Abscisic acid regulates stomatal production by imprinting a SnRK2 kinase-mediated phosphocode on the master regulator SPEECHLESS.

Stomata, the epidermal pores for gas exchange between plants and the atmosphere, are the major sites of water loss. During water shortage, plants limit the formation of new stoma via the phytohormone abscisic acid (ABA) to conserve water. However, how ABA suppresses stomatal production is largely unknown. Here, we demonstrate that three core SnRK2 kinases of ABA signaling inhibit the initiation and proliferation of the stomatal precursors in Arabidopsis. We show that the SnRK2s function within the precursors and directly phosphorylate SPEECHLESS (SPCH), the master transcription factor for stomatal initiation. We identify specific SPCH residues targeted by the SnRK2s, which mediate the ABA/drought-induced suppression of SPCH and stomatal production. This SnRK2-specific SPCH phosphocode connects stomatal development with ABA/drought signals and enables the independent control of this key water conservation response. Our work also highlights how distinct signaling activities can be specifically encoded on a master regulator to modulate developmental plasticity.

Multi-facet implications of PEGylated lysozyme stabilized-silver nanoclusters loaded recombinant PTEN cargo in cancer theranostics.

Amalgamation of delivery and tracking of therapeutically relevant moieties on a single platform is made possible by the application of metal nanoclusters, an innovative class of luminescent nanomaterials. Metal nanoclusters, possessing molecule-like attributes, display extraordinary size and shape tunable properties befitting theranostic applications. Herein, we report successful assembly of therapeutically significant phosphatase protein PTEN and fluorescent lysozyme-stabilized silver nanoclusters to accomplish delivery and tracking of the protein. Down-regulation of PTEN perturbs the cellular networking leading to copious pathological conditions. The integration of purified recombinant PTEN with silver nanoclusters was evaluated by fluorescence spectroscopy study. A key feature of this study is the use of polyethylene glycol coating that allows fabrication of the assembly into spherical nanocomposites as characterized by transmission electron microscope along with retention of both optical functionality of the cluster and biological activity of the protein. Prior to cellular application, the functional integrity of PTEN in the composite was determined in vitro, by enzymatic assay employing para-nitrophenylphosphate as substrate. Cellular internalization of the cargo was studied by confocal microscopy and flow cytometry analysis. The efficacy of the payload on modulation of cellular signaling was assessed on cell lines that expressed PTEN differentially. PTEN null U-87 MG and PTEN expressing MCF7 cell lines displayed successful alteration of AKT and FAK signaling proteins culminating in cell cycle arrest and reduced wound healing capacity. A dose dependent reduction in cell proliferation of MCF7 cells was achieved. For U-87 MG, treatment with the payload resulted in chemosensitization toward anti-cancer drug erlotinib. Thus, PEG coated GST-PTEN loaded silver nanoclusters serves as a comprehensive system encompassing cellular imaging and protein delivery with potential biomedical implications.

Retention of functional characteristics of glutathione-S-transferase and lactate dehydrogenase-A in fusion protein.

A paradigm shift toward fusion proteins to render multiple functionalities and applications on a single platform has been incurred in enzyme based diagnosis. Herein, we report development and systematic characterizations of glutathione-S-transferase (GST) and human lactate dehydrogenase A (hLDHA) in a fusion protein (GST-hLDHA) to achieve functional activities of GST and hLDHA simultaneously. The GST-pGEX-4T-2 vector system was used for cloning and purification of hLDHA, utilizing the affinity based interaction between GST and GSH in column chromatography. Bacterially purified protein was subjected to the Western blot analysis and structural analysis by circular dichroism spectroscopy, which revealed intact structural framework of the fusion construct. Kinetic characterization of the fusion GST-hLDHA protein toward GSH and NADH, suggested retention of functional activities of GST and hLDHA in fused protein as indicated by the kinetic parameters km and kcat/km. Further analysis of effect of temperature and pH on GST-hLDHA activity revealed maximum activity around human physiological conditions (37°C and pH 8). Preservation of the structural and functional characteristics of the fusion enzyme paves the way for potential application for the detection of NADH and GSH in conjunction as biomarkers for cancer diagnosis.