Nucleus

10. outlineoutline
 Introduction: ApoptosisIntroduction: Apoptosis
 NF-kB role in ApoptosisNF-kB role in Apoptosis
 Regulation of NF-kBRegulation of NF-kB
 Structure and Function: NF-kBStructure and Function: NF-kB
 ConclusionConclusion

11. General Picture of Apoptosis & NF-kB role
Anti
Apoptotic
Pro-
Apoptotic
NF-kB
(A Transcription
factor Protein)
+
Cell surface
Receptor
UV, Free
radicals,
Metals
Cytokines,
TNFα,
viral proteins,
and Toxins
mitochondria
Gene products
( proteins)
RNA Pol
m-RNA
Cytochrome C
Aptosome
Complex
APOPTOSIS
Inhibitor
(IKB)
latent
Kinase
IkB

12. Biochemical Pharmacology
Volume 68, Issue 6 , 15 September 2004, Pages 1071-1080
NF-kB role in Apoptosis
 The majority of gene products
made by NFkB prevents
apoptosis
 The dynamic bias of NF-kB for
making anti- apoptotic gene
products is critical for cell
survival.

13. General Picture of Apoptosis and NF-kB role
Anti
Apoptotic
Pro-
Apoptotic
NF-kB
(A Transcription
factor Protein)
+
Cell surface
Receptor
Metals
mitochondria
Gene products
( proteins)
RNA Pol
m-RNA
Cytochrome C
Aptosome
Complex
APOPTOSIS
Inhibitor
Inactive

14. IkB specific kinase is sequestered
in cytoplasm.
NFkB activation occurs only in
cytoplasm.
ATP is required for activation.
Ubiquitination of IkB initiates by
26s proteosome proteolysis
(Ub) n
_
_
NFkB Activation
2
2

15.  Forms a immunoglobulin like fold
(beta barrel) at N and C terminal
connected by a linker loop.
 N’ terminal Specificity Domain of
~190 amino acids region is
responsible for DNA specific
interactions
 C’ terminal dimer interface
responsible for Dimer formation and
DNA non specific binding.
NF-kB Architecture
Structure 15 February 1995, 3:135-141
DNA
Miller

16. Protein - DNA Interaction
Protein + DNANS
Protein.DNANS
Protein.DNANS + DNAS Protein.DNAS + DNANS
Here the Protein is NFkB which is a transcription factor
DNA specific contacts: Made using DNA bases (Adenine , Guanine,
thymine and cytosine).
DNA non Specific contacts:
Are made using de oxy ribose sugar and Phospho
diester backbone.

17. NF-kB Interacts specifically with
DNA using loop region.
Binds in the major groove of the
DNA.
consensus sequence:
5’GGGGACTTTC-3’
3’CCCCTGAAAG-5’
Minor groove
NF-kB Binding to DNA Specifically
Major groove
NF-kB interacting in
major groove

18. NF-kB Nucleotide Base Specific Contacts
Structure January 1999, 7:R1–R6
P65-monomer–DNA specific
Interactions
P50-monomer-DNA Specific
interactions

19. Van der Waals Interactions
Tyrosine
Gel retardation assay
Proc. Nati. Acad. Sci. USA
Vol. 91, pp. 908-912, February 1994

20. Effect of DNA on NFkB bindingEffect of DNA on NFkB binding
Structure January 1999, 7:R1–R6
P65
P50

21. p65-mono+ DNA p65-DNA
p65-p50 + DNA p50-DNA-p65
K
p50-mono + DNA p50-DNA 379
K
nM
nM
nM
210
K
K
12
app
app
app
app
The enhanced binding is
found in binding to DNA
because of dimer interface.
Dimer binds stronger than
monomer to DNA and this
can be because of reduction
in entropy.
Kapp-apparent dissociation constant
Dimerisation is critical in binding of NF-kB to DNA

22. Dimerisation is critical in binding of NF-kB to DNA
 Wild-type and Y267D/H304G
 Data sets comparing dimer and
(mutant) Y267D/H304G
on a semi-logarithmic plot of concentration
versus fraction DNA bound.
DIMER
monomer
G. Ghosh, et. al, J. Biol. Chem (2002) 277, 24694–24700.
NF-kB (nM)

23. Structure of NF-kB Dimer InteractionStructure of NF-kB Dimer Interaction
N O
H
H
H
H
H
N
O
O
H
H H
Tyr-267
Val-199
N
ON
N
H
N
OS
H
H
H
H H
H
H
H
N
O
N
N
N
H
H
H
O
N
O
O
H
H H
H
H
H
H
H
H
Arg-252
Glu65
Structure January 1999, 7:R1–R6
DIMER INTERACTION WITH DNA
DNA
p50
p65
His304Cys

24. outlineoutline
 Introduction: ApoptosisIntroduction: Apoptosis
 NF-kB role in ApoptosisNF-kB role in Apoptosis
 Structure and Function: NF-kBStructure and Function: NF-kB
Regulation of NF-kBRegulation of NF-kB
ConclusionConclusion

