Endosomal disentanglement of a transducible artificial transcription factor targeting endothelin receptor A.

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.

Constitutive androstane receptor 1 is constitutively bound to chromatin and 'primed' for transactivation in hepatocytes.

The constitutive androstane receptor (CAR) is a xenobiotic sensor expressed in hepatocytes that activates genes involved in drug metabolism, lipid homeostasis, and cell proliferation. Much progress has been made in understanding the mechanism of activation of human CAR by drugs and xenobiotics. However, many aspects of the activation pathway remain to be elucidated. In this report, we have used viral constructs to express human CAR, its splice variants, and mutant CAR forms in hepatocytes from Car-/- mice in vitro and in vivo. We demonstrate CAR expression rescued the ability of Car-/- hepatocytes to respond to a wide range of CAR activators including phenobarbital. Additionally, two major splice isoforms of human CAR, CAR2 and CAR3, were inactive with almost all the agents tested. In contrast to the current model of CAR activation, ectopic CAR1 is constitutively localized in the nucleus and is loaded onto Cyp2b10 gene in the absence of an inducing agent. In studies to elucidate the role of threonine T38 in CAR regulation, we found that the T38D mutant was inactive even in the presence of CAR activators. However, the T38A mutant was activated by CAR inducers, showing that T38 is not essential for CAR activation. Also, using the inhibitor erlotinib, we could not confirm a role for the epidermal growth factor receptor in CAR regulation. Our data suggest that CAR is constitutively bound to gene regulatory regions and is regulated by exogenous agents through a mechanism which involves protein phosphorylation in the nucleus.

Unaltered hepatic wound healing response in male rats with ancestral liver injury.

The possibility that ancestral environmental exposure could result in adaptive inherited effects in mammals has been long debated. Numerous rodent models of transgenerational responses to various environmental factors have been published but due to technical, operational and resource burden, most still await independent confirmation. A previous study reported multigenerational epigenetic adaptation of the hepatic wound healing response upon exposure to the hepatotoxicant carbon tetrachloride (CCl4) in male rats. Here, we comprehensively investigate the transgenerational effects by repeating the original CCl4 multigenerational study with increased power, pedigree tracing, F2 dose-response and suitable randomization schemes. Detailed pathology evaluations do not support adaptive phenotypic suppression of the hepatic wound healing response or a greater fitness of F2 animals with ancestral liver injury exposure. However, transcriptomic analyses identified genes whose expression correlates with ancestral liver injury, although the biological relevance of this apparent transgenerational transmission at the molecular level remains to be determined. This work overall highlights the need for independent evaluation of transgenerational epigenetic inheritance paradigms in mammals.