10 research outputs found
Strong Improvement of Interfacial Properties Can Result from Slight Structural Modifications of Proteins: The Case of Native and Dry-Heated Lysozyme
No full textAssay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles.
No full textFluorescence de-quenching can be used to analyze membrane lipid mixing during an in vitro fusion reaction. Here we describe a method to measure lipid mixing using vacuolar membranes purified from the yeast Saccharomyces cerevisiae. Labeling the isolated organelles with rhodamine-phosphatidylethanolamine allows to reveal ATP-dependent lipid mixing through fluorescence de-quenching in a spectrofluorometer. Combining this assay with content mixing indicators, such as the fusion-dependent maturation of a luminal vacuolar phosphatase, then permits the detection of hemifusion intermediates and the analysis of the requirements for fusion pore opening
Mass Spectrometric Distinction of In-Source and In-Solution Pyroglutamate and Succinimide in Proteins: A Case Study on rhG-CSF
No full textAnalysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry
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Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics
No full textPrimate lentiviruses require Inositol hexakisphosphate (IP6) or inositol pentakisphosphate (IP5) for the production of viral particles
No full textPITPs as targets for selectively interfering with phosphoinositide signaling in cells
Get PDFSec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs nor PITPs in general have been exploited as targets for chemical inhibition for such purposes. Herein, we validate what is to our knowledge the first small-molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs) and are effective inhibitors in vitro and in vivo. We further establish that Sec14 is the sole essential NPPM target in yeast and that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects and demonstrate that NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof of concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase–directed strategies
