Mislocalization of BRCA1-complex due to ABRAXAS Arg361Gln mutation

<div><p>ABRAXAS is an integral member of BRCA1-complex, which helps in its recruitment to the DNA damage site. It interacts with BRCA1 via its C-terminal phospho-peptide binding motif while the N-terminal associates with RAP80, and thereby recruits the BRCA1-complex at the site of DNA damage. Nonetheless, how ABRAXAS helps in the structural integrity of BRCA1-complex, and its DNA repair mechanism remains elusive. To elucidate the role of ABRAXAS in the DNA repair process, we characterized the ABRAXAS wild type and Arg361Gln mutant using <i>in silico</i> and <i>in vitro</i> approach. It has been observed that ABRAXAS Arg361Gln mutant is responsible for defective nuclear localization of BRCA1-complex, and hence important for DNA repair function. We found conformational changes in ABRAXAS mutant, which impaired binding to RAP80 and further disturb BRCA1-complex localization. The results presented in this paper will help to understand the cause of BRCA1 mislocalization, and various DNA repair defects that occur due to substitution. Comparative study of ABRAXAS wild type and mutant will provide helpful perspective for inhibitor designing that can potentially recompense the deleterious effect(s) of Arg361Gln mutation, and have therapeutic application.</p></div

Structural basis to characterise transactivation domain of BRCA1

<p>Familial inheritance of breast and ovarian cancer is attributed to mutations discovered in functional domains of BRCA1 gene. BRCA1 is a multifunctional protein responsible for maintaining the genomic integrity and has transcriptional regulatory function encoded in its C-terminal region. The different amino-terminal e extensions to BRCA1 BRCT domain are responsible for transcription activation. However, only BRCA1 BRCT (1649–1859) amino acids have been explored for its structural characteristics. Noting the importance of extended region to the N-terminus of BRCT different regions of BRCA1 which demonstrates maximum transactivation activity has been explored for their structure and functional activity. Secondary and tertiary structural analysis revealed a limited alpha-helical content with well-folded tertiary structure. <i>In silico</i> tools were used to corroborate the <i>in vitro</i> results. Amino acids composition and sequence analysis display a propensity for intrinsic disorder and coiled-coil formation in BRCA1 (1396–1863) (BRCA1-TAD). The results presented in this paper suggest the extreme flexibility in coiled-coil motif might be an important requirement in the establishment of protein–protein interaction networks for BRCA1.</p

Structural and functional characterization of the MERIT40 to understand its role in DNA repair

<div><p><i>MERIT40</i> (MEdiator of RAP80 Interaction and Targeting 40) is a novel associate of the BRCA1-complex and plays an essential role in DNA damage repair. It is the least characterized protein of BRCA1-complex and mainly responsible for maintaining the complex integrity. However, its structural and functional aspects of regulating the complex stability still remain elusive. Here, we carried out a comprehensive examination of MERIT40 biophysical properties and identified its novel interacting partner which would help to understand its role in BRCA1-complex. The recombinant protein was purified by affinity chromatography and unfolding pathway was determined using spectroscopic and calorimetric methods. Molecular model was generated using combinatorial approaches of modeling, and monomer–monomer docking was carried out to identify dimeric interface. Disordered region of MERIT40 was hatchet using trypsin and chymotrypsin to illustrate the existence of stable domain whose function was speculated through DALI search. Our findings suggest that MERIT40 forms a dimer in a concentration-independent manner. Its central region shows remarkable stability towards the protease digestion and has structural similarity with vWA-like region, a domain mainly present in complement activation factors. MERIT40 undergoes a three-state unfolding transition pathway with a dimeric intermediate. It interacts with adaptor molecule of BRCA1-complex, called ABRAXAS, thus help in extending the bridging interaction among various members which further stabilizes the whole complex. The results presented in this paper provide first-hand information on structural and folding behavior of MERIT40. These findings will help in elucidating the role of protein–protein interactions in stabilization of BRCA1-complex.</p></div

Binding interaction of RAP80 UIMs and ΔE81 with Di-Ub (K-63 linked).

