Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.     

Thermal denaturation of the BRCT tandem repeat region of human tumour suppressor gene product BRCA1

Reduced stability of the tandem BRCT domains of human BReast CAncer 1 (BRCA1) due to missense mutations may be critical for loss of function in DNA repair and damage-induced checkpoint control. In the present thermal denaturation study of the BRCA1 BRCT region, high-precision differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy provide evidence for the existence of a denatured state that is structurally very similar to the native. Consistency between theoretical structure-based estimates of the enthalpy (DeltaH) and heat capacity change (DeltaCp) and the calorimetric results is obtained when considering partial thermal unfolding contained in the region of the conserved hydrophobic pocket formed at the interface of the two BRCT repeats. The structural integrity of this region has been shown to be crucial for the interaction of BRCA1 with phosphorylated peptides. In addition, cancer-causing missense mutations located at the inter-BRCT-repeat interface have been linked to the destabilization of the tandem BRCT structure.     

Functional impact of missense variants in BRCA1 predicted by supervised learning

Many individuals tested for inherited cancer susceptibility at the BRCA1 gene locus are discovered to have variants of unknown clinical significance (UCVs). Most UCVs cause a single amino acid residue (missense) change in the BRCA1 protein. They can be biochemically assayed, but such evaluations are time-consuming and labor-intensive. Computational methods that classify and suggest explanations for UCV impact on protein function can complement functional tests. Here we describe a supervised learning approach to classification of BRCA1 UCVs. Using a novel combination of 16 predictive features, the algorithms were applied to retrospectively classify the impact of 36 BRCA1 C-terminal (BRCT) domain UCVs biochemically assayed to measure transactivation function and to blindly classify 54 documented UCVs. Majority vote of three supervised learning algorithms is in agreement with the assay for more than 94% of the UCVs. Two UCVs found deleterious by both the assay and the classifiers reveal a previously uncharacterized putative binding site. Clinicians may soon be able to use computational classifiers such as those described here to better inform patients. These classifiers can be adapted to other cancer susceptibility genes and systematically applied to prioritize the growing number of potential causative loci and variants found by large-scale disease association studies.     

Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2

Many sequence variants in predisposition genes are of uncertain clinical significance, and classification of these variants into high- or low-risk categories is an important problem in clinical genetics. Classification of such variants can be performed by direct epidemiological observations, including cosegregation with disease in families and degree of family history of the disease, or by indirect measures, including amino acid conservation, severity of amino acid change, and evidence from functional assays. In this study, we have developed an approach to the synthesis of such evidence in a multifactorial likelihood-ratio model. We applied this model to the analysis of three unclassified variants in BRCA1 and three in BRCA2. The evidence strongly suggests that two variants (C1787S in BRCA1 and D2723H in BRCA2) are deleterious, three (R841W in BRCA1 and Y42C and P655R in BRCA2) are neutral, and one (R1699Q in BRCA1) remains of uncertain significance. These results provide a demonstration of the utility of the model.     

BRCA1 variants in a family study of African-American and Latina women

We sequenced the entire coding region of BRCA1 to improve our understanding of the frequency and nature of BRCA1 variants in African-American and Latina women identified from a multiethnic cohort in Los Angeles, California. The study included 109 African-American and 140 Latina sibships from families with two or more cases of breast or ovarian cancer among first-degree relatives. BRCA1 was sequenced in 278 breast or ovarian cancer cases and 229 unaffected sisters. The proportion of cases with known disease-causing mutations was low (0.72, 95% confidence interval: 0–1.7%). In total, 33 sequence variants were identified, including two protein truncation mutations, one deletion, and six silent and 24 missense variants. Two novel rare variants were identified that appeared to act as benign polymorphisms. Four rare variants may be unique to women of African descent based on existing literature, and three have been described exclusively in Latina women. The frequency of common variants was similar for cases and controls, but the frequency of common variants for African-American women significantly differed from those previously described for Caucasian women. We believe this to be the largest study of high-risk African-American and Latina women sequenced for variants in the BRCA1 gene to date.     

Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy

Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single alpha-helix and two beta-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0 degrees C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some beta-strands (B3, B4, and B5 in beta-sheet B). These beta-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56 degrees C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.     

