Characterization of cancer-linked BRCA1-BRCT missense variants and their interaction with phosphoprotein targets.
The breast cancer tumor suppressor protein BRCA1 is involved in DNA repair and cell cycle control. Mutations at the two C-terminal tandem (BRCT) repeats of BRCA1 detected in breast tumor patients were identified either to lower the stability of the BRCT domain and/or to disrupt the interaction of BRCT with phoshpopeptides. The aim of this study was to analyze five BRCT pathogenic mutations for their effect on structural integrity and protein stability. For this purpose, the five cancer-associated BRCT mutants: V1696L, M1775K, M1783T, V1809F, and P1812A were cloned in suitable prokaryotic protein production vectors, and the recombinant proteins were purified in soluble and stable form for further biophysical studies. The biophysical analysis of the secondary structure and the thermodynamic stability of the wild-type, wt, and the five mutants of the BRCT domain were performed by Circular Dichroism Spectroscopy (CD) and Differential Scanning Microcalorimetry (DSC), respectively. The binding capacity of the wt and mutant BRCT with (pBACH1/BRIP1) and pCtIP were measured by Isothermal Titration Calorimetry (ITC). The experimental results demonstrated that the five mutations of the BRCT domain: (i) affected the thermal unfolding temperature as well as the unfolding enthalpy of the domain, to a varying degree depending upon the induced destabilization and (ii) altered and/or abolished their affinity to synthetic pBACH1/BRIP1 and pCtIP phosphopeptides by affecting the structural integrity of the BRCT active sites. The presented experimental results are one step towards the elucidation of the effect of various missense mutations on the structure and function of BRCA1-BRCT.
Drikos
Ioannis
I
Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15701 Athens, Hellas.
Nounesis
George
G
Vorgias
Constantinos E
CE
D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010750:Phosphoproteins / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D019313:BRCA1 Protein; D010750:Phosphoproteins