Variant of uncertain significance Arg866Cys enhances disorderedness of h-BRCA1 (759-1064) region.

High structural flexibility has been reported in the central region of BRCA1, which hinders the structural and functional evaluations of mutations identified in the domain. Additionally, the need to categorize variants of unknown significance (VUS) has increased due to the growth in the number of variants reported in clinical settings. Therefore, unraveling the disease-causing mechanism of VUS identified in different functional domains of BRCA1 is still challenging. The current study uses a multidisciplinary approach to assess the structural impact of BRCA1 Arg866Cys mutation discovered in the central domain of BRCA1. The structural alterations have been characterized using Circular-Dichroism spectroscopy, nano-DSF, and molecular-dynamics simulations. BRCA1 Arg866Cys mutant demonstrated more flexibility and lesser affinity to DNA than the wild-type protein. The BRCA1(759-1064) wild-type protein was shown to be a ßII-rich protein with an induced D-O transition in the presence of DNA and 2,2,2-Trifluoroethanol (TFE). The protein's alpha-helical composition did not significantly change in the presence of TFE, besides an increase in ß-turns and loops. Under Transmission Electron Microscopes (TEM), amyloid-like fibrils structure was detected for Arg866Cys mutant whereas the wild-type protein showed amorphous aggregates. An increased ThT fluorescence indicated ß-rich composition and aggregation-prone behaviour for BRCA1 wild-type protein, while the fluorescence intensity was significantly quenched in the Arg866Cys mutant. Furthermore, increased conformational flexibility in the Arg866Cys variant was observed by principal component analysis. This work aims to comprehend the inherently disordered region of BRCA1 as well as the impact of missense mutations on folding patterns and binding to DNA for functional aspects.

In silico and structure-based evaluation of deleterious mutations identified in human Chk1, Chk2, and Wee1 protein kinase.

Checkpoint kinases Chk1, Chk2, Wee1 are playing a key role in DNA damage response and genomic integrity. Cancer-associated mutations identified in human Chk1, Chk2, and Wee1 were retrieved to understand the function associated with the mutation and also alterations in the folding pattern. Therefore, an attempt has been made to identify deleterious effect of variants using in silico and structure-based approach. Variants of uncertain significance for Chk1, Chk2, and Wee1 were retrieved from different databases and four prediction servers were employed to predict pathogenicity of mutations. Further, Interpro, I-Mutant 3.0, Consurf, TM-align, and have (y)our protein explained were used for comprehensive study of the deleterious effects of variants. The sequences of Chk1, Chk2, and Wee1 were analyzed using Clustal Omega, and the three-dimensional structures of the proteins were aligned using TM-align. The molecular dynamics simulations were performed to explore the differences in folding pattern between Chk1, Chk2, Wee1 wild-type, and mutant protein and also to evaluate the structural integrity. Thirty-six variants in Chk1, 250 Variants in Chk2, and 29 in Wee1 were categorized as pathogenic using in silico prediction tools. Furthermore, 25 mutations in Chk1, 189 in Chk2, and 14 in Wee1 were highly conserved, possessing deleterious effect and also influencing the protein structure and function. These identified mutations may provide underlying genetic intricacies to serve as potential targets for therapeutic inventions and clinical management.

Mutations at proximal cysteine residues in PML impair ATO binding by destabilizing the RBCC domain.

Acute promyelocytic leukemia (APL) is characterized by the fusion gene promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) and is conventionally treated with arsenic trioxide (ATO). ATO binds directly to the RING finger, B-box, coiled-coil (RBCC) domain of PML and initiates degradation of the fusion oncoprotein PML-RARA. However, the mutational hotspot at C212-S220 disrupts ATO binding, leading to drug resistance in APL. Therefore, structural consequences of these point mutations in PML that remain uncertain require comprehensive analysis. In this study, we investigated the structure-based ensemble properties of the promyelocytic leukemia-RING-B-box-coiled-coil (PML-RBCC) domains and ATO-resistant mutations. Oligomeric studies reveal that PML-RBCC wild-type and mutants C212R, S214L, A216T, L217F, and S220G predominantly form tetramers, whereas mutants C213R, A216V, L218P, and D219H tend to form dimers. The stability of the dimeric mutants was lower, exhibiting a melting temperature (Tm) reduction of 30 °C compared with the tetrameric mutants and wild-type PML protein. Furthermore, the exposed surface of the C213R mutation rendered it more prone to protease digestion than that of the C212R mutation. The spectroscopic analysis highlighted ATO-induced structural alterations in S214L, A216V, and D219H mutants, in contrast to C213R, L217F, and L218P mutations. Moreover, the computational analysis revealed that the ATO-resistant mutations C213R, A216V, L217F, and L218P caused changes in the size, shape, and flexibility of the PML-RBCC wild-type protein. The mutations C213R, A216V, L217F, and L218P destabilize the wild-type protein structure due to the adaptation of distinct conformational changes. In addition, these mutations disrupt several hydrogen bonds, including interactions involving C212, C213, and C215, which are essential for ATO binding. The local and global structural features induced by these mutations provide mechanistic insight into ATO resistance and APL pathogenesis.

