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Published on: November 20, 2018
A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease
Sanjay Adhikari1, Praveen Varma Manthena, Krishna Kiran Kota
1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Abstract:
Mammalian apurinic/apyrimidinic endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are highly toxic, mutagenic, and implicated in carcinogenesis. Also, reducing the activity of APE1 protein in cancer cells and tumors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. In general, mouse models are used in studies of basic mechanisms of carcinogenesis, as well as pre-clinical studies before transitioning into humans. Human APE1 (hAPE1) has previously been cloned, expressed, and extensively characterized. However, the knowledge regarding the characterization of mouse APE1 (mAPE1) is very limited. Here we have expressed and purified full-length hAPE1 and mAPE1 in and from E. coli to near homogeneity. mAPE1 showed comparable fast reaction kinetics to its human counterpart. Steady-state enzyme kinetics showed an apparent K(m) of 91 nM and k(cat) of 4.2 s(-1) of mAPE1 for the THF cleavage reaction. For hAPE1 apparent K(m) and k(cat) were 82 nM and 3.2 s(-1), respectively, under similar reaction conditions. However, k(cat)/K(m) were in similar range for both APE1s. The optimum pH was in the range of 7.5-8 for both APE1s and had an optimal activity at 50-100 mM KCl, and they showed Mg(2+) dependence and abrogation of activity at high salt. Circular dichroism spectroscopy revealed that increasing the Mg(2+) concentration altered the ratio of "turns" to "β-strands" for both proteins, and this change may be associated with the conformational changes required to achieve an active state. Overall, compared to hAPE1, mAPE1 has higher K(m) and k(cat) values. However, overall results from this study suggest that human and mouse APE1s have mostly similar biochemical and biophysical properties. Thus, the conclusions of mouse studies to elucidate APE1 biology and its role in carcinogenesis may be extrapolated to apply to human biology. This includes the development and validation of effective APE1 inhibitors as chemosensitizers in clinical studies.
Insights
Mouse apurinic/apyrimidinic endonuclease (APE1) protein shares similar biochemical properties with human APE1. This finding supports extrapolating mouse cancer research findings to human biology and APE1 inhibitor development.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Apurinic/apyrimidinic endonuclease (APE1) repairs toxic DNA lesions and its inhibition sensitizes cancer cells to chemotherapy.
- Mouse models are crucial for cancer research, but mouse APE1 (mAPE1) characterization is limited.
- Human APE1 (hAPE1) is well-characterized, but direct comparisons with mAPE1 are scarce.
Purpose of the Study:
- To express and purify full-length human and mouse APE1 (hAPE1 and mAPE1).
- To biochemically and biophysically characterize mAPE1 and compare it with hAPE1.
- To assess the applicability of mouse models for human APE1-related cancer research.
Main Methods:
- Expression and purification of full-length hAPE1 and mAPE1 from E. coli.
- Steady-state enzyme kinetics assays to determine kinetic parameters (K(m), k(cat)).
- Circular dichroism spectroscopy to analyze protein structure and Mg(2+) ion effects.
Main Results:
- Both hAPE1 and mAPE1 were purified to near homogeneity.
- mAPE1 exhibited comparable reaction kinetics to hAPE1, with similar k(cat)/K(m) ratios.
- Kinetic parameters (K(m), k(cat)) were slightly higher for mAPE1 than hAPE1, but optimal pH, salt, and Mg(2+) dependence were similar.
- Circular dichroism indicated Mg(2+) induces conformational changes in both proteins.
Conclusions:
- Human and mouse APE1 proteins possess largely similar biochemical and biophysical properties.
- Mouse studies on APE1 biology and carcinogenesis are relevant to human biology.
- Findings support the extrapolation of mouse model conclusions to human APE1 research and therapeutic development.
