Temporal Analysis of PP2A Phosphatase Activity During Insulin Stimulation Using a Direct Activity Probe

Protein serine/threonine phosphatases (PSPs) are ubiquitously expressed in mammalian cells. In particular, PP2A accounts for up to 1% of the total protein within cells. Despite clear evidence for the role of PP2A in cellular signaling, there is a lack of information concerning the magnitude and temporal dynamics of PP2A catalytic activity during insulin stimulation. Herein, we describe the development of a direct, fluorescent activity probe capable of reporting on global changes in PP2A enzymatic activity in unfractionated cell lysates. Utilizing this new probe, we profiled the magnitude as well as temporal dynamics of PP2A activity during insulin stimulation of liver hepatocytes. These results provide direct evidence for the rapid response of PP2A catalytic activity to extracellular stimulation, as well as insight into the complex regulation of phosphorylation levels by opposing kinase and phosphatase activities within the cell. This study provides a new tool for investigating the chemical biology of PSPs

The 8-Silyloxyquinoline Scaffold as a Versatile Platform for the Sensitive Detection of Aqueous Fluoride

Utilizing a novel 8-silyloxyquinoline scaffold, we demonstrate the ability to synthesize fluorogenic probes for the sensitive and selective detection of inorganic fluoride (NaF) in aqueous samples. Our initial probe design (<b>2</b>) is capable of detecting inorganic fluoride at levels as low as 3.8 μM (72 ppb) in aqueous solutions, well below PHS recommended levels for drinking water (0.7–1.2 ppm), placing this probe among the most sensitive fluoride sensors reported to date. Furthermore, our results highlight the utility of the readily modifiable 8-silyloxyquinoline scaffold for the design of tailored fluoride sensing platforms. We demonstrate the ability to rationally tune the fluorescence and physical properties of the 8-silyloxyquinoline scaffold, producing a red-shifted fluoride probe (<b>4</b>) capable of detecting 50 μM (0.95 ppm) NaF in aqueous samples using a straightforward test-strip-based assay format. Taken together this work provides a template for the design of fluoride sensors capable of reporting on relevant concentrations of fluoride in the laboratory and in the field

Self-Assembling NanoLuc Luciferase Fragments as Probes for Protein Aggregation in Living Cells

Given the clear role of protein aggregation in human disease, there is a critical need for assays capable of quantifying protein aggregation in living systems. We hypothesized that the inherently low background and biocompatibility of luminescence signal readouts could provide a potential solution to this problem. Herein, we describe a set of self-assembling NanoLuc luciferase (Nluc) fragments that produce a tunable luminescence readout that is dependent upon the solubility of a target protein fused to the N-terminal Nluc fragment. To demonstrate this approach, we employed this assay in bacteria to assess mutations known to disrupt amyloid-beta (Aβ) aggregation as well as disease-relevant mutations associated with familial Alzheimer’s diseases. The luminescence signal from these experiments correlates with the reported aggregation potential of these Aβ mutants and reinforces the increased aggregation potential of disease-relevant mutations in Aβ_1–42. To further demonstrate the utility of this approach, we show that the effect of small molecule inhibitors on Aβ aggregation can be monitored using this system. In addition, we demonstrate that aggregation assays can be ported into mammalian cells. Taken together, these results indicate that this platform could be used to rapidly screen for mutations that influence protein aggregation as well as inhibitors of protein aggregation. This method offers a novel, genetically encodable luminescence readout of protein aggregation in living cells

Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors

We introduce a versatile approach for repurposing protein kinase chemosensors, containing the phosphorylation-sensitive sulfonamido-oxine fluorophore termed Sox, for the specific determination of endogenous protein phosphatase activity from whole cell lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for protein tyrosine phosphatase-1B (PTP1B), an established signaling node in human disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) full-length recombinant PTP1B and is remarkably selective for PTP1B among a panel of highly homologous tyrosine phosphatases. Coupling this robust activity probe with the specificity of antibodies allowed for the temporal analysis of endogenous PTP1B activity dynamics in lysates generated from HepG2 cells after stimulation with insulin. Lastly, we leveraged this assay format to profile PTP1B activity perturbations in a rat model of nonalcoholic fatty liver disease (NAFLD), providing direct evidence for elevated PTP1B catalytic activity in this disease state. Given the modular nature of this assay, we anticipate that this approach will have broad utility in monitoring phosphatase activity dynamics in human disease states