junjie chen  (Addgene inc)

Journal: Cell reports

Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers

doi: 10.1016/j.celrep.2024.113803

Figure Lengend Snippet: (A and B) BRCA1 mutations cataloged in ClinVar (accessed April 27, 2023). (A) Clinical significance of BRCA1 mutations grouped by mutation type. (B) Location of pathogenic missense mutations cataloged in ClinVar with at least a “two-gold-star” review status (n = 285). (C) C-terminal BRCA1 3×FLAG-tagged truncation construct used for LUMIER. The two BRCT subdomains are colored (orange and wheat). pSXXF denotes the BRCT binding phosphopeptide (BACH1). Protein Data Bank (PDB): 1T29. (D) LUMIER with BACON (bait control) approach to quantify chaperone interactions in HEK293T cells. Diagram was created with BioRender. (E) Chaperone binding to benign and pathogenic BRCA1-BRCT variants in ClinVar (including “likely” classifications). Z scores were calculated by averaging non-transfected wells to illustrate the significance of the data. Dashed line indicates the threshold for a statistically significant signal ( Z score > 2.5). Wild-type BRCA1-BRCT values are shown as green-filled triangles. Chaps, chaperones; Patho., pathogenic. (F) BRCA1-BRCT variant levels after pull-down detected by ELISA. (G) Variant ΔΔG values (kcal/mol) predicted by FoldX relative to the wild-type value (ΔΔG = 0). Dashed line indicates the threshold for structure disruption (ΔΔG > 2). (H) HSP70/HSP90 interaction preferences. Diagonal shows the identity line. The unknown group includes variants annotated as having uncertain significance, no significance provided, conflicting interpretations, or “one-gold-star” review status. FANCA variants and a frameshifted (fs) variant encoding an additional out-of-frame HSP70 site shown for comparison. Statistical significance was determined using two-tailed Mann-Whitney t test (E–G). ****p ≤ 0.0001. Data are presented as mean values from at least two independent experiments.

Article Snippet: Plasmids containing human BRCA1 cDNA were a gift from Junjie Chen (Addgene, Plasmid #99394).

Techniques: Mutagenesis, Construct, Binding Assay, Phospho-proteomics, Control, Transfection, Variant Assay, Enzyme-linked Immunosorbent Assay, Disruption, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers

doi: 10.1016/j.celrep.2024.113803

Figure Lengend Snippet: (A) Variants targeting secondary structure elements in the BRCA1-BRCT domain. (B) Y1845 variants that disrupt or support hydrophobic interactions. FoldX ΔΔG predictions for each variant are shown. (C) T1685 variants that disrupt or support side-chain hydrogen bonding. Two different T1685S codons were tested because these codons previously exhibited different functional effects. (D and E) Chaperone binding predictions to the BRCA1-BRCT variant library. The “disruptor” bin includes variants that introduce prolines in α-helices; truncations within the BRCT domain; buried variants that introduce a charge, decrease hydrophobicity, or create a steric clash; and charged variants that mutate residues in hydrophobic networks or disrupt side-chain hydrogen bonding. The “no effect” bin includes variants that fully delete the BRCT domain; variants outside the BRCT domain; buried variants that retain hydrophobicity; isosteric or quasi-isosteric variants; variants that maintain side-chain hydrogen bonds; and other surface variants not included prior. Dashed line indicates the cutoff for binding (HSP70 score > 0.5). (E) excluded variants previously characterized using protease sensitivity. Statistical significance was determined using a two-tailed Mann-Whitney t test. ****p ≤ 0.0001. Data are presented as mean ± standard deviation values from at least two independent experiments.

Article Snippet: Plasmids containing human BRCA1 cDNA were a gift from Junjie Chen (Addgene, Plasmid #99394).

Techniques: Variant Assay, Functional Assay, Binding Assay, Introduce, Two Tailed Test, MANN-WHITNEY, Standard Deviation

Journal: Cell reports

Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers

doi: 10.1016/j.celrep.2024.113803

Figure Lengend Snippet: (A and B) Correlation of HSP70 binding with BRCA1-BRCT variant stability. Horizontal dashed line is the average of four double variants that combine strongly chaperone-bound single variants. Vertical dashed line reflects the upper limit of stability measurements owing to variant insolubility. The correlations were fit to a linear regression within the linear regime (FoldX: < 10 kcal/mol, empirical: < 5 kcal/mol). G1788V was not fit because the stability measurement for this variant was previously reported as contradictory. (C and D) HSP70 binding binned according to the functional effect class measured using aggregation/degradation or transcriptional activation assays. (E) Linear correlation of HSP70 binding and variant levels (ELISA) at different concentrations of cell lysates pulled down. Dashed lines indicate the average LUMIER and ELISA signals observed under our standard assay conditions for variants that bound strongly to HSP70. (F and G) BRCA1 variant function in HAP1 (cell fitness) or HeLa (HDR) cells binned according to the degree of HSP70 binding. (H) Integrated functional data from neXtProt binned by the magnitude of HSP70 binding. Percentages calculated using the percentage of variants associated with each HSP70 binding magnitude and phenotype intensity. Func., functional. Statistical significance was determined using Kruskal-Wallis ANOVA test (C, D, F, and G) or chi-squared test (H). ****p ≤ 0.0001, **p ≤ 0.01, and *p ≤ 0.05. Data are presented as mean ± standard deviation values from at least two independent experiments.

