Comparative analysis of the primary structure and production of recombinant poly(ADP-ribose)polymerase 1 of long-lived Heterocephalus glaber

Abstract

DNA repair is a most important cellular process that helps maintain the integrity of the genome and is currently considered by researchers as one of the factors determining the maximum lifespan. The central regulator of the DNA repair process is the enzyme poly(ADP-ribose)polymerase 1 (PARP1). PARP1 catalyzes the synthesis of poly(ADP-ribose) polymer (PAR) upon DNA damage using nicotinamide adenine dinucleotide (NAD+) as a substrate. This polymer covalently attaches to PARP1, which leads to its dissociation from the complex with damaged DNA and stimulation of the repair process. Despite intensive research on PARP1, its properties as an isolated protein have not been practically studied in mammals that demonstrate a long maximum lifespan, such as, for example, the naked mole rat (Heterocephalus glaber). High activity of DNA repair systems is observed in the cells of the naked mole rat, which ensures their high resistance to oxidative stress, as well as to genotoxic effects. The revealed features may be due to the high activity of PARP1 in the cells of the naked mole rat; however, this issue remains poorly understood and, thus, requires more detailed research, including one with the use of isolated protein PARP1 of the naked mole rat, the isolation and characterization of which have not been carried out before. In the present work, the amino acid sequence of PARP1 of the naked mole rat is compared with the amino acid sequences of orthologous proteins of other mammals. In contrast to human PARP1, 13 evolutionarily conservative amino acid substitutions in various functional domains of the protein have been identified in the amino acid sequence of naked mole rat PARP1. Using the cDNA of the naked mole rat's Parp1 gene, a vector was created for the expression of the target protein in Escherichia coli cell culture. For the first time, a detailed description of the procedure for the expression and purification of the recombinant protein PARP1 of the long-lived naked mole rat is presented. In addition, poly(ADP-ribose)polymerase activity of the obtained protein was evaluated. The results presented in this paper are the basis for further detailed characterization of the properties of purified recombinant naked mole rat PARP1.

Keywords: DNA repair; Heterocephalus glaber; poly(ADP-ribose)polymerase 1.

Table 1.. Sequences of oligodeoxynucleotides and primers used in the work

Fig. 1.. Evolutionary analysis of the primary structure of the PARP1 protein.

а – list of mammal species included in the analysis and their divergence; b – amino acid substitutions in PARP1 unique to the naked mole rat. * – substitutions also present in the Damaraland mole rat; ** – substitution options from the NCBI and Ensembl databases.

Fig. 2.. Analysis of PARP1 content in lysates of transformed E. coli cells.

Cells were cultured in an autoinduction system at 37 °C with and without lactose induction. The position of the PARP1 protein is indicated by red arrows. Control – recombinant human PARP1 protein.

Fig. 3.. Elution profiles of the PARP1 (H. glaber) protein from Ni-NTAagarose (a), heparin-Sepharose (b), ssDNA-cellulose (c).

Blue line – optical density of the solution at a wavelength of 280 nm (mAU); red line – solution conductivity (mS/cm).

Fig. 4.. Electropherogram showing the purification stages of recombinant naked mole rat PARP1.

1 – control protein sample; 2–4 – application, breakthrough and elution from Ni-NTA-agarose, respectively; 5 – breakthrough from heparin-Sepharose; 6, 7 – elution from heparin-Sepharose 0.3 M and 1 M NaCl, respectively; 8 – breakthrough from ssDNA cellulose; 9, 10 – elution of the target protein from ssDNA cellulose with 0.3 M and 1 M NaCl, respectively.

Fig. 5.. Analysis of the activity of recombinant naked mole rat PARP1 in the autoPARylation reaction.

а – autoradiography of 10 % SDS-PAGE, in which the separation of protein modification products was carried out; b – diagram summarizing the results of three independent experiments performed using the TCA target method (see Materials and methods).