Antibody-free time-resolved terbium luminescence assays designed for cyclin-dependent kinase 5 (CDK5).

Novel time-resolved terbium luminescence assays were developed for CDK5 and CDK2 by designing synthetic substrates which incorporate phospho-inducible terbium sensitizing motifs with kinase substrate consensus sequences. Substrates designed for CDK5 showed no phosphorylation by CDK2, opening the possibility for CDK5-specific assay development for selective drug discovery.

Novel Substrate Prediction for the TAM Family of RTKs Using Phosphoproteomics and Structure-Based Modeling.

The TAM family of receptor tyrosine kinases is implicated in multiple distinct oncogenic signaling pathways. However, to date, there are no FDA-approved small molecule inhibitors for the TAM kinases. Inhibitor design and screening rely on tools to study the kinase activity.

Novel Bruton's Tyrosine Kinase (BTK) Substrates for Time-Resolved Luminescence Assays.

Bruton's tyrosine kinase (BTK) is a well-documented target for cancer therapeutics due to its role in B-cell signaling pathways. However, inhibitor design is hindered by lack of tools to assess kinase activity. We used in vitro phosphoproteomics to determine BTK's substrate preferences and applied this information to our updated data processing pipeline, KINATEST-ID 2.

Lipid Droplet-Derived Monounsaturated Fatty Acids Traffic via PLIN5 to Allosterically Activate SIRT1.

Lipid droplets (LDs) provide a reservoir for triacylglycerol storage and are a central hub for fatty acid trafficking and signaling in cells. Lipolysis promotes mitochondrial biogenesis and oxidative metabolism via a SIRT1/PGC-1α/PPARα-dependent pathway through an unknown mechanism. Herein, we identify that monounsaturated fatty acids (MUFAs) allosterically activate SIRT1 toward select peptide-substrates such as PGC-1α.

Substrate specificity of an actively assembling amyloid catalyst.

In the presence of Zn , the catalytic, amyloid-forming peptide Ac-IHIHIQI-NH , was found to exhibit enhanced selectivity for hydrophobic p-nitrophenyl ester substrates while in the process of self-assembly. As opposed to the substrate p-nitrophenyl acetate, which was more effectively hydrolyzed with Ac-IHIHIQI-NH in its fully fibrillar state, the hydrophobic substrate Z-L-Phe-ONp was converted with a second-order rate constant more than 11-times greater when the catalyst was actively assembling. Under such conditions, Z-L-Phe-ONp hydrolysis proceeded at a greater velocity than the more hydrophilic and otherwise more labile ester Boc-L-Asn-ONp.

Tailored Presentation of Carbohydrates on a Coiled Coil-Based Scaffold for Asialoglycoprotein Receptor Targeting.

The coiled-coil folding motif represents an ideal scaffold for the defined presentation of ligands due to the possibility of positioning them at specific distances along the axis. We created a coiled-coil glycopeptide library to characterize the distances between the carbohydrate-binding sites of the asialoglycoprotein receptors (ASGPR) on hepatocytes. The components of the glycopeptide library vary for the number of displayed ligands (galactose), their position on the peptide sequence, and the space between peptide backbone and carbohydrate.