Post-translational regulation of Nanog and Nanog-interacting proteins in mouse embryonic stem cells
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Date
30/11/2012Author
Roy, Marcia Michelle
Metadata
Abstract
Pluripotent embryonic stem cells (ESCs) possess an unlimited capacity for
self-renewal. This property of ES cells is both defining and unique. Harnessing this
potential of ESCs would provide tremendous opportunity in the field of regenerative
medicine and its attempts to combat degenerative diseases such as Parkinson’s,
muscular dystrophy, etc.
In 2006, Shinya Yamanaka was able to demonstrate that the ectopic
expression of four proteins could reverse the process of differentiation and provide
somatic cells with the characteristics ESCs. One year later, James Thompson’s group
proved the same feat could be accomplished in human somatic cells using a different
set of four proteins, including Nanog. The prospect of converting one’s own cells
into a stem cell which could subsequently differentiate and repopulate an area of the
body afflicted by gross degeneration was revolutionary. In the years following
Yamanaka’s and Thompson’s discoveries, however, there has been little insight
gained into how these proteins are regulated post-translationally.
In this study, four proteins which had previously been identified by
Yamanaka as being ‘pluripotency factors’ were used as baits in order to ascertain a
protein-protein interaction network. This network was subsequently interrogated
using various chemical compounds and small molecules in order to dissect the signal
transduction pathways feeding into pluripotency, as well as, post-translational
modifications regulating the factors themselves.
In this way, the chemical inhibitor H89 was found to decrease the presence of
Nanog phosphorylation and possibly its dimerization resulting in the Nanog protein
being destabilized and targeted for degradation. Inversely, the pan-cullin inhibitor
MLN4924 was identified to increase the abundance of both phosphorylated Nanog
and total Nanog protein. In an attempt to identify the Cullin Ring Ligase (CRL)
responsible for the degradation of Nanog protein in ESCs, each cullin identified in
the protein interaction network was inhibited using specific shRNAs. Quantitative
fluorescence microscopy was performed and identified that inhibition of CUL3
increases Nanog protein levels, suggesting that a CUL3-based CRL may be
responsible for the post-translation regulation of Nanog.
Additionally, the quantitation of Sox2 protein levels in CUL4B shRNA cell
line demonstrates that Sox2 protein levels may be regulated by a CUL4B-based
CRL. Further studies will reveal whether or not CUL4A depletion also results in
elevated Sox2 protein levels. If not, this would include the pluripotency factor Sox2
among the recently identified CUL4B-isoform-specific substrates for degradation
and possibly provide the basis for a hypothesis of developmentally regulated
substrate specificity.
In addition to MLN4924, several other small molecules were identified as
being able to increase phospho-Nanog protein levels in this study. Among them were
the cell permeable peptides Ht-31 and PKI (14-22) amide. These peptides were found
to both stabilize phospho-Nanog and produce ES cell colonies that uniformly express
the Nanog protein. The development of a growth medium containing these peptides
in order to maintain homogeneous pluripotent ES cells is currently in progress and
received backing for a patent application by the University of Edinburgh on February
23, 2012.