Article Snippet: Plasmids containing human BRCA1 cDNA were a gift from Junjie Chen (Addgene, Plasmid #99394).

Techniques: Binding Assay, Variant Assay, Functional Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cell reports

Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers

doi: 10.1016/j.celrep.2024.113803

Figure Lengend Snippet: (A) HSP70 interaction scores for natural human variants observed in patients with cancer (ClinVar and cBioPortal/TCGA , ) or the general population (gno-mAD ). Unknown (unk.) includes variants annotated as having uncertain significance, no significance provided, conflicting interpretations, or one-gold-star review status. (B) The functional severity , of natural BRCA1-BRCT variants grouped by the degree of HSP70 binding. LoF, loss of function. (C) HSP70 binding to natural BRCA1-BRCT human variants binned by the predicted effect on domain structure. Variants were ranked using an informed hierarchical approach (see ). Moderate HSP70-bound variants are colored magenta and overlaid onto the BRCT crystal structure. An example long-range interaction that coordinates the two BRCT subdomains is indicated with an arrow. Struct., structure. (D and E) ROC curves using pathogenicity annotated in ClinVar (D) or mode phenotypic intensity annotated in neXtProt (E) as the target datasets. The neXtProt curves designate mild, moderate, and ambiguous (multimodal) variants as pathogenic. (F) The observed AUCs when mild, moderate, and ambiguous variants in the neXtProt target dataset were designated benign or pathogenic. Statistical significance was determined using Kruskal-Wallis ANOVA test (A and C) or chi-squared test (B). ****p ≤ 0.0001 and **p ≤ 0.01. ns, not significant. Data are presented as mean values from at least two independent experiments.

Article Snippet: Plasmids containing human BRCA1 cDNA were a gift from Junjie Chen (Addgene, Plasmid #99394).

Techniques: Functional Assay, Binding Assay

Journal: Cell reports

Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers

doi: 10.1016/j.celrep.2024.113803

Figure Lengend Snippet: (A) BRCA1 mutation penetrance likelihood binned by the magnitude of HSP70 binding. Data shown were obtained from the Leiden Open Variation Database (LOVD) (accessed April 24, 2023). (B–D) Age of first cancer diagnosis for patients carrying BRCA1 mutations binned by mutation type (B), missense mutations in domains (C), and the degree of HSP70 binding (D). Statistical significance was determined using two-tailed (A) or one-tailed (B–D) Mann-Whitney t test. ****p ≤ 0.0001, **p ≤ 0.01, and *p ≤ 0.05. The number of variants in each bin is shown in parentheses. Data are presented as mean values from at least two independent experiments.

Article Snippet: Plasmids containing human BRCA1 cDNA were a gift from Junjie Chen (Addgene, Plasmid #99394).

Techniques: Mutagenesis, Binding Assay, Biomarker Discovery, Two Tailed Test, One-tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers

doi: 10.1016/j.celrep.2024.113803

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmids containing human BRCA1 cDNA were a gift from Junjie Chen (Addgene, Plasmid #99394).

Techniques: Recombinant, Luciferase, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot, Mutagenesis, Variant Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Down‐regulation of BRCA1 in EBV‐associated NPCs. (A) The expression of BRCA1, ATM and PARP1 proteins in immortalized normal NP (NP69), four NPC cell lines and four NPC patient‐derived xenografts (PDXs) were analysed by immunoblotting. Actin was probed as the loading control. (B) The expression levels of BRCA1 mRNA in the cell lines were measured using RT‐qPCR. The relative BRCA1 mRNA expression was calculated using 2(∆∆−Ct) method, and the expression in NP69 was set as 1 for comparison. The data shown is the mean + SD. (C) The whiskers 10‐90 percentiles plot shows the relative BRCA1 mRNA expression in primary samples. The BRCA1 mRNA was significantly up‐regulated in NPCs (n = 55) when compared with the NPs (n = 22). (D) Immunohistochemistry staining of BRCA1 protein in primary samples (number of NPs = 30 and NPCs = 41). The representative images of negative and positive BRCA1 stain in NP and NPC specimens are shown (original magnification X400). (E) The dot plot shows the total expression levels of miR‐BART2‐3p, BART12, BART17‐5p and BART19‐3p in 20 NPC biopsies, in which the BRCA1 protein expression status was analysed in IHC. The expression of miR‐BARTs was normalized to EBNA1. The median values of each group are shown by the dash line and the Mann‐Whitney test was used for the statistical analysis

