Journal: PLoS ONE

Article Title: NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16

doi: 10.1371/journal.pone.0130416

Figure Lengend Snippet: A. Domain structure of NLRP7. Location of analyzed HYDM1-causing mutations L398R, R693P, R693W and non-synonymous variant (NSV) K511R are marked with orange arrowheads. Apart from a full-length construct and the four individual NLRP7 domains, five additional deletion constructs were generated, either lacking one or two domains at a time. B. Domain structure of the transcription factor ZBTB16 identified as interaction partner by the yeast two-hybrid screening. Apart from the full-length construct, containing an N-terminal BTB/POZ, a RD2 and nine zinc-fingers, five additional ZBTB16 deletion constructs were generated (ZBTB16_del1-5). ZBTB16_del4 (aa 268–673) corresponds to “prey#1” identified by the yeast two-hybrid screen.

Article Snippet: The full-length cDNA for human NLRP7, ZBTB16 (pCMV-SPORT6 IMAGE ID 4944546; Imagenes), KHDC3L (pCR4-TOPO CloneID 40146866; ThermoScientific), the NLRP7 individual domains PYD, NACHT, NAD, LRR and the NLRP7 deletion constructs ΔNAD/LRR, ΔLRR, ΔPYD/LRR, ΔPYD, ΔNACHT ( ) were cloned by the Gateway system (Invitrogen) into GAL4 yeast expression vectors pGBKT7/pGADT7 (Clontech) and by restriction-free cloning into Flag- or Myc-containing pcDNA3.1 vector (Invitrogen) as previously reported [ ].

Techniques: Variant Assay, Construct, Generated, Two Hybrid Screening, Zinc-Fingers

Journal: PLoS ONE

Article Title: NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16

doi: 10.1371/journal.pone.0130416

Figure Lengend Snippet: A. Yeast two-hybrid screen against the different NLRP7 constructs (fused to the GAL4 activation domain; pGADT7) using full-length ZBTB16 as bait (fused to the GAL4 binding domain; pGBKT7) performed with the Gal4-System (Clontech). ZBTB16 interacted with the highly reactive NAD domain and the LRR deleted constructs. An interaction of ZBTB16 with full-length NLRP7 failed. The self-interaction between full-length ZBTB16 served as positive control as it is already known that ZBTB16 forms a dimer by its N-terminal POZ/BTB domain . B. Yeast two-hybrid mutation screen using ZBTB16 as bait (pGBKT7) against full-length NLRP7 containing one of the three HYDM1-causing mutations L398R (NACHT), R693P (LRR), R693W (LRR) or the NSV K511R (all pGADT7). The NACHT domain-associated mutation L398R resulted in the same strong interaction between full-length NLRP7 and full-length ZBTB16 as seen between NAD:ZBTB16 and ΔLRR:ZBTB16.

Article Snippet: The full-length cDNA for human NLRP7, ZBTB16 (pCMV-SPORT6 IMAGE ID 4944546; Imagenes), KHDC3L (pCR4-TOPO CloneID 40146866; ThermoScientific), the NLRP7 individual domains PYD, NACHT, NAD, LRR and the NLRP7 deletion constructs ΔNAD/LRR, ΔLRR, ΔPYD/LRR, ΔPYD, ΔNACHT ( ) were cloned by the Gateway system (Invitrogen) into GAL4 yeast expression vectors pGBKT7/pGADT7 (Clontech) and by restriction-free cloning into Flag- or Myc-containing pcDNA3.1 vector (Invitrogen) as previously reported [ ].

Techniques: Two Hybrid Screening, Construct, Activation Assay, Binding Assay, Positive Control, Mutagenesis

Journal: PLoS ONE

Article Title: NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16

doi: 10.1371/journal.pone.0130416

Figure Lengend Snippet: A. Co-immunoprecipitation of transiently transfected ZBTB16 (Myc-tagged) and NLRP7 (Flag-tagged) in HEK293T cells. Immunoprecipitation was done using an anti-Flag specific antibody. Quantification of interaction between NLRP7 and ZBTB16 was determined relative to NLRP7 full-length. B. Co-immunoprecipitation of five ZBTB16 deletion constructs del1-5 (Myc-tagged) by immunoprecipitation of full-length NLRP7 (Lane 1) or one of four NLRP7 deletion constructs (lane 2–5; Flag-tagged) using an anti-Flag specific antibody. C. Blue Native gel electrophoresis of NLRP7 (Flag-tagged) and ZBTB16 (Myc-tagged) after transient transfection in HEK293T cells. Native protein extraction was performed using different concentrations of digitonin that is indicated above the picture. Upper panels: In the first dimension, the native Flag-NLRP7 appears as a smeary oligomer in a broad shift from 480–1000 kD (left side), while native ZBTB16-Myc was detected as a sharp band at 480 kD (right side). Lower panels: In the second dimension, the monomeric Flag-NLRP7 is visible as a thin line at 113kD, while ZBTB16-Myc occurs as individual spot at its expected size of 75 kD.

