Arrhythmogenic Cardiomyopathy Related DSG2 Mutations Affect Desomosmal Binding Kinetics

Abstract

whereas effects of TMAO on nonpolar residues lead to peptide swelling. This suggests competing mechanisms of TMAO on proteins which would accounts for swelling of hydrophobic cores and stabilization of charge-charge interactions. These mechanisms are studied from replica exchange molecular dynamics simulations of the Trp cage miniprotein. In many cases, dissecting biophysical processes in cells requires tools to elicit time-dependent gene expression and/or protein localization. However, many of the drugs used in presently available inducible systems possess endogenous cellular targets, which can cause undesirable side-effects that make them incompatible for use in therapeutic applications. With the increasing prospect of the use of gene modulation in human therapies (i.e., gene therapy, cellbased therapies, etc.), orthogonal drug-inducible systems that use safe ligand molecules will be needed. Here we present a novel method for druginducible gene expression control using existing (FDA-approved) anti-viral drug compounds that are able to bind and inhibit the cis-proteolytic activity of the Hepatitis C virus (HCV) protease NS3/4a. We show that the protease can be used to preserve an artificial transcription factor based on dCas9 subject to drug control via insertion of the viral enzyme between the dCas9 scaffold and a C-terminal transactivation domain. In the absence of drug, the protease serves as a self-immolating linker that leads to dismemberment of the chimera. Upon exposure to drug, the protease is inhibited and intact chimera are preserved. Intact copies of the protein are able to translocate to the nucleus to activate the expression of sgRNA-specified target genes. Use of NS3/4a and its anti-viral inhibitors as a preserveable linker have proven to be highly modular and can be inserted into a host of other chimera, including alternate RNA hairpin binding transcription factors. Overall, these results demonstrate the versatility of using the HCV NS3/4a domain as a drug-sensitive module for regulating the activity and localization of engineered transcription factors. The aggregation of the 42-residue form of the amyloid-beta peptide (amyloid-beta42) is a pivotal event in Alzheimer's Disease (AD). Here, we describe a chemical kinetics-based approach that allows us to quantify precisely the effects of small molecules on the aggregation of amyloid-beta42. We thus report the identification of a library of compounds that can target specific microscopic steps in amyloid-beta42 aggregation. By validating the effects of these compounds in human cerebrospinal fluid, and in a Caenorhabditis elegans model of AD, we show that this approach provides us a diverse pool of small molecules that can be taken forward in drug discovery programs for AD. This systematic concept also offers an exciting opportunity in enabling us to rationally design candidate molecules that can both inhibit specific microscopic steps of the protein aggregation process, and at the same time, possess good drug pharmocokinetic characteristics. Cadherins...