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Quantitative RT-PCR, Comparison, Immunohistochemistry, Staining, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The BRCA1 is the potential target of miR‐BARTs. (A) The relative luciferase activity of the reporter plasmids harbouring a full length of BRCA1‐3’UTR (sFL‐3’UTR) or a full length of BRCA1‐3’UTR in reversed orientation (asFL‐3’UTR) was co‐transfected together with the indicated miRNAs. The luciferase signal with the co‐transfection of negative miRNA mimic control (miR‐NEG) was set at 1 for comparison. (B) The direct interaction between the putative binding sites on BRCA1 and miR‐BARTs were demonstrated in the reporter assays. The firefly luciferase reporter activity was normalized to the Renilla luciferase control. The data shown is the mean + SD from three independent experiments. The result with the co‐transfection of miR‐NEG and pMIR‐CTL was set at 1. pMIR‐CTL = pMIR‐REPORTTM vectors containing unrelated sequences; pMIR‐B = pMIR‐REPORTTM vector harbouring the predicted miR‐BART binding site, pMIR‐CDS = predicted binding site on CDS (Table ). B2‐3p = BART2‐3p; B12 = BART12; B17‐5p = BART17‐5p; B19‐3p = BART19‐3p. * P < 0.05, ** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Control, Comparison, Binding Assay, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Regulation of BRCA1 expression by miR‐BARTs (A) Western blot of BRCA1 in NPC cell lines. Actin was probed as the protein‐loading control, and the expression level was compared with NP69 (set as 1). (B) The total expression of viral BART2‐3p, BART12, BART17‐5p and BART19‐3p (upper panel) and the expression of cellular miR‐146a (lower panel) in the cell lines were assayed by RT‐qPCR. The expression values of total miR‐BARTs and miR‐146a were calculated using the 2(‐∆Ct) and 2(∆∆‐Ct) methods, respectively. The analysis of each sample was performed in triplicate with mean + SD shown. (C) In the EBV‐negative epithelial cells, the BRCA1 level was suppressed by the transfection of the indicated miRNA mimics, BART2‐3p (B2‐3p), BART12 (BT12), BART17‐5p (BT17‐5p) and BART19‐3p (BT19‐3p). (D) The BRCA1 protein expression in C666‐1 cells was regained by suppressing the endogenous miR‐BARTs activities with specific miR‐BART inhibitors for 48 h. The negative control mimic/inhibitor (Inh‐Ctl) transfection was used for comparison. Either actin or vinculin was probed as the loading control

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Transfection, Negative Control, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The CDDP and DOX sensitivity in HK1 and NP69 cells. (A) Western blot of p53 and p21 in NPC cell lines were analysed. (B) Transfection of either BRCA1‐specific siRNA, BART17‐5p or BART19‐3p mimics increased CDDP‐ and DOX‐mediated S phase or G2/M phase cell‐cycle arrest in the HK1 and NP69 cells. The transfected cells were incubated with either the control buffer or the indicated chemotherapeutic agent for 24 h. Subsequently, the cells were fixed for DNA content analysis with BD FACSCalibur flow cytometry system. (C) Protein lysate from the treated cells were harvested for phosphor‐CHK1 (p‐CHK1) expression analysis. (D) The suppression of BRCA1 sensitized HK1 cells to CDDP and DOX treatment. HK1 cells were transfected with BRCA1‐specific siRNAs (si‐BRCA1) or siRNA control (si‐NEG) and the protein lysates were collected for BRCA1 expression analysis 24 h after transfection (left panel). The transfected HK1 cells were incubated with different concentrations of CDDP or DOX for 48 h before CCK‐8 analysis. The IC50 value was determined by fitting a sigmoidal dose‐response curve to the data using GraphPad Prism 5 program. Sum‐of‐squares F‐test was used as the comparison method (right panel). (E) Clonogenic survival assays. Approximately 500 or 1000 transfected cells were seeded into the 6‐well plate and treated with CDDP or DOX for 24 h. The cells were cultured for 14‐18 d in normal medium before staining, and colonies containing more than 30 cells were counted. The number of colonies generated from the mock treatment was compared (set as 100%). All the experiments were performed in triplicate and the Student's t ‐test was conducted, compared with the control transfected cells. * P < 0.05; ** P < 0.01

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Western Blot, Transfection, Incubation, Control, Flow Cytometry, Expressing, CCK-8 Assay, Comparison, Cell Culture, Staining, Generated

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The EBV‐miRNAs impair cisplatin‐ and doxorubicin‐induced DNA damage response in nasopharyngeal epithelial cells. The representative images of the RAD51 foci staining in HK1 cells (upper left panel) and NP69 cells (upper right panel) are shown. The cells transfected with either siRNA control (si‐NEG), BRCA1‐specific siRNA (si‐BRCA1) or miR‐BARTs mimics were treated with cisplatin (CDDP) and doxorubicin (DOX), followed by immunostaining with the RAD51 antibody. At least 100 nuclei were randomly selected for counting, and the cells containing more than five apparent RAD51 foci in the nucleus were considered positive. The percentage of the RAD51‐positive cells with mean + SD from three independent experiments are shown in the lower panel. Student's t ‐test was used to compare them with the control transfected cells (miR‐NEG) in each set of experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Staining, Transfection, Control, Immunostaining