3 research outputs found
A Chimeric p53 Evades Mutant p53 Transdominant Inhibition in Cancer Cells
Because
of the dominant negative effect of mutant p53, there has
been limited success with wild-type (wt) p53 cancer gene therapy.
Therefore, an alternative oligomerization domain for p53 was investigated
to enhance the utility of p53 for gene therapy. The tetramerization
domain of p53 was substituted with the coiled-coil (CC) domain from
Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains
proper nuclear localization in breast cancer cells detected via fluorescence
microscopy and shows a similar expression profile of p53 target genes
as wt-p53. Additionally, similar tumor suppressor activities of p53-CC
and wt-p53 were detected by terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD),
and colony-forming assays. Furthermore, p53-CC was found to cause
apoptosis in four different cancer cell lines, regardless of endogenous
p53 status. Interestingly, the transcriptional activity of p53-CC
was higher than wt-p53 in 3 different reporter gene assays. We hypothesized
that the higher transcriptional activity of p53-CC over wt-p53 was
due to the sequestration of wt-p53 by endogenous mutant p53 found
in cancer cells. Co-immunoprecipitation revealed that wt-p53 does
indeed interact with endogenous mutant p53 via its tetramerization
domain, while p53-CC escapes this interaction. Therefore, we investigated
the impact of the presence of a transdominant mutant p53 on tumor
suppressor activities of wt-p53 and p53-CC. Overexpression of a potent
mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53,
p53-CC retains the same level of tumor suppressor activity. Finally,
viral transduction of wt-p53 and p53-CC into a breast cancer cell
line that harbors a tumor derived transdominant mutant p53 validated
that p53-CC indeed evades sequestration and consequent transdominant
inhibition by endogenous mutant p53
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. An optimal reporter for this purpose would be small to negligibly
influence intracellular processes, be readily linked to the endogenous
genes with minimal experimental effort, and be sensitive enough to
detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9,
we demonstrate that HiBiT can be rapidly and efficiently integrated
into the genome to serve as a reporter tag for endogenous proteins.
Without requiring clonal isolation of the edited cells, we were able
to quantify changes in abundance of the hypoxia inducible factor 1A
(HIF1α) and several of its downstream transcriptional targets
in response to various stimuli. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1α levels with
the hydroxyproline modification that mediates its degradation. These
results demonstrate the ability to efficiently tag endogenous proteins
with a small luminescent peptide, allowing sensitive quantitation
of the response dynamics in their regulated expression and covalent
modifications
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. An optimal reporter for this purpose would be small to negligibly
influence intracellular processes, be readily linked to the endogenous
genes with minimal experimental effort, and be sensitive enough to
detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9,
we demonstrate that HiBiT can be rapidly and efficiently integrated
into the genome to serve as a reporter tag for endogenous proteins.
Without requiring clonal isolation of the edited cells, we were able
to quantify changes in abundance of the hypoxia inducible factor 1A
(HIF1α) and several of its downstream transcriptional targets
in response to various stimuli. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1α levels with
the hydroxyproline modification that mediates its degradation. These
results demonstrate the ability to efficiently tag endogenous proteins
with a small luminescent peptide, allowing sensitive quantitation
of the response dynamics in their regulated expression and covalent
modifications