DYRKs are kinases that self-activate in vitro by auto-phosphorylation of a YTY motif in the kinase domain, but their regulation in vivo is not well understood. In C. elegans zygotes, MBK-2/DYRK phosphorylates oocyte proteins at the end of the meiotic divisions to promote the oocyte-to-embryo transition. Here we demonstrate that MBK-2 is under both positive and negative regulation during the transition. MBK-2 is activated during oocyte maturation by CDK-1-dependent phosphorylation of Serine 68, a residue outside of the kinase domain required for full activity in vivo. The pseudo-tyrosine phosphatases EGG-4 and EGG-5 sequester activated MBK-2 until the meiotic divisions by binding to the YTY motif and inhibiting MBK-2’s kinase activity directly, using a novel mixed-inhibition mechanism that does not involve tyrosine dephosphorylation. Our findings link cell cycle progression to MBK-2/DYRK activation and the oocyte-to-embryo transition.
We suggest that successful transition from an oocyte to a zygote depends on the cell cycle-regulated relocalization of key regulators from the cortex to the cytoplasm of the egg.
Natural
products and their derivatives continue to be wellsprings
of nascent therapeutic potential. However, many laboratories have
limited resources for biological evaluation, leaving their previously
isolated or synthesized compounds largely or completely untested.
To address this issue, the Canvass library of natural products was
assembled, in collaboration with academic and industry researchers,
for quantitative high-throughput screening (qHTS) across a diverse
set of cell-based and biochemical assays. Characterization of the
library in terms of physicochemical properties, structural diversity,
and similarity to compounds in publicly available libraries indicates
that the Canvass library contains many structural elements in common
with approved drugs. The assay data generated were analyzed using
a variety of quality control metrics, and the resultant assay profiles
were explored using statistical methods, such as clustering and compound
promiscuity analyses. Individual compounds were then sorted by structural
class and activity profiles. Differential behavior based on these
classifications, as well as noteworthy activities, are outlined herein.
One such highlight is the activity of (−)-2(S)-cathafoline, which was found to stabilize calcium levels in the
endoplasmic reticulum. The workflow described here illustrates a pilot
effort to broadly survey the biological potential of natural products
by utilizing the power of automation and high-throughput screening.
Methods to identify the bioactive diversity within natural product extracts (NPEs) continue to evolve. NPEs constitute complex mixtures of chemical substances varying in structure, composition and abundance. NPEs can therefore be challenging to evaluate efficiently with high throughput screening approaches designed to test pure substances. Here we facilitate the rapid identification and prioritization of anti-malarial NPEs using a pharmacologically-driven, quantitative high throughput screening (qHTS) paradigm. In qHTS each NPE is tested across a concentration range from which sigmoidal response, efficacy and apparent EC50s can be used to rank order NPEs for subsequent organism re-culture, extraction and fractionation. Using an NPE library derived from diverse marine microorganisms we observed potent antimalarial activity from two Streptomyces sp. extracts identified from thousands tested using qHTS. Seven compounds were isolated from two phylogenetically related Streptomyces species; Streptomyces ballenaensis collected from Costa Rica and Streptomyces bangulaensis collected from Papua New Guinea. Among them we identified actinoramides A and B belonging to the unusually elaborated non-proteinogenic amino acid-containing tetrapeptide series of natural products. In addition, we characterized a series of new compounds, including an artifact 25-epi-actinoramide A, and actinoramides D, E, F which are closely related biosynthetic congeners of the previously reported metabolites.
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