Title
Site-specific amino acid substitution in dodecameric peptides determines the stability and unfolding of c-MYC quadruplex promoting apoptosis in cancer cells
Author
Pallabi Sengupta, Nilanjan Banerjee, Tanaya Roychowdhury, Anindya Dutta, Samit Chattopadhyay, Subhrangsu Chatterjee
Year
2018
Journal
Nucleic Acids Research
Abstract
c-MYC proto-oncogene harbours a transcriptioninhibitory
quadruplex-forming scaffold (Pu27) upstream
P1 promoter providing anti-neoplastic therapeutic
target. Previous reports showed the binding
profile of human Cathelicidin peptide (LL37)
and telomeric G-quadruplex. Here, we truncated
the quadruplex-binding domain of LL37 to prepare
a small library of peptides through site-specific
amino acid substitution. We investigated the intracellular
selectivity of peptides for Pu27 over
other oncogenic quadruplexes and their role in cMYC
promoter repression by dual-luciferase assays.
We analysed their thermodynamics of binding
reactions with c-MYC quadruplex isomers (Pu27,
Myc22, Pu19) by Isothermal Titration Calorimetry.
We discussed how amino acid substitutions and
peptide helicity enhanced/weakened their affinities
for c-MYC quadruplexes and characterized specific
non-covalent inter-residual interactions determining
their selectivity. Solution NMR structure indicated
that KR12C, the best peptide candidate,
selectively stabilized the 5
-propeller loop of cMYC
quadruplex by arginine-driven electrostaticinteractions
at the sugar-phosphate backbone while
KR12A peptide destabilized the quadruplex inducing
a single-stranded hairpin-like conformation. Chromatin
immunoprecipitations envisaged that KR12C
and KR12A depleted and enriched Sp1 and NM23-
H2 (Nucleoside diphosphate kinase) occupancy at
Pu27 respectively supporting their regulation in stabilizing
and unfolding c-MYC quadruplex in MCF7
cells. We deciphered that selective arresting of
c-MYC transcription by KR12C triggered apoptoticsignalling
pathway via VEGF-A-BCL-2 axis.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, DNA binding, Thermal stability, Thermodynamics, Biochemistry