A luminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions (2018)
Figure 6

Figure 6: Disease-causing mutations influence the association of CSPα with partner proteins.

A Overview of the targeted PPI screen. CSPα-mCit-PA was screened in two independent experiments against a focused library of 125 NL-tagged presynaptic proteins. Venn diagram depicting the 42 identified CSPα interaction partners. Domain structure of CSPα. The J-domain and the cysteine-string domain (CSD) are depicted. The ANCL-causing mutations ΔL116 and L115R are indicated.

B,C Tukey box plots of the mean cBRET (B) and cLuC ratios (C) obtained from two independent PPI experiments with wild-type CSPα-mCit-PA and its mutant variants. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test. *p<0.05.

D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal. Number of proteins identified with wild-type (wt) CSPα-mCit-PA or its mutant variants (ΔL116 and L115R) are indicated.

E Influence of mutations in CSPα (ΔL116, L115R) on the interactions with ZDHHC17 in primary hippocampal neurons. BRET and LuC ratios were systematically quantified for interactions with wild-type and mutant CSPα fusion proteins. Data are representative of three independent experiments. All values are mean ± sd. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; **p<0.005; ***p<0.001.

F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.

G Immunofluorescence image of SHEP cells co-transfected with NL-ZDHHC17 and CSPα-mCit-wt, -ΔL116 or -L115R constructs lacking the PA-tag. NL-ZDHHC17 was stained with an anti NanoLuc antibody. Scale bar: 20 µm.

Appendix Figure Content

Appendix Figure S1: Requirements for LuTHy assays.

Appendix Figure S2: LuTHy assay principle.

Appendix Figure S3: Expression values for the interaction PA-mCit-BAD and NL-BCL2L1.

Appendix Figure S4: Quantification of cBRET and cLuC ratios for binary interactions in hPRS.

Appendix Figure S5: Quantification of cBRET and cLuC ratios for binary interactions in hRRS.

Appendix Figure S6: Comparision of the double-readout LuTHy method with single-readout PPI detection methods.

Appendix Figure S7: Quantification of cBRET and cLuC ratios for binary intractions in AIRS.

Appendix Figure S8: Analysis of the interaction between RNASE1 and RNH1 with LuTHy, FRET and DULIP.

Appendix Figure S9: Investigating the binding strengths of interactions from the AIRS with donor saturation BRET assays.

Appendix Figure S10: Analyzing the effects of missense mutations on the interaction between Munc18 and Syntaxin-1 with BRET and BLI.

Appendix Figure S11: Detecting the interaction between NL-VCP and UBXD9-mCit with BRET at near endogenous levels.

Appendix Figure S12: Reproducibility of the targeted CSPα interaction screen.

Appendix Figure S13: Quantification of cBRET and cLuC ratios from the primary screen of CSPα against the synaptic library.

Appendix Figure S14: Investigation of CSPα and PICK1 interactions with LuTHy and proximity ligation assays.

Appendix Figure S15: Effects of disease-causing mutations on the detection of CSPα interactions with LuTHy.

Dataset Content

Dataset EV1: Used plasmids.

Dataset EV2: Detection of PPIs in hPRS by quantification of cBRET and cLuC ratios.

Dataset EV3: Detection of PPIs in hRRS by quantification of cBRET and cLuC ratios.

Dataset EV4: Detection of PPIs in the AIRS by quantification of cBRET and cLuC ratios.

Dataset EV5: Detection of PPIs between VCP and ten UBX-domain containing proteins by quantification of cBRET and cLuC ratios.

Dataset EV6: LuTHy results from an interaction screen of a synaptic library with the molecular chaperone CSPα. Primary hits were validated in a secondary screen by quantification of cBRET and cLuC ratios.

Dataset EV7: Published interaction partners of CSPα.

Dataset EV8: Functional enrichment analysis of identified CSPα interactions from the synaptic protein set.

Dataset EV9: LuTHy results from membrane associated interaction partners of CSPα, tested against PICK1.

Dataset EV10: LuTHy results from an interaction screen of a non-synaptic library with the molecular chaperone CSPα. Primary hits were validated in a secondary screen by quantification of cBRET and cLuC ratios.

Dataset EV11: Quantification of the effect of ANCL disease mutations on CSPα interactions by quantification of cBRET and cLuC ratios.

Dataset EV12: Used primers.