4 DISCUSSION
In the present study, we have predicted the possible interaction of
lamin A with trf2 and telomeric repeats, thereby contribute in the
structural integrity of the nucleus. First, we studied in detail about
the wild type and mutant lamin A and its phenotypes. Based on the
literature, we hypothesized that wild type lamin A has an interaction
with telomeric repetitive sequences (TTAGGG)n. Hence, we performed
bioinformatics analysis to prove our hypothesis. Our data suggest that
the DP-bind simulation-based bioinformatics approach confirms that lamin
A has strong interaction with telomere repeats (TTAGGG)n in addition to
trf2. To support the claim, we performed similar analysis with trf2
protein, which has 542 amino acid residues27. Previous
reports clearly suggested that trf2 has strong interaction with
telomeric repeats28,29. Specifically, trf2 has DNA
binding domain at 484 to 541 amino acids. We have chosen trf2 as a
positive control for DP-bind bioinformatic tool to study the lamin A –
DNA interaction. Our data confirms that lamin A has strong interaction
with telomeric repeats.
To validate the bioinformatic data, we performed experiments to check
the lamin A and telomere interaction using DNA binding assay. According
to the report, DNA coating solution was used to anti-dsDNA measurement
for calf thymus DNA30. Our data suggest that lamin A
binds specifically to repetitive telomere DNA sequences. Similarly, as a
positive control, trf2 shows signal, which illustrates that trf2 has
capable of binding with telomere region31.
Interestingly, lamin A not only binds to telomeric repeats rather, it
interacts with trf2 of telomeric complex called as ‘shelterin’. To
confirm the data, we performed co-immunoprecipitation assay with lamin A
and trf2 antibodies, vice versa. Our data confirms that protein lysate
immunoprecipitated with lamin A antibody shows signal for both lamin A
as well as trf2. Similarly, protein lysate immunoprecipitated with trf2
antibody shows signal for both trf2 and lamin A. The data clearly
indicates that lamin A and trf2 has closely interacted with each other
in the shelterin complex. Lamin C is the splicing variant of
lamin A32, which has only 572 amino acid residues and
it lacks carboxyl terminal domain of last 98 amino acid residues of
mature lamin A. Our results show that co-immunoprecipitation of wild
type lamin A with trf2 and vice versa confirms that lamin A binds with
trf2 and not lamin C. Because as mentioned earlier, lamin C lacks the
carboxyl terminal domain of 98 amino acid residues of mature lamin A.
Based on the observation, it is clear that tail domain of lamin A plays
a crucial role in binding with shelterin complex.
Based on the data, lamin A binds with both trf2 and telomeric DNA
repeats, but specifically it is not known that whether lamin A binds to
minor or major groove of the telomeric repetitive sequences (TTAGGG)n.
Interestingly, it is known that trf2 specifically binds with telomeric
repetitive DNA sequences at major as well as minor
groove33. Hence, it may be a chance that lamin A binds
at major groove of telomeric DNA repetitive sequences. Because,
N-terminal region of trf2 binds to minor groove of telomeric repetitive
DNA sequences33. At specific stage of cell cycle, the
chromosome arrangement looks like a ring structure, which is due to the
interaction of lamin A with trf2 and telomere25. We
have performed experiments and our results conclude that lamin A binds
with trf2 rather; it is not known whether lamin A binds with any of the
sheltron complex proteins.
Fusion, degradation, recombination in chromosomes can be protected
specifically by telomere repeats34. Telomere repeats
are generated by telomerase reverse transcriptase (TERT), which
recognizes chromosome ends and synthesizes telomere repeats via de novo
using a telomerase-associated RNA molecule (TERC) as a
template35. But in the case of HGPS mutant lamin A,
premature aging occurs frequently suggest that lamin A has interactive
role with telomere. Bioinformatic data confirms that wild-type lamin A,
especially C-terminal end of 39 amino acid residues have strong
interaction with telomere region and thereby holds the aging process.
But, in the case of HGPS mutant lamin A, 39 amino acids residues were
not present and hence premature aging begins frequently. The complete
mechanism behind the premature aging is not clearly studied.
Loss of 39 amino acid residues in HGPS mutant lamin A not only cause
premature aging, but also impairs the nuclear structural integrity and
induce chromosomal fusion. The reason for chromosome fusion might be due
to; 1. Mutation in the lamin A results in loss of C terminal 39 amino
acid residues and hence, it is not specifically binding with telomeric
complexes (specifically trf2 and telomeric repetitive sequences
(TTAGGG)n); and 2. Loss of C terminal 39 amino acid residues in HGPS
mutant lamin A results in impairing in the nuclear structural integrity.
Transfection of wild type and HGPS mutant lamin A into fibroblast cells
shows that, compare to control cell, mutated cell nuclear morphology was
impaired with irregular lobulated nuclei. Loss of C terminal 39 amino
acid residues of lamin A results in structural deformities of the
nucleus. Reason for the nuclear structural impair might be due to; 1.
Loss of C terminal 39 amino acid residues of lamin A; and 2. Disturb in
the tail domain of lamin A has impact in coiling process of forming
nuclear lamina along with lamin C. Interestingly, E145K progeria patient
fibroblast cells morphology shows lobulated nucleus36.
LMNAp.R388P mutation patient cells show specific deficiencies in lamina
organization and cell explosion ability37. Similarly,
fibroblast cells overexpressing R133L and L140R mutant lamin A displayed
numerous degrees of asymmetrical nuclei [38]. Based on the above
reports, it is clear that any mutation in lamin A results in irregular
lobulated or asymmetrical nuclei, not specifically, loss of C terminal
39 amino acid residues of HGPS mutant lamin A. Based on the data, we
conclude that C-terminal 39 amino acids from tail domain of mature lamin
A possibly interact with trf2 and telomere, not lamin C and HGPS mutant
lamin A.