Journal: Communications Biology

Article Title: Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells

doi: 10.1038/s42003-020-0851-2

Figure Lengend Snippet: a Domain organization of human SSSCA1, aligned with a disorder probability plot and localization of the three domains. Numbers denote amino acid positions. Zn, N-terminal zinc-binding domain (residues 20–86). Pro, proline-rich region (residues 86–148). The C-terminal helical domain can be subdivided into two predicted α-helices H1 (150–167) and H2 (173–195). b Schematic domain organization of representative SSSCA1 orthologs. Zn, N-terminal zinc-binding domain. Pro, proline-rich region. H, C-terminal helical domain. Percentage of positive identities to human SSSCA1 is indicated. c Phylogenetic tree of SSSCA1. Orthologs’ sequences retrieved from Ensembl, Genbank, or UniProt were aligned and a phylogenetic tree was constructed using the neighbor-joining tree method. Kingdoms, major phyla, and taxonomical groups are indicated. For clarity, vertebrate and invertebrate species are shown in red and blue nuances, respectively.

Article Snippet: Nonlinear regression with FP values was performed in GraphPad Prism 7.00 (GraphPad Software, La Jolla, California, USA) using a one-site total binding model. All conjugated peptides were purchased from GenScript: Axin1 positive control (EDAPRPPVPGEEG), Axin1 G>R negative control (EDAPRPPVPREEG), SSSCA1-H1 (DVMACTQTALLQKLTWASAELGSS), and SSSCA1-H2 (TSLETSIQLCGLIRACAEALRSLQQL).

Techniques: Binding Assay, Construct

Journal: Communications Biology

Article Title: Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells

doi: 10.1038/s42003-020-0851-2

Figure Lengend Snippet: a Flow chart of the tandem-affinity purification coupled to mass spectrometry (TAP–MS) technique. b Venn diagram representing the significantly enriched proteins when purifying His-GFP-SSSCA1 in three different cancer cell lines. c Identification of five potential targets of SSSCA1 in the three cancer cell lines. d Confirmation of the identification of TNKS1 as SSSCA1’s binding partner by immunoblotting. e Co-localization of His-GFP-SSSCA1 and endogenous TNKS1 during mitosis in HeLa cells. Two representative examples are shown. Arrows indicate co-localization. AF AlexaFluor. Scale bar: 10 µm.

Article Snippet: Nonlinear regression with FP values was performed in GraphPad Prism 7.00 (GraphPad Software, La Jolla, California, USA) using a one-site total binding model. All conjugated peptides were purchased from GenScript: Axin1 positive control (EDAPRPPVPGEEG), Axin1 G>R negative control (EDAPRPPVPREEG), SSSCA1-H1 (DVMACTQTALLQKLTWASAELGSS), and SSSCA1-H2 (TSLETSIQLCGLIRACAEALRSLQQL).

Techniques: Affinity Purification, Mass Spectrometry, Binding Assay, Western Blot

Journal: Communications Biology

Article Title: Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells

doi: 10.1038/s42003-020-0851-2

Figure Lengend Snippet: a Schematic representation of His-GFP-tagged SSSCA1 constructs. FL full length. b Different constructs of human SSSCA1 were expressed in HeLa cells. After TAP, the binding of each construct to endogenous TNKS1 was assessed by western blotting. Green stars indicate the constructs of interest. β-actin was chosen for input control because of the very low and barely detectable endogenous expression of TNKS1. c Schematic representation of His-GFP-tagged TNKS1 constructs. FL full length. d Different constructs of human TNKS1 were expressed in HeLa cells. After TAP, the binding of each construct to endogenous SSSCA1 was assessed by western blotting. Green stars indicate the constructs of interest. e TNKS1 ARC1–3 was expressed and purified from E. coli . Schematic representation of the construct and Coomassie stained SDS-gel control after purification. f TNKS1 ARC1–3 binding to FAM-tagged peptides (positive control Axin1 in black, negative control Axin1 G>R in gray, SSSCA1-H1 in pink, and SSSCA1-H2 in purple) was assessed by fluorescence polarization. Affinities of each peptide for TNKS1 ARC1–3 are indicated. n = 4; error bars, SEM; K d error values, standard error of the fit; n.q. not quantifiable.

Article Snippet: Nonlinear regression with FP values was performed in GraphPad Prism 7.00 (GraphPad Software, La Jolla, California, USA) using a one-site total binding model. All conjugated peptides were purchased from GenScript: Axin1 positive control (EDAPRPPVPGEEG), Axin1 G>R negative control (EDAPRPPVPREEG), SSSCA1-H1 (DVMACTQTALLQKLTWASAELGSS), and SSSCA1-H2 (TSLETSIQLCGLIRACAEALRSLQQL).

