RAP80 is a critical component of the BRCA1 complex, essential for the DNA damage response and homologous recombination repair. It contains two tandem ubiquitin-interacting motifs (UIM1 and UIM2) at its N-terminus that recognize K63-linked polyubiquitin chains on histone H2AX, facilitating recruitment of the BRCA1 complex to double-strand break sites. The RAP80 DE81 mutation, resulting from an in-frame deletion of glutamic acid at position 81 within UIM1, has been identified in familial breast cancer cases and is highly conserved across vertebrates. This mutation disrupts the structural integrity of RAP80, impairing its ability to bind polyubiquitin chains effectively. Our study employed a multi-model approach combining in silico modeling, biophysical analysis, and in vitro binding assays to investigate the functional consequences of this mutation.
Circular dichroism and thermal denaturation experiments revealed that RAP80 DE81 exhibits significantly reduced secondary structure stability compared to wild-type RAP80. The melting temperature (Tm) of DE81 was measured at 22°C versus 29°C for the wild type, indicating substantial destabilization. Fluorescence spectroscopy using ANS confirmed this trend, with Tm values of 23°C for DE81 and 30°C for wild type. Differential scanning calorimetry further supported these findings, showing a well-defined transition for wild-type RAP80 but no clear unfolding peak for DE81 due to its inherent instability. These results suggest that the DE81 mutation induces a conformational shift from helical to random structure, particularly affecting the UIM domain.1,7-Dibromoheptane References
Proteolytic resistance assays demonstrated that RAP80 DE81 is more susceptible to trypsin and chymotrypsin digestion than the wild type, reinforcing the notion of structural fragility. Molecular modeling revealed that Glu81 plays a key role in stabilizing the UIM1 helix through hydrogen bonding with Arg42 and hydrophobic interactions with Leu73 of ubiquitin. Its deletion disrupts these intermolecular contacts, leading to misfolding and loss of binding affinity. Surface plasmon resonance (SPR) analysis quantified this defect: the dissociation constant (KD) for DE81 was 0.459 µM, over ten times higher than the wild type’s KD of 36.5 nM. Association rate constants were also markedly reduced in DE81, confirming impaired kinetics of interaction.
GST pull-down assays corroborated SPR data, showing diminished binding between DE81 and di-ubiquitin (K63-linked). Consequently, the recruitment of the RAP80-BRCA1 complex to DNA damage foci is defective, resulting in impaired homologous recombination repair. This dysfunction contributes to genomic instability, chromosomal aberrations, and increased susceptibility to tumorigenesis.Ixekizumab Immunology/Inflammation The dynamic equilibrium between helical and random conformations in UIM domains likely enables flexible recognition of ubiquitin chains during repair processes.PMID:35169866 However, the DE81 mutation shifts this balance toward a disordered state, compromising the avidity-based cooperative binding mechanism.
In summary, the RAP80 DE81 mutation destabilizes the UIM1 domain, disrupts ubiquitin chain recognition, and prevents proper localization of the BRCA1 complex at DNA damage sites. This leads to defective DNA repair and accumulation of genetic errors. Understanding these molecular mechanisms provides insight into the pathogenesis of hereditary cancers and opens avenues for developing therapeutic strategies targeting protein stability or enhancing residual function in mutant carriers.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com