UHRF1 is an essential epigenetic regulator implicated in the maintenance of DNA
methylation. While its functional state has been suggested to be allosterically regulated
by phosphatidylinositol 5-phosphate and dependent on purification conditions and tags
coupled to the protein, the expression system might have a broader impact on UHRF1s
interaction properties. We hypothesized that the translation kinetics defined by the
expression host has an impact on the folding process of the protein, which ultimately
affects its structure and function. To test this idea, the cDNA of UHRF1 was recoded in
order to generate optimized and harmonized sequences that were expected to alter the
overall translation speed. Both proteins were expressed in Escherichia coli BL21-DE3 and
their interaction profiles with H3K9me3 and unmodified H3 peptides were determined
by microscale thermophoresis assays. The dissociation constants were compared by ttests in order to evaluate a possible change in the interaction properties of the
optimized and harmonized proteins, compared to non-optimized UHRF1 expressed in E.
coli BL21-DE3. While no difference was found for the interaction of optimized UHRF1
with the H3K9me3 peptide, a significant difference was found for its interaction with the
unmodified H3 peptide. Moreover, both the interactions of harmonized UHRF1 with
H3K9me3 and unmodified H3 peptides were determined to change. For this reason, we concluded that translation kinetics dependent on the expression system impacts the
functional state of UHRF1.
To further study this phenomenon, we expressed the consensus sequence of
UHRF1 in Escherichia coli BL21-Codon Plus-(DE3)-RIL, a bacterial strain that is enriched
with arginine, isoleucine, and leucine tRNA isoacceptors. Differences in its interaction
profile with histone peptides were found when compared with UHRF1 expressed in
Escherichia coli BL21-DE3. Since the major difference between these strains is the
abundance of tRNAs, we obtained further findings that support our initial hypothesis.
Additionally, the interaction profiles from the consensus UHRF1 protein were
determined in the presence of PI5P to get an insight into how this phosphoinositide
might impact the final structure and function of UHRF1. MST measurements and limited
proteolysis assays led us to the idea of a partially open conformation for the UHRF1
expressed in E. coli BL21-DE3 and E.coli Codon Plus-(DE3)-RIL.
|Date of Award||May 2018|
- Biological, Environmental Science and Engineering
|Supervisor||Wolfgang Fischle (Supervisor)|