Structural implications of amyloidogenic rare variants Ser282Leu and Gln356Arg identified in h-BRCA1.

Preliminary studies have shown BRCA1 (170-1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h-BRCA1(260-553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small-angle X-ray scattering analysis revealed WT scores Rg -32 Å, Dmax -93 Å, and Rflex-51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder-to-order transition in the presence of cruciform DNA and 2,2,2-Trifluoroethanol (TFE). An increased alpha-helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT-DNA complex compared to the Gln356Arg and Ser282Leu protein-DNA complex. Moreover, mature amyloid-like fibrils were observed with 30 µM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher-order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

In-silico and structure-based assessment to evaluate pathogenicity of missense mutations associated with non-small cell lung cancer identified in the Eph-ephrin class of proteins.

Ephs belong to the largest family of receptor tyrosine kinase and are highly conserved both sequentially and structurally. The structural organization of Eph is similar to other receptor tyrosine kinases; constituting the extracellular ligand binding domain, a fibronectin domain followed by intracellular juxtamembrane kinase, and SAM domain. Eph binds to respective ephrin ligand, through the ligand binding domain and forms a tetrameric complex to activate the kinase domain. Eph-ephrin regulates many downstream pathways that lead to physiological events such as cell migration, proliferation, and growth. Therefore, considering the importance of Eph-ephrin class of protein in tumorigenesis, 7,620 clinically reported missense mutations belonging to the class of variables of unknown significance were retrieved from cBioPortal and evaluated for pathogenicity. Thirty-two mutations predicted to be pathogenic using SIFT, Polyphen-2, PROVEAN, SNPs&GO, PMut, iSTABLE, and PremPS in-silico tools were found located either in critical functional regions or encompassing interactions at the binding interface of Eph-ephrin. However, seven were reported in nonsmall cell lung cancer (NSCLC). Considering the relevance of receptor tyrosine kinases and Eph in NSCLC, these seven mutations were assessed for change in the folding pattern using molecular dynamic simulation. Structural alterations, stability, flexibility, compactness, and solvent-exposed area was observed in EphA3 Trp790Cys, EphA7 Leu749Phe, EphB1 Gly685Cys, EphB4 Val748Ala, and Ephrin A2 Trp112Cys. Hence, it can be concluded that the evaluated mutations have potential to alter the folding pattern and thus can be further validated by in-vitro, structural and in-vivo studies for clinical management.

Structural dynamics of clinically-reported VUS in the BARD1 ARD-BRCT region to predict the molecular basis of alterations.

The functional domains of BARD1, comprise the Ankyrin Repeat Domain (ARD), C-Terminal domains (BRCTs), and a linker between ARD and the BRCTs, which are known to bind to Cleavage stimulation Factor complex-subunit of 50 kDa (CstF-50). The pathogenic mutation Q564H in the BARD1 ARD-linker-BRCT region has been reported to abrogate the binding between BARD1 and CstF-50. Intermediate penetrance variants of BARD1 are associated with the occurrence of breast cancer. Therefore, seven missense variants of unknown significance (VUS), L447V, P454L, N470S, V507M, I509T, C557S, and Q564H of BARD1, reported in the ARD domain and the linker region were evaluated via molecular dynamics (MD) simulations. The mutants revealed statistically significantly different distributions of RMSD (root mean square deviation), residuewise RMSF (root mean square fluctuation), Rg (radius of gyration), SASA (solvent accessible surface area), and COM (centre of mass)-to-COM distance between the ARD and the BRCT repeat, between the wild type and each mutant. The secondary structural composition of the mutants was slightly altered relative to that of the wild type. However, the reported in-silico based prediction require further validation using in-vitro, biophysical and structure-based approachCommunicated by Ramaswamy H. Sarma.