Techniques: Construct, Binding Assay, Western Blot, Expressing, Purification, Staining, SDS-Gel, Positive Control, Negative Control, Fluorescence

Journal: Communications Biology

Article Title: Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells

doi: 10.1038/s42003-020-0851-2

Figure Lengend Snippet: a SEC profiles of the N-terminal domain of SSSCA1 (black) and a set of three standard proteins (gray). The purified SSSCA1 N-terminal Auto_anti-p27 domain was subjected to SDS–PAGE and stained with Coomassie blue. b Crystal structure of the Auto_anti-p27 domain of human SSSCA1 at 2.3 Å resolution representing a new class of Zn-binding ribbon domain. Zinc ions are represented as orange spheres. Coordinating cysteine residues of each protomer are represented as sticks (wheat color). c Highly conserved cysteine residues are indicated as wheat colored sticks on the magnification of the Zn-binding site (orange sphere). The anomalous difference density map (orange map) is contoured at 5 σ and cover the Zn position. Lysine residues involved with ubiquitination are shown in gray and the conserved tyrosine cluster in dark cyan. An electron density map (2Fo-Fc, purple map) contoured at 1 σ is covering the tyrosine positions. The closest structure homologs of the core antiparallel β-sheet of the zinc-binding domain are two WW(Y) domains with conserved Trp and Tyr residues (sticks) highlighted: ( d ) The E3-ubiquitin-protein ligase SMURF1 (PDB ID 2LAZ) in yellow, and ( e ) the neuronal protein FE65 (PDB ID 2IDH) in magenta. f The C4 zinc-binding loops of the related Zn-binding ribbon domain 1 (ZNRD1) (gray) only have the position of the cysteines in common.

Article Snippet: Nonlinear regression with FP values was performed in GraphPad Prism 7.00 (GraphPad Software, La Jolla, California, USA) using a one-site total binding model. All conjugated peptides were purchased from GenScript: Axin1 positive control (EDAPRPPVPGEEG), Axin1 G>R negative control (EDAPRPPVPREEG), SSSCA1-H1 (DVMACTQTALLQKLTWASAELGSS), and SSSCA1-H2 (TSLETSIQLCGLIRACAEALRSLQQL).

Techniques: Purification, SDS Page, Staining, Binding Assay

Journal: Communications Biology

Article Title: Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells

doi: 10.1038/s42003-020-0851-2

Figure Lengend Snippet: a Alignment of human ( Homo sapiens ), mouse ( Mus musculus ), zebrafish ( Danio rerio ), frog ( Xenopus laevis ), round worm ( Caenorhabditis elegans ) and fission yeast ( Sch. pombe ) SSSCA1 C-termini. Leucine and isoleucine residues are highlighted in green. WebLogo of SSSCA1 orthologs C-termini (human residues 156-198). b NetNES prediction plot of SSSCA1 orthologs. The gray bar indicates the residues of the alignment in a . c Confocal imaging of endogenous SSSCA1 and different GFP-tagged constructs of human SSSCA1 transiently expressed in HeLa cells. Scale bar: 10 µm.

Article Snippet: Nonlinear regression with FP values was performed in GraphPad Prism 7.00 (GraphPad Software, La Jolla, California, USA) using a one-site total binding model. All conjugated peptides were purchased from GenScript: Axin1 positive control (EDAPRPPVPGEEG), Axin1 G>R negative control (EDAPRPPVPREEG), SSSCA1-H1 (DVMACTQTALLQKLTWASAELGSS), and SSSCA1-H2 (TSLETSIQLCGLIRACAEALRSLQQL).

Techniques: Imaging, Construct

Journal: Communications Biology

Article Title: Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells

doi: 10.1038/s42003-020-0851-2

Figure Lengend Snippet: a Schematic representation of His-GFP-tagged SSSCA1 constructs. FL full length. b Different constructs of human SSSCA1 were expressed in HeLa cells. After TAP, the binding of each construct to endogenous TNKS1 was assessed by western blotting. Green stars indicate the constructs of interest. β-actin was chosen for input control because of the very low and barely detectable endogenous expression of TNKS1. c Schematic representation of His-GFP-tagged TNKS1 constructs. FL full length. d Different constructs of human TNKS1 were expressed in HeLa cells. After TAP, the binding of each construct to endogenous SSSCA1 was assessed by western blotting. Green stars indicate the constructs of interest. e TNKS1 ARC1–3 was expressed and purified from E. coli . Schematic representation of the construct and Coomassie stained SDS-gel control after purification. f TNKS1 ARC1–3 binding to FAM-tagged peptides (positive control Axin1 in black, negative control Axin1 G>R in gray, SSSCA1-H1 in pink, and SSSCA1-H2 in purple) was assessed by fluorescence polarization. Affinities of each peptide for TNKS1 ARC1–3 are indicated. n = 4; error bars, SEM; K d error values, standard error of the fit; n.q. not quantifiable.

Article Snippet: Nonlinear regression with FP values was performed in GraphPad Prism 7.00 (GraphPad Software, La Jolla, California, USA) using a one-site total binding model. All conjugated peptides were purchased from GenScript: Axin1 positive control (EDAPRPPVPGEEG), Axin1 G>R negative control (EDAPRPPVPREEG), SSSCA1-H1 (DVMACTQTALLQKLTWASAELGSS), and SSSCA1-H2 (TSLETSIQLCGLIRACAEALRSLQQL).

Techniques: Construct, Binding Assay, Western Blot, Control, Expressing, Purification, Staining, SDS-Gel, Positive Control, Negative Control, Fluorescence