Thermal unfolding and aggregation of actin

Actin is one of the most abundant proteins in nature. It is found in all eukaryotes and plays a fundamental role in many diverse and dynamic cellular processes. Also, actin is one of the most ubiquitous proteins because actin-like proteins have recently been identified in bacteria. Actin filament (F-actin) is a highly dynamic structure that can exist in different conformational states, and transitions between these states may be important in cytoskeletal dynamics and cell motility. These transitions can be modulated by various factors causing the stabilization or destabilization of actin filaments. In this review, we look at actin stabilization and destabilization as expressed by changes in the thermal stability of actin; specifically, we summarize and analyze the existing data on the thermal unfolding of actin as measured by differential scanning calorimetry. We also analyze in vitro data on the heat-induced aggregation of actin, the process that normally accompanies actin thermal denaturation. In this respect, we focus on the effects of small heat shock proteins, which can prevent the aggregation of thermally denatured actin with no effect on actin thermal unfolding. As a result, we have proposed a mechanism describing the thermal denaturation and aggregation of F-actin. This mechanism explains some of the special features of the thermal unfolding of actin filaments, including the effects of their stabilization and destabilization; it can also explain how small heat shock proteins protect the actin cytoskeleton from damage caused by the accumulation of large insoluble aggregates under heat shock conditions.     

Thermal unfolding studies of a phytocyanin

The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 degrees C at pH 7.5 and between 50.7 and 57.4 degrees C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry-Eyring unfolding model Native<-->Unfolded-->Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (DeltaG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character.     

Thermal versus mechanical unfolding of ubiquitin

The authors studied the temperature-induced unfolding of ubiquitin by all-atom Monte Carlo simulations. The unfolding behavior is compared with that seen in previous simulations of the mechanical unfolding of this protein, based on the same model. In mechanical unfolding, secondary-structure elements were found to break in a quite well-defined order. In thermal unfolding, the authors saw somewhat larger event-to-event fluctuations, but the unfolding pathway was still far from random. Two long-lived secondary-structure elements could be identified in the simulations. These two elements have been found experimentally to be the thermally most stable ones. Interestingly, one of these long-lived elements, the first beta-hairpin, was found to break early in the mechanical unfolding simulations. Their combined simulation results thus enable the authors to predict in detail important differences between the thermal and mechanical unfolding behaviors of ubiquitin.     

Thermal unfolding simulations of a multimeric protein--transition state and unfolding pathways

The folding of an oligomeric protein poses an extra challenge to the folding problem because the protein not only has to fold correctly; it has to avoid nonproductive aggregation. We have carried out over 100 molecular dynamics simulations using an implicit solvation model at different temperatures to study the unfolding of one of the smallest known tetramers, p53 tetramerization domain (p53tet). We found that unfolding started with disruption of the native tetrameric hydrophobic core. The transition state for the tetramer to dimer transition was characterized as a diverse ensemble of different structures using Phi value analysis in quantitative agreement with experimental data. Despite the diversity, the ensemble was still native-like with common features such as partially exposed tetramer hydrophobic core and shifts in the dimer-dimer arrangements. After passing the transition state, the secondary and tertiary structures continued to unfold until the primary dimers broke free. The free dimer had little secondary structure left and the final free monomers were random-coil like. Both the transition states and the unfolding pathways from these trajectories were very diverse, in agreement with the new view of protein folding. The multiple simulations showed that the folding of p53tet is a mixture of the framework and nucleation-condensation mechanisms and the folding is coupled to the complex formation. We have also calculated the entropy and effective energy for the different states along the unfolding pathway and found that the tetramerization is stabilized by hydrophobic interactions.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR).To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.     

BRCA1 and BRCA2 missense variants of high and low clinical significance influence lymphoblastoid cell line post-irradiation gene expression

The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82–94%), poor for BRCAX with an LCS (40–50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71–100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.     

Thermal unfolding simulations of apo-calmodulin using leap-dynamics

The simulation method leap-dynamics (LD) has been applied to protein thermal unfolding simulations to investigate domain-specific unfolding behavior. Thermal unfolding simulations of the 148-residue protein apo-calmodulin with implicit solvent were performed at temperatures 290 K, 325 K, and 360 K and compared with the corresponding molecular dynamics trajectories in terms of a number of calculated conformational parameters. The main experimental results of unfolding are reproduced in showing the lower stability of the C-domain: at 290 K, both the N- and C-domains are essentially stable; at 325 K, the C-domain unfolds, whereas the N-domain remains folded; and at 360 K, both domains unfold extensively. This behavior could not be reproduced by molecular dynamics simulations alone under the same conditions. These results show an encouraging degree of convergence between experiment and LD simulation. The simulations are able to describe the overall plasticity of the apo-calmodulin structure and to reveal details such as reversible folding/unfolding events within single helices. The results show that by using the combined application of a fast and efficient sampling routine with a detailed molecular dynamics force field, unfolding simulations of proteins at atomic resolution are within the scope of current computational power.