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. 2018 Jul 11;14(7):e8071.
doi: 10.15252/msb.20178071.

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells

Philipp Trepte  1 , Sabrina Kruse  1 , Simona Kostova  1 , Sheila Hoffmann  2 , Alexander Buntru  1 , Anne Tempelmeier  1 , Christopher Secker  1   3 , Lisa Diez  1 , Aline Schulz  1 , Konrad Klockmeier  1 , Martina Zenkner  1 , Sabrina Golusik  1 , Kirstin Rau  1 , Sigrid Schnoegl  1 , Craig C Garner  2 , Erich E Wanker  4
Affiliations

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells

Philipp Trepte et al. Mol Syst Biol. .

Abstract

Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC). The double-readout procedure detects interactions with higher sensitivity than traditional single-readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease-causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult-onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease-causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease-associated mutations impair protein activity in biological systems.

Keywords: CSPα; LuTHy; missense mutations; protein–protein interactions; quantitative.

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Figures

Figure 1
Figure 1. The LuTHy procedure

  1. Schematic representation of the workflow of the LuTHy method. Expression vectors encoding NL and PA‐mCit‐tagged hybrid proteins are cloned and co‐transfected into mammalian cells. Binary interactions are detected with a double readout; first, in vivo with in‐cell bioluminescence resonance energy transfer (BRET) and second, ex vivo with a luminescence‐based co‐precipitation (LuC).

  2. Schematic representation of control proteins and fusion proteins of interest for investigating the interaction between PA‐mCit‐BAD and NL‐BCL2L1 in proof‐of‐principle LuTHy experiments.

  3. Calculated BRET ratios for the indicated protein pairs. The positive control protein PA‐mCit‐NL and the interacting fusion proteins NL‐BCL2L1 and PA‐mCit‐BAD show high BRET ratios.

  4. Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA‐NL and PA‐mCit‐NL and the interacting proteins NL‐BCL2L1 and PA‐mCit‐BAD show high LuC ratios.

Data information: Data are representative of more than three independent experiments. All values are mean ± s.d. Significance was calculated by one‐way ANOVA followed by Dunnett's multiple comparisons post hoc test; ***P < 0.001.
Figure 2
Figure 2. Evaluation of assay performance with positive and negative binary reference sets
  1. A, B

    Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cBRET (A) and cLuC (B) ratios from two independent experiments (Exp 1 and Exp 2), and the calculated two‐tailed Pearson correlation coefficient is indicated; ***P < 0.001.

  2. C

    Receiver operating characteristic (ROC) analysis of cBRET (AUC = 0.80 ± 0.04) and cLuC (AUC = 0.72 ± 0.04) data determines the thresholds to define true positive binary interactions.

  3. D

    Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS.

Figure 3
Figure 3. Systematic analysis of interacting proteins with known binding affinities

  1. Selection of PPIs for the affinity‐based interaction reference set (AIRS) from the PDBbind (Wang et al, 2004) and the Protein–Protein‐Interaction Affinity Database 2.0 (Kastritis et al, 2011).

  2. Selected protein pairs cover a broad range of published binding affinities; PPIs with low (> 10−6 M), medium (> 10−8 M), and high binding affinities (≤ 10−8 M) were sub‐grouped in the AIRS.

  3. Recovery rates of PPIs from affinity‐based sub‐groups with BRET and LuC assays. Significance was calculated by two‐sided Fisher's exact test; *P < 0.05.

  4. Success of PPI detection from AIRS sub‐groups considering single and double LuTHy readouts.

  5. Scatter plot depicting the relationships between published binding affinities (KD) and in‐cell BRET50 measurements for 25 interactions in AIRS; BRET50 values are the mean from two independent experiments. Significance was calculated by two‐tailed Spearman correlation; ***P < 0.001.

Figure 4
Figure 4. Effects of missense mutations and small molecules on PPI detection with LuTHy

  1. Live‐cell bioluminescence imaging (BLI) of HEK293 cells co‐producing the indicated fusion proteins. mCitrine was excited at 500 nm, and emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band‐pass (BP) filters in a dual‐view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm.

  2. Quantification of the rapamycin‐induced interaction between NL‐FKBP12 and PA‐mCit‐FRB. Plasmids encoding the fusion proteins were co‐transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h, BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with nonlinear curve fitting. Data are representative of at least two independent experiments. All values are mean ± s.d.

  3. Effects of Nutlin‐3 on the interaction between NL‐MDM2 and PA‐mCit‐p53. Same procedure as in (B) except for 6 h treatment with Nutlin‐3. Data are representative of at least two independent experiments. All values are mean ± s.d.

  4. Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co‐producing NL‐HSF1 and PA‐mCit‐HSF1 for 24 h were transferred to 384‐well plates containing ganetespib or geldanamycin to reach a final concentration of 1 μM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± s.e.m. Significance was calculated by two‐way ANOVA followed by Dunnett's multiple comparison's post hoc test. *P < 0.05; **P < 0.005; ***P < 0.001.

  5. Schematic depiction of a heat shock experiment. HEK293 cells co‐producing NL‐HSF1/PA‐mCit‐HSF1 for 48 h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24 h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180, and 360 min, and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat‐treated. All values are mean ± s.e.m. from three independent experiments. Significance was calculated by one‐way ANOVA followed by Dunnett's multiple comparisons post hoc test; *P < 0.05.

Figure 5
Figure 5. Systematic investigation of the impact of fusion‐tag orientation
  1. A

    Domain overview of VCP and 10 UBX‐domain‐containing proteins. The UBX proteins bind with their C‐terminally located UBX‐domains to the N‐domain in VCP, with the exception of UBXD1 that binds to the C‐terminus of VCP via a PUB domain.

  2. B, C

    VCP and the UBX‐domain‐containing proteins were co‐expressed as N‐ and C‐terminal NL and PA‐mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥ 0.01 and ≥ 0.03, respectively, are colored in cyan.

  3. D

    The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.

Figure 6
Figure 6. Disease‐causing mutations influence the association of CSPα with partner proteins
  1. 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.

  2. 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.

  3. 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.

  4. 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 ± s.d. Significance was calculated by two‐way ANOVA followed by Dunnett's multiple comparisons post hoc test; **P < 0.005; ***P < 0.001.

  5. 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.

  6. 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.

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