32 research outputs found
Structure-Based Design of an Intramolecular Proton Transfer Site in Murine Carbonic Anhydrase V â€
Structural Insights into Ternary Complex Formation of Human CARM1 with Various Substrates
CARM1 Preferentially Methylates H3R17 over H3R26 through a Random Kinetic Mechanism
CARM1
is a type I arginine
methyltransferase involved in the regulation of transcription, pre-mRNA
splicing, cell cycle progression, and the DNA damage response. CARM1
overexpression has been implicated in breast, prostate, and liver
cancers and therefore is an attractive target for cancer therapy.
To date, little about the kinetic properties of CARM1 is known. In
this study, substrate specificity and the kinetic mechanism of the
human enzyme were determined. Substrate specificity was examined by
testing CARM1 activity with several histone H3-based peptides in a
radiometric assay. Comparison of <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> values reveals that methylation of H3R17 is
preferred over that of H3R26. These effects are <i>K</i><sub>M</sub>-driven as <i>k</i><sub>cat</sub> values remain
relatively constant for the peptides tested. Shortening the peptide
at the C-terminus by five amino acid residues greatly reduced binding
affinity, indicating distal residues may contribute to substrate binding.
CARM1 appears to bind monomethylated peptides with an affinity similar
to that of unmethylated peptides. Monitoring of the CARM1-dependent
production of monomethylated and dimethylated peptides over time by
self-assembled monolayer and matrix-assisted laser desorption ionization
mass spectrometry revealed that methylation by CARM1 is distributive.
Additionally, dead-end and product inhibition studies suggest CARM1
conforms to a random sequential kinetic mechanism. By defining the
kinetic properties and mechanism of CARM1, these studies may aid in
the development of small molecule CARM1 inhibitors
Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP
The
protein methyltransferase (PMT) SETDB1 is a strong candidate
oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine
9 of histone 3 (H3K9), utilizing <i>S</i>-adenosylmethionine
(SAM) as the methyl donor and its catalytic activity, has been reported
to be regulated by a partner protein ATF7IP. Here, we examine the
contribution of ATF7IP to the <i>in vitro</i> activity and
substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed
and 1:1 stoichiometric complexes were purified for comparison against
SETDB1 enzyme alone. We employed both radiometric flashplate-based
and SAMDI mass spectrometry assays to follow methylation on histone
H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated,
or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex
efficiently catalyze both monomethylation and dimethylation of H3K9
peptide substrates. The activity of the binary complex was 4-fold
lower than SETDB1 alone. This difference was due to a decrease in
the value of <i>k</i><sub>cat</sub> as the substrate <i>K</i><sub>M</sub> values were comparable between SETDB1 and
the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in
a distributive manner, and this too was unaffected by the presence
of ATF7IP. This finding is important as H3K9 can be methylated by
HMTs other than SETDB1 and a distributive mechanism would allow for
interplay between multiple HMTs on H3K9. Our results indicate that
ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting
alternate roles, such as affecting cellular localization or mediating
interaction with additional binding partners
Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: Molecular basis of isozyme-drug discrimination
Identification of a peptide inhibitor for the histone methyltransferase WHSC1
<div><p>WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.</p></div
Crystallographic data collection and refinement statistics for WHSC1L1 1054–1285 with PTD2 and SAM.
<p>Crystallographic data collection and refinement statistics for WHSC1L1 1054–1285 with PTD2 and SAM.</p
Norleucine-containing peptides can inhibit WHSC1 and WHSC1L1 activity in vitro.
<p>Representative peptide inhibitor biochemical dose-response curves for (A) WHSC1 941–1240 and (B) WHSC1L1 1054–1285. Error bars represent the standard deviation of three independent replicates. Resulting IC<sub>50</sub> values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#pone.0197082.t002" target="_blank">Table 2</a>.</p
Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285<sup>a</sup>.
<p>Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#t002fn001" target="_blank"><sup>a</sup></a>.</p
Representative SPR sensorgrams for PTD2 binding to Avi-tagged WHSC1 941–1240 in the absence or presence of SAM analogs.
<p>WHSC1 was immobilized on a streptavidin-coated chip and peptide inhibitor was either injected in the absence of cofactor (left panel), co-injected with SAH (middle panel), or co-injected with SFG (right panel) utilizing a 2-fold, 10-point dilution series ending at a 100 μM top concentration.</p