2 research outputs found
p53 oligomerization status modulates cell fate decisions between growth, arrest and apoptosis
<p>Mutations in the oligomerization domain of p53 are genetically linked to cancer susceptibility in Li-Fraumeni Syndrome. These mutations typically alter the oligomeric state of p53 and impair its transcriptional activity. Activation of p53 through tetramerization is required for its tumor suppressive function by inducing transcriptional programs that lead to cell fate decisions such as cell cycle arrest or apoptosis. How p53 chooses between these cell fate outcomes remains unclear. Here, we use 5 oligomeric variants of p53, including 2 novel p53 constructs, that yield either monomeric, dimeric or tetrameric forms of p53 and demonstrate that they induce distinct cellular activities and gene expression profiles that lead to different cell fate outcomes. We report that dimeric p53 variants are cytostatic and can arrest cell growth, but lack the ability to trigger apoptosis in p53-null cells. In contrast, p53 tetramers induce rapid apoptosis and cell growth arrest, while a monomeric variant is functionally inactive, supporting cell growth. In particular, the expression of pro-arrest <i>CDKN1A</i> and pro-apoptotic <i>P53AIP1</i> genes are important cell fate determinants that are differentially regulated by the oligomeric state of p53. This study suggests that the most abundant oligomeric species of p53 present in resting cells, namely p53 dimers, neither promote cell growth or cell death and that shifting the oligomeric state equilibrium of p53 in cells toward monomers or tetramers is a key parameter in p53-based cell fate decisions.</p
Expression, Purification, and Properties of a Human Arachidonoyl-Specific Isoform of Diacylglycerol Kinase
Diacylglycerol
kinase ε (DGKε) catalyzes the phosphorylation
of diacylglycerol, producing phosphatidic acid. DGKε demonstrates
exquisite specificity for the acyl chains of diacylglycerol. This
contributes to the enrichment of particular acyl chains within the
lipids of the phosphatidylinositol cycle. Phosphatidylinositol is
highly enriched with 1-stearoyl-2-arachidonoyl, which is important
for maintaining cellular health. Dysregulation of DGKε perturbs
lipid signaling and biosynthesis, which has been linked to epilepsy,
Huntington’s disease, and heart disease. Recessive loss-of-function
mutations in the DGKε gene cause atypical hemolytic uremic syndrome.
Because DGKε has never been purified, little is known about
its molecular properties. We expressed human DGKε and a truncated
version lacking the first 40 residues (DGKεΔ40) and purified
both proteins to near homogeneity using nickel affinity chromatography.
Kinase activity measurements showed that both purified constructs
retained their acyl chain specificity for diacylglycerol with an activity
level comparable to that of N-terminally FLAG epitope-tagged forms
of these proteins expressed in COS7 cells. Both constructs lost activity
upon being stored, particularly upon freezing and thawing, which was
minimized by the addition of glycerol. Circular dichroism revealed
that DGKε and DGKεΔ40 both contain significant amounts
of α-helical and β structure and exhibit biphasic thermal
denaturations. The loss of secondary structure upon heating was irreversible
for both constructs, with relatively little effect of added dioleoylphosphatidylcholine.
The addition of 50% glycerol stabilized both constructs and facilitated
refolding of their secondary structures after heating. This is the
first successful purification and characterization of DGKε’s
enzymatic and conformational properties. The purification of DGKε
permits detailed analyses of this unique enzyme and will improve our
understanding of DGKε-related diseases