Photoswitchable cell-penetrating PNAs for the manipulation of quiescence during regenerative myogenesis
1) Olalla Vázquez (PI; University of Marburg), coworker:
2) Olivier Kassel (PI; Karlsruhe Institute of Technology), coworker:
The repopulation of stem cell niches is essential to maintain the regeneration capacity of many organs and tissues. In skeletal muscle, during the so-called regenerative myogenesis, this is achieved in part by the process of reserve cells formation. In this process, some progenitor cells (myoblasts) that were already engaged in differentiation change their fate and return to quiescence by poorly understood mechanisms. We have identified nTRIP6, the nuclear isoform of the LIM domain protein TRIP6, as a candidate regulator of this process. Indeed, this transcriptional co-regulator prevents myoblast differentiation and is enriched in reserve cells. However, the study of its role in reserve cell formation in vitro and in vivo requires tools to selectively block its expression or function with high spatial and temporal precision, i.e. only in reserve cells, and only at the time of their fate decision. We have shown that nTRIP6 is generated by alternative translation at an internal AUG in Trip6 mRNA, and that a cell-penetrating PNA (PNA-CPP) targeting this AUG inhibits the translation of nTRIP6 without affecting that of TRIP6. Based on our previous work on the synthesis and optimisation of PNAs, CPPs and chemical photoswitches, our aim I to develop novel optochemical tools in the form of photoswitchable PNA-CPPs that target translation. These will be applied to investigate the role of nTRIP6 in the formation of reserve cells during myogenesis in vitro and muscle regeneration in zebrafish embryos, an animal model particularly amenable to optical methods. Furthermore, based on the experience we will gain in the photocontrol of translation, we will develop a generic optochemical genetic tool to rapidly induce the translation of any protein of interest (POI) in a reversible manner. This tool, which we call SPRINT for Switchable PNAs for the Rapid INduction of Translation, is a 2-component system: a photoswitchable PNA-CPP (optochemical component) and an expression vector for the POI (genetic component), which contains cis-acting elements that repress the translation of the POI. These cis-acting elements are the target of the PNA. Thus, light exposure of selected cells expressing the construct and treated with the photoswitchable PNA-CPP will lead to the rapid and reversible induction of the POI translation. In proof of principle experiments, SPRINT will be applied to the manipulation of nTRIP6 levels and function during myoblast return to quiescence in vitro and in the zebrafish embryo. Thus, this collaborative project between a biologist and a chemist will shed new light onto the mechanism of reserve cell formation. Furthermore, SPRINT will represent an important addition to the current toolbox for the manipulation of biological systems with very high spatial and temporal precision.
Logic of interactions within the joint project:
The Kassel lab investigates the mechanisms of myogenic differentiation. The challenge of this project is to specifically inhibit the translation of candidate regulators of quiescence with high spatial and temporal precision. The Vázquez lab develops optochemical tools, in particular photoswitchable cell-penetrating PNAs that would be ideal to tackle this question. Both labs work together to adapt and further develop these tools for the particular biological question, as well as to develop generic optochemical genetic tools to rapidly induce the translation of any protein of interest in a reversible manner.
Publications Kassel/ Vázquez relevant for the proposal:
Albert L, Peñalver A, Djokovic N, Were L, Hoffarth M, Ruzic D, Xu J, Essen L-O, Nikolic K, Dou Y, Vázquez O (2019) Modulating Protein?Protein Interactions with Visible?Light Responsive Peptide Backbone Photoswitches. ChemBioChem, 20: 1417-1429.
Heinrich B, Bouazoune K, Wojcik M, Bakowsky U., Vázquez O (2019) ortho-Fluoroazobenzene derivatives as DNA intercalators for photocontrol of DNA and nucleosome binding by visible light. Org Biomol Chem 17: 1827-1833
Albert L, Xu J, Wan R, Srinivasan V, Dou Y & Vázquez O (2017) Controlled Inhibition of Methyltransferases Using Photoswitchable Peptidomimetics: towards an Epigenetic Regulation of Leukemia. Chem Sci 8: 4612-4618.
Vázquez O & Seitz O (2014) Cytotoxic peptide-PNA conjugates by RNA-programmed peptidyl transfer with turnover. Chem Sci 5: 2850-2854.
Vázquez O, Blanco J B, Vázquez M E, Martinez-Costas J, Castedo L, & Mascareñas JL (2008) Efficient DNA Binding and Nuclear Uptake by Distamycin Derivatives Conjugated to Octa- arginine Sequences. Chembiochem, 9: 2822-2829.
Kemler D, Dahley O, Roßwag S, Litfin M & Kassel O (2016) The LIM domain protein nTRIP6 acts as a co-repressor for the transcription factor MEF2C in myoblasts. Sci Rep 6:7746.
Diefenbacher ME, Reich D, Dahley O, Kemler D, Litfin M, Herrlich P & Kassel O (2014) The LIM Domain Protein nTRIP6 Recruits the Mediator Complex to AP-1-Regulated Promoters. PLoS ONE 9: e97549
Röder IV, Strack S, Reischl M, Dahley O, Khan MM, Kassel O, Zaccolo M & Rudolf R (2012) Participation of Myosin va and pka type I in the regeneration of neuromuscular junctions. PLoS ONE 7: e40860
Diefenbacher ME, Litfin M, Herrlich P & Kassel O (2010) The nuclear isoform of the LIM domain protein Trip6 integrates activating and repressing signals at the promoter-bound glucocorticoid receptor. Mol Cell Endocrinol 320: 58-66
Kassel O, Schneider S, Heilbock C, Litfin M, Göttlicher M & Herrlich P (2004) A nuclear isoform of the focal adhesion LIM-domain protein Trip6 integrates activating and repressing signals at AP-1- and NF-kappaB-regulated promoters. Genes Dev 18: 2518-2528