Evaluation of Pathogenicity and Structural Alterations for the Mutations Identified in the Conserved Region of the C-Terminal Kinase Domain of Human-Ribosomal S6 Kinase 1.

Human-ribosomal s6 kinase 1 (h-RSK1) is an effector kinase of the Ras/MAPK signaling pathway, which is involved in the regulation of the cell cycle, proliferation, and survival. RSKs comprise two functionally distinct kinase domains at the N-terminal (NTKD) and C-terminal (CTKD) separated by a linker region. The mutations in RSK1 may have the potential to provide an extra benefit to the cancer cell to proliferate, migrate, and survive. The present study focuses on evaluating the structural basis for the missense mutations identified at the C-terminal kinase domain of human-RSK1. A total of 139 mutations reported on RSK1 were retrieved from cBioPortal, where 62 were located at the CTKD region. Furthermore, 10 missense mutations Arg434Pro, Thr701Met, Ala704Thr, Arg725Trp, Arg726Gln, His533Asn, Pro613Leu, Ser720Cys, Arg725Gln, and Ser732Phe were predicted to be deleterious using in silico tools. To our observation, these mutations are located in the evolutionarily conserved region of RSK1 and shown to alter the inter- and intramolecular interactions and also the conformational stability of RSK1-CTKD. The molecular dynamics (MD) simulation study further revealed that the five mutations Arg434Pro, Thr701Met, Ala704Thr, Arg725Trp, and Arg726Gln showed maximum structural alterations in RSK1-CTKD. Thus, based on the in silico and MD simulation analysis, it can be concluded that the reported mutations may serve as potential candidates for further functional studies.

In Silico-Based Structural Evaluation to Categorize the Pathogenicity of Mutations Identified in the RAD Class of Proteins.

RAD genes, known as double-strand break repair proteins, play a major role in maintaining the genomic integrity of a cell by carrying out essential DNA repair functions via double-strand break repair pathways. Mutations in the RAD class of proteins show high susceptibility to breast and ovarian cancers; however, adequate research on the mutations identified in these genes has not been extensively reported for their deleterious effects. Changes in the folding pattern of RAD proteins play an important role in protein-protein interactions and also functions. Missense mutations identified from four cancer databases, cBioPortal, COSMIC, ClinVar, and gnomAD, cause aberrant conformations, which may lead to faulty DNA repair mechanisms. It is therefore necessary to evaluate the effects of pathogenic mutations of RAD proteins and their subsequent role in breast and ovarian cancers. In this study, we have used eight computational prediction servers to analyze pathogenic mutations and understand their effects on the protein structure and function. A total of 5122 missense mutations were identified from four different cancer databases, of which 1165 were predicted to be pathogenic using at least five pathogenicity prediction servers. These mutations were characterized as high-risk mutations based on their location in the conserved domains and subsequently subjected to structural stability characterization. The mutations included in the present study were selected from clinically relevant mutants in breast cancer pedigrees. Comparative folding patterns and intra-atomic interaction results showed alterations in the structural behavior of RAD proteins, specifically RAD51C triggered by mutations G125V and L138F and RAD51D triggered by mutations S207L and E233G.

In silico structural analysis of secretory clusterin to assess pathogenicity of mutations identified in the evolutionarily conserved regions.

Clusterin (CLU) is a secreted glycoprotein, heterodimeric in nature, and is expressed in a wide variety of tissues and body fluids such as serum and plasma. CLU has also been known to be a promising biomarker for cell death, malignancy, cancer progression, and resistance development. However, the lack of a CLU crystal structure obstructs understanding the possible role of reported mutations on the structure, and the subsequent effects on downstream signaling pathways and cancer progression. Considering the importance of crystal structure, a model structure of the pre-secretory isoform of CLU was built to predict the effect of mutations at the molecular level. Ab initio model was built using RaptorX, and loop refinement and energy minimization were carried out with ModLoop, ModRefiner, and GalaxyWeb servers. The cancer associated mutational spectra of CLU was retrieved from the cBioPortal server and 117 unique missense mutations were identified. Evolutionarily conserved regions and pathogenicity of mutations identified in CLU were analyzed using ConSurf and Rhapsody, respectively. Furthermore, sequence and structure-based mutational analysis were carried out with iSTABLE, DynaMut and PremPS servers. Molecular dynamics simulations were carried out with GROMACS for 50 ns to determine the stability of the wild type and mutant protein structures. A dynamically stable model structure of pre-secretory CLU (psCLU) which has high concurrence with the sequence based secondary structure predictions has been explored. Changes in the intra-atomic interactions and folding pattern between wild type and mutant structures were observed. To our conclusion, eleven mutations with the highest structural and functional significance have been predicted to have pathogenic and deleterious effects.Communicated by Ramaswamy H. Sarma.

