5 research outputs found
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
“Addition” and “Subtraction”: Selectivity Design for Type II Maternal Embryonic Leucine Zipper Kinase Inhibitors
While <i>adding</i> the
structural features that are
more favored by on-target activity is the more common strategy in
selectivity optimization, the opposite strategy of <i>subtracting</i> the structural features that contribute more to off-target activity
can also be very effective. Reported here is our successful effort
of improving the kinase selectivity of type II maternal embryonic
leucine zipper kinase inhibitors by applying these two complementary
approaches together, which clearly demonstrates the powerful synergy
between them
Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight
MELK
kinase has been implicated in playing an important role in
tumorigenesis. Our previous studies suggested that MELK is involved
in the regulation of cell cycle and its genetic depletion leads to
growth inhibition in a subset of high MELK-expressing basal-like breast
cancer cell lines. Herein we describe the discovery and optimization
of novel MELK inhibitors <b>8a</b> and <b>8b</b> that
recapitulate the cellular effects observed by short hairpin ribonucleic
acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered
a novel fluorine-induced hydrophobic collapse that locked the ligand
in its bioactive conformation and led to a 20-fold gain in potency.
These novel pharmacological inhibitors achieved high exposure in vivo
and were well tolerated, which may allow further in vivo evaluation