11 research outputs found
Pleiotropic and Novel Phenotypes in The \u3cem\u3eDrosophila\u3c/em\u3e Gut Caused by Mutation of \u3cem\u3eDrop-Dead\u3c/em\u3e
Normal gut function is vital for animal survival, and deviations from such function can contribute to malnutrition, inflammation, increased susceptibility to pathogens, diabetes, neurodegenerative diseases, and cancer. In the fruit fly Drosophila melanogaster, mutation of the gene drop-dead (drd) results in defective gut function, as measured by enlargement of the crop and reduced food movement through the gut, and drd mutation also causes the unrelated phenotypes of neurodegeneration, early adult lethality and female sterility. In the current work, adult drd mutant flies are also shown to lack the peritrophic matrix (PM), an extracellular barrier that lines the lumen of the midgut and is found in many insects including flies, mosquitos and termites. The use of a drd-gal4 construct to drive a GFP reporter in late pupae and adults revealed drd expression in the anterior cardia, which is the site of PM synthesis in Drosophila. Moreover, the ability of drd knockdown or rescue with several gal4 drivers to recapitulate or rescue the gut phenotypes (lack of a PM, reduced defecation, and reduced adult survival 10–40 days post-eclosion) was correlated to the level of expression of each driver in the anterior cardia. Surprisingly, however, knocking down drd expression only in adult flies, which has previously been shown not to affect survival, eliminated the PM without reducing defecation rate. These results demonstrate that drd mutant flies have a novel phenotype, the absence of a PM, which is functionally separable from the previously described gut dysfunction observed in these flies. As the first mutant Drosophila strain reported to lack a PM, drd mutants will be a useful tool for studying the synthesis of this structure
Characterization of Protease-Activated Receptor (PAR) Ligands: Parmodulins are Reversible Allosteric Inhibitors of PAR1-Driven Calcium Mobilization in Endothelial Cells
Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation
The Parmodulin NRD-21 is an Allosteric Inhibitor of PAR1 Gq Signaling with Improved Anti-Inflammatory Activity and Stability
Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications
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Development of Chemical and Protein Tools to Interrogate and Harness Biomolecular Interactions
When we consider the roles biopolymers play in our cells, the central dogma of biology generally comes to mind: DNA stores information to be read out by cellular machinery transcribing it into RNA which encodes proteins responsible for catalysis. While these functions are critical, they do not account for the numerous secondary and tertiary phenomena responsible for the bulk of cellular function: all three biopolymers can be decorated with various modifications, and, in addition to or in concert with secondary structural changes, interact transiently with each other through protein-protein, RNA-protein, and DNA-protein interactions. These dynamic processes dictate whether DNA is accessible for transcription, whether resultant RNA will be processed normally or degraded/upregulated, and which competition-mediated signaling pathways might be activated downstream, to name a few. Due to this complexity, it has been historically challenging to measure or harness such interactions. Therefore, in this thesis we will cover the development of several tools designed to tackle these challenges. The first exploits the RNA output created by two small molecule-sensing T7 polymerase-based biosensors in order to control Cas9 activity in cells. The second harnesses the interchangeability-potential of ester-caged imaging and bioactive molecules using a split esterase biosensor to study protein-protein interactions. The third further employs the esterase-based technology through the creation of an RNA proximity labeling method via a unique ester-masked enol ester acylating reagent. Finally, we will briefly discuss efforts toward an esterase-based “synthetic” bioluminescent sensitive in vivo imaging system. Taken together, this work generated technologies that will help better understand biomolecular interactions and harness them to control cellular processes
Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors
Systems to control Cas9 with spatial
and temporal precision offer
opportunities to decrease side effects, protect sensitive tissues,
and create gene therapies that are only activated at defined times
and places. Here, we present the design of new Cas9 controllers based
on RNA polymerase (RNAP)-based biosensors that produce gRNAs, thereby
regulating target knockout. After development and validation of a
new abscisic acid-inducible biosensor to control Cas9, we lowered
the background of the system using continuous evolution. To showcase
the versatility of the approach, we designed biosensors that measure
medically relevant protein–protein interactions to drive knockout.
Finally, to test whether orthogonal RNAP biosensors could integrate
multiple input signals to drive multiple gRNA-based outputs with a
single Cas9 protein, we designed an “on-switch/off switch”
controller. The addition of one input activates the “on switch”
and induces knockout, while the addition of a second input activates
the “off switch” and produces a gRNA that directs the
Cas9 protein to degrade the “on switch” gRNA vector,
thereby deactivating it. This combined activation and deactivation
system displayed very low background and inducible target knockout
using different combinations of small-molecule treatment. Our results
establish engineered RNAP biosensors as deployable Cas9 control elements
and open up new opportunities for driving genetic editing technologies
by diverse input signals
Development of a Split Esterase for Protein-Protein Interaction Dependent Small Molecule Activation
Development of a Split Esterase for Protein-Protein Interaction Dependent Small Molecule Activation
Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications
Characterization of Protease-Activated Receptor (PAR) Ligands: Parmodulins Are Reversible Allosteric Inhibitors of PAR1-Driven Calcium Mobilization in Endothelial Cells
We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of different PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation. Detailed synthetic protocols are also provided for several known and novel PAR ligands.<br /
Characterization of Protease-Activated Receptor (PAR) Ligands: Parmodulins Are Reversible Allosteric Inhibitors of PAR1-Driven Calcium Mobilization in Endothelial Cells
We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of different PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation. Detailed synthetic protocols are also provided for several known and novel PAR ligands.<br /