<p>(<b>A</b>) Structure of Di-Ub (K-63 linked)-RAP80 UIMs (79–124) wild type (PDB ID: 2RR9), and (<b>B</b>) Di-Ub (K-63 linked)-RAP80 (79–124) UIMs ΔE81. Wild type and Di-Ub (K-63 linked) complex is stabilized by weak intermolecular interactions. α-helix of RAP80 (79–124) UIM ΔE81 was found to be distorted. (<b>C</b>) multiple sequence alignment of UIMs region showed it's highly conserved nature in various species. Glu 81 residue is highlighted in red color.</p

Molecular weight estimation of purified protein.

<p>Ve/Vo: Elution volume/Void volume ratio in gel filtration chromatography (superdex 200 16/60).</p>a<p>Determined from Protparam, Expasy.</p>b<p>Determined from standard myoglobin, ovalbumin, albumin, IgG, Ferritin.</p

Structure and stability analysis of RAP80 wild type and ΔE81.

<p>Secondary structural components and thermal stability of RAP80 wild type and ΔE81. (<b>A</b>) Overlay of Far-UV Circular Dichroism spectrum of wild type and ΔE81. Wild-type showed well-defined α/β characteristics compared to a random structure pattern of ΔE81. Thermal stability of RAP80 wild type. (<b>B</b>) Thermal denaturation of RAP80 wild type and ΔE81 using Circular Dichroism and (<b>C</b>) using ANS as extrinsic fluorophore in Fluorescence. Unfolded fractions were calculated and plotted against different temperatures. (<b>D</b>) Differential Scanning Calorimetry profile of RAP80 wild type. Protein showed a well-defined transition around 28°C.</p

Resistivity profile of RAP80 wild type and ΔE81 towards Protease digestion.

<p>Limited proteolysis of RAP80 wild type (<b>A, C</b>) and ΔE81 (<b>B, D</b>) using trypsin (<b>A, B</b>) and Chymotrypsin (<b>C, D</b>) as proteases. Wild type showed relatively high resistance towards proteolysis as indicated by less rate of decrease of band intensity. This suggests a well-folded structure of wild type compared to ΔE81. Ctl- control was taken as untreated with proteases.</p

Expression and purification profile of RAP80 wild type and ΔE81.

<p>(<b>A</b>) Whole-cell lysate, and supernatant obtained after sonication and centrifugation were heated with Laemmli buffer and loaded onto SDS-PAGE. Similarly, protein was eluted from beads by heating with Laemmli buffer and loaded on gel. Lane 1-Total protein, 2-soluble protein, 3-fusion protein bound on beads, 4- protein after on beads cleavage, 5-elution fraction of affinity purified proteins. Single arrow - RAP80 wild type protein (<b>B</b>) Purified protein after gel filtration chromatography on SDS-PAGE. Lane 1- RAP80 ΔE81, 2- RAP80 wild type (<b>C</b>) Overlay of gel filtration spectra of RAP80 wild type and ΔE81 (Superdex 200). Elution profiles of both the protein were similar and suggest their monomeric nature.</p

Anticipated mechanism of consequence due to RAP80 ΔE81.

<p>The model elucidate a possible mechanism of chromosomal aberration due to RAP80 ΔE81 mutation. In the wild-type RAP80: <b>Step1</b>, showed the intact nucleosome complex; <b>Step 2</b>, double strand break due to ionization radiation; <b>Step 3</b>, ATM/ATR kinase activation and assembly of various damage repair proteins at DNA double strand break (DSB) site followed by formation of polyubiquitin chain(s) on histone(s) (H2AX). The polyubiquitin chain(s) are recognized by RAP80 UIMs motif thereby recruiting the BRCA1 complex to the DNA damage site. However, in case of ΔE81 mutation, interaction between polyubiquitin chain and RAP80 UIM altered due to structural distortion in α-helix which further leads to defective recruitment of the BRCA1 complex. Error-prone DNA damage repair increases the chances of chromosomal aberration and hence the tumorigenesis.</p

Thermal parameters of protein unfolding.

<p>T_m Melting Temperature.</p