Article Snippet: The full-length cDNA for human NLRP7, ZBTB16 (pCMV-SPORT6 IMAGE ID 4944546; Imagenes), KHDC3L (pCR4-TOPO CloneID 40146866; ThermoScientific), the NLRP7 individual domains PYD, NACHT, NAD, LRR and the NLRP7 deletion constructs ΔNAD/LRR, ΔLRR, ΔPYD/LRR, ΔPYD, ΔNACHT ( ) were cloned by the Gateway system (Invitrogen) into GAL4 yeast expression vectors pGBKT7/pGADT7 (Clontech) and by restriction-free cloning into Flag- or Myc-containing pcDNA3.1 vector (Invitrogen) as previously reported [ ].

Techniques: Immunoprecipitation, Transfection, Construct, Nucleic Acid Electrophoresis, Protein Extraction

Journal: PLoS ONE

Article Title: NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16

doi: 10.1371/journal.pone.0130416

Figure Lengend Snippet: A. Cellular localization of transiently transfected EGFP- (N-terminal) and DsRed2- (C-terminal) tagged NLRP7 or ZBTB16 in HEK293T cells, respectively. Nucleus was counterstained with ToPro3. ZBTB16 localizes in the nuclear speckles, while NLRP7 is distributed in the cytoplasm with occasional accumulation near the nucleus. B. Upper row: Co-localization of transiently transfected N-terminal EGFP-tagged NLRP7 and C-terminal DsRed2-tagged ZBTB16 in HEK293T cells. Both proteins co-localize in the cytoplasm near the nucleus. ZBTB16 is re-localized from the nucleus (stained with ToPro3) to the cytoplasm. Lower row: Co-localization of transiently transfected C-terminal DsRed2-tagged NLRP7 and N-terminal EGFP-tagged ZBTB16 in HEK293T. Again, both proteins co-localize in the cytoplasm in a juxtanuclear aggregate while some amounts of EGFP-tagged ZBTB16 remain in the nucleus (stained with ToPro3). C. Single left panel: Staining of endogenous ZBTB16 in non-transfected HEK293T cells. The protein shows its typical nuclear localization in the nuclear speckles. Right row: Transient transfection of N-terminal EGFP-tagged NLRP7 in HEK293T cells results in nuclear export of endogenous ZBTB16 and shows their co-localization in a cytoplasmic juxtanuclear aggregate. D. Single left panel: Transient transfection of C-terminal DsRed2-tagged KHDC3L in HEK293Tcells. The protein shows a diffuse distribution in the cytoplasm with occasional accumulation near the nucleus. Right row: Transient transfection of N-terminal EGFP-tagged NLRP7 and C-terminal DsRed2-tagged KHDC3L in HEK293T cells. Both proteins co-localize diffusely in the cytoplasm and in juxtanuclear aggregates.

Article Snippet: The full-length cDNA for human NLRP7, ZBTB16 (pCMV-SPORT6 IMAGE ID 4944546; Imagenes), KHDC3L (pCR4-TOPO CloneID 40146866; ThermoScientific), the NLRP7 individual domains PYD, NACHT, NAD, LRR and the NLRP7 deletion constructs ΔNAD/LRR, ΔLRR, ΔPYD/LRR, ΔPYD, ΔNACHT ( ) were cloned by the Gateway system (Invitrogen) into GAL4 yeast expression vectors pGBKT7/pGADT7 (Clontech) and by restriction-free cloning into Flag- or Myc-containing pcDNA3.1 vector (Invitrogen) as previously reported [ ].

Techniques: Transfection, Staining

Journal: PLoS ONE

Article Title: NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16

doi: 10.1371/journal.pone.0130416

Figure Lengend Snippet: The figure is split into three panels, each representing the ZBTB16/NLRP7 dock in differently colored formats. The models are depicted in stick form. A. The first panel shows the proteins ZBTB16 and NLRP7 in yellow and green. The interface regions are marked and specified. The red residues on the interface regions belong to ZBTB16, while the blue ones belong to NLRP7. B. and C . Both panels show the same docks but with the individual domains of ZBTB16 and NLRP7, respectively, colored according to the code described below. The apposing protein in each panel is uniformly colored in grey.

Article Snippet: The full-length cDNA for human NLRP7, ZBTB16 (pCMV-SPORT6 IMAGE ID 4944546; Imagenes), KHDC3L (pCR4-TOPO CloneID 40146866; ThermoScientific), the NLRP7 individual domains PYD, NACHT, NAD, LRR and the NLRP7 deletion constructs ΔNAD/LRR, ΔLRR, ΔPYD/LRR, ΔPYD, ΔNACHT ( ) were cloned by the Gateway system (Invitrogen) into GAL4 yeast expression vectors pGBKT7/pGADT7 (Clontech) and by restriction-free cloning into Flag- or Myc-containing pcDNA3.1 vector (Invitrogen) as previously reported [ ].

Techniques: