Protein:protein interaction between Interferon Regulatory Factor (IRF-1) and necleophosmin (NPM1)
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Date
02/07/2016Item status
Restricted AccessEmbargo end date
31/12/2100Author
Neelagandan, Kalainanghi S.
Metadata
Abstract
Interferon Regulatory Factor -1 (IRF-1) is a transcription factor that acts as a tumour
suppressor in cancer cells. The inactivation or deletion of IRF-1 either in one or both
allele has been frequently reported in leukaemia and myelodysplasia (MDS). On the
other hand nucleophosmin (NPM), a nucleo-cytoplasmic shuttling phosphoprotein is
also known to be aberrant in some form of leukaemia. NPM was first proposed as a
binding partner of IRF-1 in 1997 and suggested to inactivate IRF-1 by inhibiting its
DNA binding ability. No further researches on the interaction between IRF-1 and
NPM1 was reported prior to the start of my PhD. In the research presented here the
interaction and mechanism by which IRF-1 might be inactivated by NPM was studied.
Under the context of both NPM and IRF-1 being frequently associated with leukaemia
and MDS, the study was done to determine the role of NPM under its naïve condition
and a most frequent mutated condition (NPMc+), where the C-terminal of NPM was
frequently mutated to give rise to a cytoplasmic NPM in certain leukaemia.
In this current research, the direct interaction between IRF1 and NPM was further
confirmed both in vitro as well as within the cells. Following this, the effect of
this interaction in respect to the leukaemic condition having NPMc+ mutation was
done, by comparing the end results on AML2 (leukaemic cells with intact wild type
NPM) and AML3 (leukaemic cells having a single NPM allele mutated to form
NPMc+) cells. In this research, overexpression of wild type NPM (NPMwt) was found
to increase IRF-1 transcriptional activity. On further analysis, the DNA binding activity
of IRF-1 due to the presence of NPMwt or NPMc+ was not always inhibited, instead
it shows a change in binding specificity, where NPMwt bound IRF-1, lacks DNA
binding ability and DNA bound IRF-1 has a reduced binding towards NPM. This is
being studied further in terms of NPM overexpression and increased IRF-1 transcriptional activity, as the order of addition (order of interaction in vivo) plays a major role
in activating or deactivating IRF-1. This along with the increased transcriptional activity
of IRF-1 suggests a novel function of NPM, where it could act in favour of IRF-
1 activity. Additionally, the NPM induced change in IRF-1 localisation was confirmed
by the cytoplasmic localised IRF-1 in NPMc+ expressing cells and nucleolar sequestration
in NPMwt overexpressing cells. This gives a novel mechanism by which NPM
regulates IRF-1. Further, the NPMc+ specific colocalisation of IRF-1 urges to study
the other proteins that may have been re-localised in AML cells due to the NPMc+
specific interaction. A mass spectrometric analysis on the cellular distribution of total
proteins were analysed between AML2 (cells with NPMwt) and AML3 (cells containing
NPMc+). Specific proteins related to cancer have been identified to be differentially
distributed rather than being a random translocation.
With this being said, a peptide phage display technology coupled with next
generation sequencing was done to identify NPMwt binding peptides that can be used
in drug discovery process or as small molecule inhibitors or activators. Three different
peptides were selected at the end of the study that bind very effectively to NPMwt.
These peptide can either aid or restrict NPM activity and need to be validated and
studied in the future.