25. Nuclear Membrane Transport
Anti
Apoptotic
IK-Kinases
IkB
Nuclear
trans port
protein
GTP
GTP ase
GTP ase
GDP
Cytoplasm
Nucleus
RNAPOL
m-RNA

26. Nuclear Membrane Transport
Anti
Apoptotic
IK-Kinases
IkB
Nuclear
trans port
protein
GTP
GTP ase
GTP ase
GDP
Nucleus
Cytoplasm
NFkBNFkB.IkB

27. Cytoplasm Nucleus
NFkB-NLS-carrier protein
Conf. 2
NFkB-NLS-carrier protein
Conf. 1
GTP
Carrier protein-GTP
Active
NFkB-IkB-NES
NFkB
NFkB-IkB-NES-(Ser)2Carrier protein -GTP
2ATP
Inactive 2ADP
Kinase
NFkB-IkB-NES-(Ser)2
Carrier protein -GTP
Carrier protein
NFkB-IkB
GDP
Regulation of Transport
kinase
IkB-(Ser)2
P03
-
P03
-
P03
-
Active
2ATP
2ADP

28. NFkB-IkB +
Conf.1
Regulation of Activation : cytoplasm to nucleusRegulation of Activation : cytoplasm to nucleus
2ATP
IkB-(UB)n-(OPO3)2
Active
proteolysis by
Ubiquitin path
way
Carrier protein + NFkB
Conf. B
Conf.2
Conf.2
Carrier protein-NFkB
Nucleus
NFkB + IkB-(OPO3
-
)2
IkB-(OPO3
-
)2+ n (UB)
+ 2ADP +2H2O
GTP + Carrier protein-NFkB Carrier protein-GTP + NFkB
Conf.2
Active
Cytoplasm
Conf. B
Conf.2
Conf.2
Conf. B Conf. A
Transcription of anti
apoptotic protein
Kinases

29. NFkB-IKB + Carrier protein + GTP NFkB-IKB-Carrier
protein -GTP
NFkB-IKB –Carrier protein –GTP NFkB-IkB + Carrier protein+ GDP
GTP ase
activity
Regulation of Inactivation: Nucleus to CytoplasmRegulation of Inactivation: Nucleus to Cytoplasm
NFkB-IKB –Carrier
protein -GTP Nuclear pore
complex
NFkB-IKB –Carrier protein -GTP
Nucleus
cytoplasm
GTP + H20 GDP + Pi

30. Cell (1998) 95, 729–731
Nuclear localization signal masking by IkBNuclear localization signal masking by IkB
NFkB-IKB
PyMOL – PDB -1NFI
IkB Prevents the export of NF-kB from
cytoplasm to nucleus by binding to
nuclear localization signal.
Ankyrin repeat
 NLS -nuclear localizing signal
 NES - Nuclear export signal
1
2
3
4
5
6
NLS
NES

31. (SER)2-IkB-NFkB
2 ATP
2ADP
NFkB-IKB-(SER)2
(Ubiquitin) n
NLS-NFkB-IKB- (SER)2
Ub-Ub-Ub-Ub
proteolysis
DNA + NFkB
ACTIVE
IN-ACTIVE
2 ATP
2ADP
IkB
DNA
I K Kinase
Regulation of NF-kB TransportRegulation of NF-kB Transport
Cytoplasm
Nucleus
NES
NLS
Transcription of
IkB as gene
products
DNA- NFkB
CII-kinase
P03
-
GTP + carrier protein
IKB-NFkB-GTP-carrier protein
IKB-NFkB
GTPase
carrier protein
Carrier protein
IKB
P03
-
P03
-
P03
-

32. IkB an allosteric inhibitor of (NFkB-DNA)
NFkB DNA+ NFkB.DNA
+
IkB
NFkB.IkB + DNA No Reaction
Transcription
Gene Products for
the inhibition of
apoptotic path way
CELL SURVIVAL
Kd=~12nM
Kd=~ 3.0nM

33. IN ACTIVE
Structure January 1999, 7:R1–R6
Conformational change in DNA
binding site of N’ terminal p65
subunit of NF-kB is regulated
allosterically by IkB
ACTIVE
Cell, Vol. 95, 759–770, December 11, 1998
IkB an allosteric inhibitor of (NFkB-DNA)
Structure alignment of NF-kB

34. Conclusion
 NF-kB plays a critical role in deciding the cell fate.
 Regulates many of the gene products which are potential
inhibitors of apoptosis.
 Dimerisation is playing a vital role in NFkB specific recognition of
DNA.
 DNA specific interactions are contributed by loop regions.
 IkB is important inhibitor of NFkB.

35. THANK YOU
•Dr. David Petering
•Dr. Niloofar Tabatabai
•Sue Krezoski
•Jeff Meeusen
•Chris Oberle
•Ujala
•Cristin
•Wanrong