In silico and structure-based assessment to classify VUS identified in the α-helical domain of BRCA2.

Breast cancer type 2 susceptibility (BRCA2) protein plays a crucial role in DNA double-strand breaks repair mechanism by homologous recombination. Pathogenic mutations in the BRCA2 gene confer an increased risk of hereditary breast and ovarian cancer (HBOC). Different missense mutations are identified from a larger cohort of patient populations in the BRCA2. However, most missense mutations are classified as 'Variants of Uncertain Significance' (VUS) due to a lack of data from structural, functional, and clinical assessments. Therefore, this study focused on assessing VUS identified in the α-helical domain of h-BRCA2 using different in silico tools and structure-based molecular dynamics simulation. A total of 286 identified VUS were evaluated using Align-GVGD, PROVEAN and PANTHER servers and 18 variants were predicted to be pathogenic. Further, out of 18 variants analyzed using the ConSurf server, 16 variants were found to be evolutionary conserved. These 16 conserved variants were submitted to PremPS and Dynamut server to assess the effect of the mutation at the protein structure level; 12 mutations were predicted to have a destabilizing effect on the native protein structure. Finally, molecular dynamics simulations revealed 5 variants BRCA2 Cys2646Tyr, Asp2665Val, Trp2619Arg, Trp2619Ser and Tyr2660Cys can alter the folding pattern and need further validation using in vitro, structural and in vivo studies to classify as pathogenic.Communicated by Ramaswamy H. Sarma.

In Silico and Structure-Based Assessment of Similar Variants Discovered in Tandem Repeats of BRCT Domains of BRCA1 and BARD1 To Characterize the Folding Pattern.

BRCA1 and BARD1 are important proteins in the homologous DNA damage repair pathways. Different genetic variants identified in these proteins have been clinically correlated with the occurrence of hereditary breast and ovarian cancer (HBOC). Variants of unknown significance (VUS) reported in the BRCT domains of BRCA1 and BARD1 substantiate the importance of BRCT domain-containing proteins for genomic integrity. To classify the pathogenicity of variants, in silico, structural and molecular dynamics (MD)-based approaches were explored. Different variants reported in the BRCT region were retrieved from cBioPortal, LOVD3, BRCA Exchange, and COSMIC databases to evaluate the pathogenicity. Multiple sequence alignment and superimposition of the structures of BRCA1 BRCT and BARD1 BRCT domains were performed to compare alterations in folding patterns. From 11 in silico predictions servers, variants reported to be pathogenic by 70% of the servers were considered for structural analysis. To our observations, four residue pairs of both the proteins were reported, harboring 11 variants, H1686Y, W1718L, P1749L, P1749S, and W1837L variants for BRCA1 BRCT and H606D, H606N, W635L, P657L, P657S, and W762F for BARD1 BRCT. MD simulations of the BRCT repeat regions of these variants and wild-type proteins were performed to evaluate the differences of folding patterns. Root mean square deviation (RMSD), R g, solvent-accessible surface area (SASA), and root mean square fluctuation (RMSF) of variants showed slight differences in the folding patterns from the wild-type proteins. Furthermore, principal components analysis of H1686Y, P1749S, and W1718L variants of BRCA1 showed less flexibility than the wild type, whereas that of H606D, W635L, and W762F of BARD1 showed more flexibility than the wild type. Normal mode analysis of the energy minima from the simulation trajectories revealed that most of the variants do not show much differences in the flexibility compared to the wild-type proteins, except for the discrete regions in the BRCT repeats, most prominently in the 1798-1801 amino acid region of BRCA1 and at the residue 744 in BARD1.

Multimodal approach to characterize the tetrameric form of human PML-RBCC domain and ATO-mediated conformational changes.

