Breast cancer susceptibility gene 2 (BRCA2)—a human tumor suppressor gene—is related to various malignancies such as breast and ovarian cancer. This gene can induce the key protein RAD51 recombinase, which is involved in homologous recombination with single-stranded DNA in the human body and can regulate RAD51 to complete the repair of damaged double-stranded DNA. Eight highly conserved BRC repeat motifs in BRCA2 protein serve as sites for the interaction between BRCA2 and RAD51. BRCA2 regulates RAD51 through these motifs. However, the mechanism of this interaction still requires further research. In this study, the BRC4 motif that demonstrated strong interaction with RAD51 was selected as template peptide. On the basis of known data regarding the crystal structure of the BRC4-RAD51(191–220) complex, a series of BRC4 mutants was designed using PyMOL software based on the sequence of BRC4, and polypeptides were synthesized by the Fmoc solid-phase method. After purification by reversed-phase high-performance liquid chromatography, the purity of the polypeptides reached >95 %. The primary determination of circular dichroism spectra showed that the polypeptides exhibited slight changes in secondary structure, which indicated that mutation on the non-conserved sites in BRC4 probably affected the interaction with BRC4. These findings will facilitate research on the interaction between targeting peptides and BRC4 mutants, as well the basic rules covering this interaction.
Pan Boyuan,Fu Linna,Du Heng,Liu Guangbin,Duan Bingchao,Lu Kui
RAD51 is a core factor for homologous recombination to repair DNA double-strand breaks and overexpressed in breast cancer cells. Truncated peptide BRC4 (1523-1537) was obtained by computer simulation which had the highest binding free energy targeting RAD51. To enhance the binding affinity to the target protein, six nicotinic acid derivatives were modified at the N -terminal of BRC4 (1523-1537) by Fmoc solid-state synthesis to obtain nicotinamide-modified peptides. The interaction of RAD51 (181-200) with BRC4 (1523-1537) and nicotinamide-modified peptides was verified by circular dichroism (CD) spectroscopy and fluorescence spectroscopy. In conclusion, modifying small molecule pharmacophores can improve binding ability. According to spectral results, 2-chloro-5-fluoronicotinic acid modified BRC4 (1523-1537) has the most significant influence on the secondary structure of RAD51 (181-200); binding constant is 1.1×10 4 L·mol -1 . Cell experiments showed that BRC4 (1523-1537) modified with nicotinic acid N-oxide had the best inhibitory effect on the proliferation of MDA-MB-231 cells.
Li Jinlei,Lu Kui,Lv Mingxiu,Liu Guangbin,Ma Li,Qi Jianzhao
Breast cancer susceptibility gene 2 ( BRCA2 ) is an important tumor suppressor, which is participated in repair of damaged DNA by its highly conserved BRC repeat motifs regulating RAD51 protein homologous recombination and thereby preventing cell carcinogenesis. In this study, the BRCA2(1524–1548)–RAD51(241–260) complex structure was obtained based on PDB bank data 1N0W, which provided the basis for site-specific mutation of BRCA2(1524–1548). The BRC4 and BRC4 analogous peptides were synthesized, and the interaction between BRC peptide and RAD51(241–260) was studied by fluorescence spectroscopy, circular dichroism spectroscopy and microscale thermophoresis (MST). The results of circular dichroism showed that the changes in secondary structures of RAD51(241–260) occurred after adding BRC4 analogous peptides, and the α-helix content increased significantly. Fluorescence spectral data demonstrated that the model of BRC peptide binding to RAD51(241–260) was static quenching, and the binding constants of BRC4, P1, P2, P4 with RAD51(241–260) were 1.647 × 10 −4 L mol −1 , 2.532 × 10 −4 L mol −1 , 3.161 × 10 −4 L mol −1 , 1.705 × 10 −4 L mol −1 , respectively. The results of MST indicated that P2 and RAD51(241–260) have better affinity for dissociation constant 44.286 μM. The strongest affinity between P2 and RAD51(241–260) indicated that the mutation of amino acid residue constituting BRC α-helix affects the structure and interaction of BRC peptide and RAD51(241–260).
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