Accepted_test

Luciferases based biosensors for detecting chaperone activity and monitoring mitochondrial bioenergetics
by Rahaf Al Ebrahim | Maria G. Alekseeva | Sergey V. Bazhenov | Vadim V. Fomin | A.G. Kessenikh | Dilara A. Mavletova | Andrey A. Nesterov | Elena U. Poluektova | Valeriy N. Danilenko | Ilya V. Manukhov | Moscow Institute of Physics and Technology | Laboratory of Genetics of Microorganisms, Vavilov Institute of General Genetics Russian Academy of Sciences | Moscow Institute of Physics and Technology | Moscow Institute of Physics and Technology, Laboratory of Microbiology BIOTECH University | Laboratory of Microbiology BIOTECH University | Laboratory of Genetics of Microorganisms, Vavilov Institute of General Genetics Russian Academy of Sciences | Laboratory of Genetics of Microorganisms, Vavilov Institute of General Genetics Russian Academy of Sciences, Institute of Environmental Engineering, RUDN University | Laboratory of Genetics of Microorganisms, Vavilov Institute of General Genetics Russian Academy of Sciences | Laboratory of Genetics of Microorganisms, Vavilov Institute of General Genetics Russian Academy of Sciences | Moscow Institute of Physics and Technology
Abstract ID: 778
Event: BGRS-abstracts
Sections: [Sym 8] Section “Biotechnology through the lens of the microbiome”

In this study, we developed a series of constructs with luciferase genes from various sources. Recipient cells with these constructs allow for the analysis of intracellular chaperone activity and assessment of the reduced equivalents pool.
A pair of luciferase genes differing in thermostability, were used to investigate the disaggregase activity of Limosilactobacillus fermentum U-21 strain. This strain secretes chaperones into the medium and is considered a candidate for the development of disaggregase drugs for Parkinson's disease. We showed that the secreted protein encoded by C0965_000195 locus, according to sequence and structural analysis, is ClpL. Using the above-mentioned pair of luciferases, it was shown that ClpL, can compensate for the clpB deficiency and improve protein refolding. In vitro experiments demonstrated that the culture medium containing ClpL, can prevent the thermal denaturation of luciferases. These results suggest that ClpL, possessing disaggregase properties, can significantly contribute to the pharmabiotic properties of L. fermentum U-21.
The luciferase genes from the firefly L. mingrelica and the bacterium P. luminescens, optimized for expression in mammals, were transfected into HEK 293T cells. Constructs with the luc gene from L. mingrelica allowed for the analysis of the intracellular ATP pool. Constructs with the luxAB genes from P. luminescens enable the analysis of the intracellular reduced FMN pool. The LuxG oxidoreductase allows for the assessment of NADH pool. Localization of bacterial and firefly luciferases in the cytoplasm and mitochondrial matrix makes it possible to evaluate the effects of various bioactive substances on the energy processes in different cell compartments.