RING-B box-coiled coil (RBCC) domain of promyelocytic leukemia (PML) comprises a zinc finger motif that is targeted by arsenic trioxide (ATO) to treat acute promyelocytic leukemia (APL) pathogenesis. Preliminary evidence suggests that the PML-RBCC has different functional characteristics, but no structural details have been reported despite its importance in differential expression and cell-cycle regulation. Therefore, the recombinant h-PML-RBCC protein was purified to its homogeneity, and characterized for oligomeric behaviour which indicated that RBCC domain exists as a tetramer in solution. Furthermore, nano-DSF and circular-dichroism demonstrated that the tetrameric form preserves its native conformation along with thermal stability (Tm = 83.2 °C). In-silico-based PML-RBCC structure was used to perform the molecular dynamics simulation for 300 ns in the presence of zinc atoms, which demonstrated the differential dynamic of PML-RBCC tetrameric chains. MMPBSA analysis also indicated the role of hydrophobic interactions that favours stable tetrameric structure of PML-RBCC. ATO-induced secondary and tertiary structure changes were observed in PML-RBCC using circular dichroism and fluorescence spectroscopy. Dynamic light scattering and transmission electron microscopy revealed ATO-induced higher-order oligomerization and aggregation of PML-RBCC. The unique oligomeric nature of the h-PML-RBCC protein and its interactions with ATO will help to understand the mechanism of APL pathogenesis and drug resistance.

Evaluation of conformational transitions of h-BRCA2 functional domain and unclassified variant Arg2502Cys using multimodal approach.

Breast cancer type 2 susceptibility (BRCA2) protein plays an essential role in the repair mechanism of DNA double-strand breaks and interstrand cross-links by Homologous recombination. Germline mutations identified in the BRCA2 gene confer an increased risk of hereditary breast and ovarian cancer. Missense mutations are identified all over the gene, including the DNA binding region of BRCA2 that interacts with FANCD2. However, the majority of these missense mutations are classified as 'Variants of Uncertain Significance' due to a lack of structural, functional and clinical correlations. Therefore, multi-disciplinary in-silico, in-vitro and biophysical approaches have been explored to characterize an unclassified missense mutation, BRCA2 Arg2502Cys, identified from a case-control study. Circular-dichroism and Fluorescence spectroscopy show that the Arg2502Cys mutation in hBRCA2 (residues 2350-2545) decreases the α-helical/ß-sheet propensity of the wild-type protein and perturb the tertiary structure conformation. Molecular dynamics simulations revealed alteration in the intramolecular H-bonds, overall compactness and stability of the hydrophobic core were observed in the mutant protein. Principle component analysis indicated that Arg2502Cys mutant exhibited comparatively large conformational transitions and periodic fluctuation. Therefore, to our conclusion, BRCA2 Arg2502Cys mutant perturbed the structural integrity and conformational dynamics of BRCA2.

Crystal structure of clinically reported mutations Gly656Arg, Gly656Glu and Asp751His identified in the kinase domain of EphA7.

Erythropoietin producing hepatocellular (Eph) forms the largest family of receptor tyrosine kinases (RTK). As a family, Eph regulates physiological events such as cell-cell interaction, cell migration, and adhesion. The Kinase domain is the catalytic core of the Eph receptor and is highly conserved sequentially. EphA7 has been recently regarded as a cancer driver gene and comprises several clinically important mutations. Three of the EphA7 mutations Gly656Glu, Gly656Arg, and Asp751His, present in the kinase domain, are predicted to be highly pathogenic. Furthermore, Gly656Glu and Gly656Arg are reported to be hotspot mutations. Considering the importance of mutations, crystals structure of EphA7 Gly656Glu, Gly656Arg, and Asp751His mutants has been explored. Changes in folding pattern and intramolecular interactions were observed in mutant structures. Secondary structural changes were observed in the hinge region of EphA7 Gly656Arg and Asp751His structure, affecting the transition of kinase domain between open and closed conformations. EphA7 Asp751His mutant structure shows a distorted nucleotide-binding groove. Differences were observed in hydrogen bonding and hydrophobic interactions between the catalytic and highly conserved DFG motif in the EphA7 mutants, which may influence the catalytic activity of kinase domain.

Genetic ablation of pregnancy zone protein promotes breast cancer progression by activating TGF-ß/SMAD signaling.

