Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia

Genetically encodable bioluminescent system from fungi

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.This research was supported by Planta LLC and Evrogen JSC. IVIS imaging and animal experiments were carried out using the equipment of the Center for Collective Usage “Medical Nanobiotechologies” located in the Russian National Research Medical University. Experiments were partially carried out using the equipment provided by the Institute of Bioorganic Chemistry of the Russian Academy of Sciences Сore Facility (CKP IBCH; supported by Russian Ministry of Education and Science Grant RFMEFI62117X0018). T.G. and M.M.-H. acknowledge support from Spanish Ministry of Economy and Competitiveness Grant BFU2015-67107 cofounded by the European Regional Development Fund, European Research Council (ERC) Grant ERC-2012-StG-310325 under the European Union’s Seventh Framework Programme FP7/2007-2013, and the European Union’s Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie Grant H2020-MSCA-ITN-2014-642095. F.A.K. acknowledges the support of HHMI International Early Career Scientist Program 55007424, the Spanish Ministry of Economy and Competitiveness (MINECO) Grants BFU2012-31329 and BFU2015-68723-P, MINECO Centro de Excelencia Severo Ochoa 2013-2017 Grant SEV-2012-0208, Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat’s Agency for Management of University and Research Grants Program 2014 SGR 0974, the Centres de Recerca de Catalunya Programme of the Generalitat de Catalunya, and ERC Grant 335980_EinME under the European Union’s Seventh Framework Programme FP7/2007-2013. H.E.W., A.G.O., and C.V.S. acknowledge support from São Paulo Research Foundation Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 11/10507-0 (to H.E.W.), 10/11578-5 (to A.G.O.), and 13/16885-1 (to C.V.S.)