PURPOSE: Pregnancy zone protein (PZP) is best known as protease inhibitor and its concentration in human blood plasma increases dramatically during pregnancy. Recent investigation revealed a role of PZP inactivating germ-line mutation in breast cancer predisposition, and therefore we designed a study to evaluate functional involvement of this protein in tumor pathogenesis. METHODS: PZP knockout cells were generated utilizing the CRISPR-Cas9 approach in MCF7 and T47D (breast cancer) cell lines, and colony formation, cell proliferation, and migration assays carried out. TGF-ß and SMAD expression studies were performed using qRT-PCR and Western blot. PZP expression in tumor vs normal tissue was compared using meta-analyses of data records of breast cancer patients (n = 1211) included in the TCGA consortium registry as well as in independent cohorts of hormone receptor-positive (n = 118) and triple-negative breast cancer (TNBC) patients (n = 116). RESULTS: We demonstrated that genetic ablation of PZP efficiently inhibits tamoxifen-induced apoptosis and enhances cell proliferation, migration, and colony-forming capacity. We found a significant increase in survival fraction of CRISPR/Cas9-mediated PZP knockout clones compared to wild-type counterpart after tamoxifen treatment (p < 0.05). The PZP knockout significantly promoted breast cancer cell migration (p < 0.01) in vitro. We observed high expression of TGF-ß2 ligand, TGF-ß- receptor 2, and upregulation of phosphorylated regulatory-SMADs (pSMAD2 and pSMAD3) activating the pro-survival function of TGF-ß/SMAD signaling in PZP knockout clones. Meta-analyses of data records of breast cancer patients indicated that low PZP expression is associated with poor overall survival at 6 years (51.7% vs 62.9% in low vs high expressers, respectively; p = 0.026). We also observed a significantly lower PZP mRNA expression in TNBC as compared with hormone receptor-positive tumors (p = 0.019). CONCLUSION: Taken together, our results suggest that genetic ablation of PZP results in tumor progression and low expression of PZP is associated with poor survival of breast cancer patients.

Comprehensive Analysis of Low Molecular Weight Serum Proteome Enrichment for Mass Spectrometric Studies.

RATIONALE: The low molecular weight (LMW) proteins present in circulating body fluids, such as serum and plasma, hold biological significance as possible biomarkers. A major obstacle in mass spectrometry-based proteomics of serum is the presence of abundant high molecular weight proteins which mask the identification and quantitation of lower molecular weight proteins. Traditional methods involve the use of affinity resins to remove high molecular weight proteins, such as albumin and immunoglobulin G, with concomitant loss of lower molecular weight proteins. Considering the importance of depleting high molecular proteins, this paper compares an affinity resin, a gel-filter, and an acetonitrile (ACN) precipitation method to achieve successful removal of high molecular weight proteins and recovery of lower molecular weight proteins. METHODS: Serum enrichment was carried out by multiple methods such as with the commercially available serum protein mini kit, ACN precipitation, and a gel filter method. Mass spectrometric runs were carried out on an AB SCIEX ESI QTOF 5600 mass spectrometer. Mass spectrometry analysis of the enriched serum obtained by ACN precipitation and gel filter method was performed for global proteome profiling. Quantitative mass spectrometry using isobaric tags for relative and absolute quantitation (iTRAQ) for ACN-precipitated enriched serum was also carried out. RESULTS: The gel filter method, though allowing for the resolution and identification of LMW proteins, was better suited for global proteome analysis and not preferred for quantitative proteomic experiments. In contrast, enrichment by the ACN precipitation method allowed for the reproducible identification and quantitation of LMW proteins having molecular weight ≥4 kDa. CONCLUSIONS: Using only chilled ACN and centrifugation, most of the highly abundant proteins were successfully removed from the serum, while recovering a significant portion of the LMW proteome. A more rapid protocol, which is compatible with iTRAQ labeling, to achieve improved results has been elucidated, thus allowing for better screening and identification of potential biomarkers.

miRNA-mRNA Profiling Reveals Prognostic Impact of SMC1A Expression in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) with NPM1 mutation is a disease driving genetic alteration with good prognosis. Although it has been suggested that NPM1 mutation induces chemosensitivity in leukemic cells, the underlying cause for the better survival of NPM1 mutated patients is still not clear. Mutant NPM1 AML has a unique microRNA and their target gene (mRNA) signature compared to wild-type NPM1. Dynamic regulation of miRNA-mRNA has been reported to influence the prognostic outcome. In the present study, in silico expression data of miRNA and mRNA in AML patients was retrieved from genome data commons, and differentially expressed miRNA and mRNA among NPM1 mutated (n = 21) and NPM1 wild-type (n = 162) cases were identified to establish a dynamic association at the molecular level. In vitro experiments using high-throughput RNA sequencing were performed on human AML cells carrying NPM1 mutated and wild-type allele. The comparison of in vitro transcriptomics data with in silico miRNA-mRNA expression network data revealed downregulation of SMC1A. On establishing miRNA-mRNA interactive pairs, it has been observed that hsa-mir-215-5p (logFC: 0.957; p = 0.0189) is involved in the downregulation of SMC1A (logFC: -0.481; p = 0.0464) in NPM1 mutated AML. We demonstrated that transient expression of NPM1 mutation upregulates miR-215-5p, which results in downregulation of SMC1A. We have also shown using a rescue experiment that neutralizing miR-215-5p reverses the effect of NPM1 mutation on SMC1A. Using the leukemic blasts from AML patients, we observed higher expression of miR-215-5p and lower expression of SMC1A in NPM1 mutated patients compared to wild-type cases. The overall survival of AML patients was significantly inferior in SMC1A high expressers compared to low expressers (20.3% vs. 58.5%, p = 0.018). The data suggest that dynamic miR-215-SMC1A regulation is potentially modulated by NPM1 mutation, which might serve as an explanation for the better outcome in NPM1 mutated AML.

Exome sequencing study of Russian breast cancer patients suggests a predisposing role for USP39.

PURPOSE: Germline variants in known breast cancer (BC) predisposing genes explain less than half of hereditary BC cases. This study aimed to identify missing genetic determinants of BC. METHODS: Whole exome sequencing (WES) of lymphocyte DNA was performed for 49 Russian patients with clinical signs of genetic BC predisposition, who lacked Slavic founder mutations in BRCA1, BRCA2, CHEK2, and NBS1 genes. RESULTS: Bioinformatic analysis of WES data was allowed to compile a list of 229 candidate mutations. 79 of these mutations were subjected to a three-stage case-control analysis. The initial two stages, which involved up to 797 high-risk BC patients, 1504 consecutive BC cases, and 1081 healthy women, indicated a potentially BC-predisposing role for 6 candidates, i.e., USP39 c.*208G > C, PZP p.Arg680Ter, LEPREL1 p.Pro636Ser, SLIT3 p.Arg154Cys, CREB3 p.Lys157Glu, and ING1 p.Pro319Leu. USP39 c.*208G > C was strongly associated with triple-negative breast tumors (p = 0.0001). In the third replication stage, we genotyped the truncating variant of PZP (rs145240281) and the potential splice variant of USP39 (rs112653307) in three independent cohorts of Russian, Byelorussian, and German ancestry, comprising a total of 3216 cases and 2525 controls. The data obtained for USP39 rs112653307 supported the association identified in the initial stages (the combined OR 1.72, p = 0.035). CONCLUSIONS: This study suggests the role of a rare splicing variant in BC susceptibility. USP39 encodes an ubiquitin-specific peptidase that regulates cancer-relevant tumor suppressors including CHEK2. Further epidemiological and functional studies involving these gene variants are warranted.

Biophysical evaluation to categorize pathogenicity of cancer-predisposing mutations identified in the BARD1 BRCT domain.

The BRCT domain of BARD1 (BARD1 BRCT) is involved in many cellular processes such as DNA damage repair (DDR) and cell-cycle checkpoint regulation. BARD1 BRCT performs tumor suppressor function by recruiting BRCA1 at DNA damage site via interactions with other DNA damage repair (DDR) proteins. Considering the importance of the BRCT domain in genomic integrity, we decided to evaluate reported mutations of BARD1 BRCT Cys645Arg, Val695Leu, and Ser761Asn for their pathogenicity. To explore the effect of the mutation on the structure and function, BARD1 BRCT wild-type proteins and the mutant proteins were studied using different biochemical, biophysical and in silico techniques. Comparative fluorescence, circular dichroism (CD) spectroscopy and limited proteolysis studies demonstrate the well-folded structural conformation of wild-type and mutant proteins. However, thermal and chemical denaturation studies revealed similarity in the folding pattern of BARD1 BRCT wild-type and Cys645Arg mutant proteins, whereas there was a significant loss in the thermodynamic stability of Val695Leu and Ser761Asn mutants. Molecular dynamics (MD) simulation studies on wild-type and mutant protein structures indicate the loss in structural integrity of mutants compared with the wild-type protein.