Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers
- University of Texas Southwestern Medical Center at Dallas, United States
- University of Texas Health Science Center, United States
- University of Texas Health Cancer Center, United States
- University of Texas at Dallas, United States
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 3 supplements
Derivation and characterization of ERX-11.
Structure of ERX-11, as a derivative of the D2 peptidomimetic with a hydroxyethyl moiety in the flanking position to mimic a Serine (A, left panel). Effect of 500 nM of each peptidomimetic on the …
Figure 1—figure supplement 1
Derivation of the α-helix mimetic ERX-11 and structural design, synthesis and activity of the α-helix mimetic.
(A) String analyses depicting the interactome of PELP1, especially with ER and AR using string-db.org software v 10.4. (B) Effect of increasing concentrations of D1 and D2 peptidomimetic on the cell …
Figure 1—figure supplement 2
Characterization of ERX-11 activity.
(A) The lowest energy conformation of ERX-11 (left), Superimposition of the energy-minimized ERX-11 (green) on an α-helix (orange) (middle) and an α-helical LXXLL motif containing a Ser at its …
Figure 1—figure supplement 3
Derivation of the α-helix mimetic ERX-11 and synthesis of tris-benzamide peptidomimetics.
(A) Synthesis of 3-alkoxy-4-nitrobenzoic acids. (B) Solid-phase synthesis of tris-benzamdies (Reagents and conditions: (a) 3d, HATU, DIEA, DMF, rt, 24 hr; (b) SnCl2⋅2H2O, AcOH/HCl/THF, rt, 24 hr; (c)…
Figure 2 with 7 supplements
ERX-11 interacts with ER and blocks its interactome.
Interaction with endogenous ER was evaluated in nuclear lysates prepared from ZR-75 cells stimulated with E2, incubated with biotin-control or biotin-ERX-11 and analyzed by avidin pull-down assay (A)…
Figure 2—figure supplement 1
Pathways analysis in terms of either biological processes or molecular functions revealed that ERX-11-binding proteins were involved in the activation of multiple pathways leading to transcriptional regulation.
https://doi.org/10.7554/eLife.26857.008
Figure 2—figure supplement 2
Characterization of ERX-11 interactions with ER.
(A) Nuclear lysates from E2-stimulated MCF-7 cells treated with vehicle or ERX-11 were subjected to immunoprecipitation with ER antibody. The immunoprecipates were analyzed by mass spectroscopy. The …
Figure 2—figure supplement 3
Analyses of the ER-binding proteins blocked by ERX-11 or Tamoxifen.
https://doi.org/10.7554/eLife.26857.010
Figure 2—figure supplement 4
Effect of ERX-11 on inhibition of ER -coregulators interactions.
(A) Quantification of the effect of ERX-11 on the proximity ligation between ER and PELP1 or SRC1 or SRC3 in MCF-7 and ZR-75 cells. (B) Quantification of the effect of ERX-11 on the proximity …
Figure 2—figure supplement 5
Effect of SERDs or tamoxifen on ERX-11 interactions with ER.
(A) Effect of 1 μM of a SERD, GDC-0810, on the ability of ERX-11 to pulldown ER in MDA-MB-231 cells stably transfected with WT-ER, L540Q ER or ▲12 ER. Input is shown on the right. (B) Effect of 1 μM …
Figure 2—figure supplement 6
Models showing the putative interactions of ERX-11 with residues in the ER protein.
(A) Docking studies of ERX-11 on different ER crystal structures shows that ERX-11 (green) can interact with ER, either in the presence of agonist diethylstilbesterol (red) (A: 3ERD.pdb), an …
Figure 2—figure supplement 7
Model describing interaction between ER (purple) and ERX-11 (green) in presence of agonist (yellow) (A), SERD (orange) (B) or tamoxifen (red)(C).
Note that in the presence of tamoxifen, ERX-11 binds to a secondary weaker affinity site on ER. Similarly, the interaction between ER▲12 (blue) and ERX-11 is modeled in the absence of agonist (D), …
Figure 3 with 1 supplement
ERX-11 globally disrupts ER-mediated transcriptome.
Total RNA was isolated from the ZR-75 cells that were treated with either vehicle or ERX-11 for 48 hr and subjected to RNA sequencing. The heat map of differentially expressed genes between vehicle …
Figure 3—figure supplement 1
ERX-11 treatment has potential to promote apoptosis.
(A) Volcano plot of statistical significance (adjusted p value) against RPKM fold-change (FC) between vehicle and ERX-11 treated ZR-75 cells. Only significantly changed genes (p<0.01 and FC > 1.5) …
Figure 4 with 1 supplement
ERX-11 affects ER ligand-dependent and independent transcriptional activity.
ZR-75 cells stably transfected with ER and ERE-Luc vectors were treated with E2 (10−8M) in the presence of indicated concentrations of ERX-11 or tamoxifen. After 24 hr, the reporter gene activity …
Figure 4—figure supplement 1
Effect of ERX-11 on AR functions, E2 mediated non-genomic actions and ER stability.
(A) Evaluation of the effect of ERX-11 on AR DNA binding to its target sequences. (B) The effect of ERX-11 on AR dimerization as evaluated by the NanoBiT luciferase assay. (C) Evaluation of the …
Figure 5 with 2 supplements
ERX-11 inhibits the growth of ER-positive, syngeneic and coregulator-driven breast tumors in vivo.
ER-positive ZR-75 cells were injected into the mammary fat pads of nude mice implanted subcutaneously with E2 pellet. After 2 weeks, mice with xenografts were treated with vehicle or 10 mg/kg/day of …
Figure 5—figure supplement 1
Characterization of ERX-11 treated tumors.
(A) Body weights of mice implanted with ZR-75 xenograft tumors treated with vehicle or ERX-11. (B) Characterization of D2A1 model cells: (A) Western blot analysis of D2A1 cell lysates. Murine …
Figure 5—figure supplement 2
Normal tissues collected from mice that were treated with vehicle or ERX-11 were examined for toxicity.
(A) Effect of ERX-11 on various tissues as seen by H and E. (B) Effect of ERX-11 on Ki67 staining. (C) Effect of ERX-11 on Ki67 with quantitation. (D) Effect of ERX-11 on ER staining in the ovary.
Figure 6 with 1 supplement
ERX-11 reduces the growth of ER positive and ER-MT endocrine-therapy-resistant tumors.
Cell viability assays evaluated the effect of ERX-11 on Tamoxifen-resistant MCF-7-TamR cells (A), tamoxifen-resistant MCF-7/HER2 cells (B) and letrozole-resistant MCF-7-LTLT cells (C). ICI was used …
Figure 6—figure supplement 1
ERX-11 reduces the growth of ER-positive and ER-MT endocrine-therapy-resistant tumors.
(A) MCF-7-LTLT xenografts were treated with vehicle or ERX-11 or Fulvestrant. Tumor volume, tumor weights and body weights are shown****p<0.0001. (B) MCF-7-LTLT tumors treated with or without ERX-11 …
Figure 7 with 1 supplement
ERX-11 decreases the growth of patient-derived explants (PDEx): Schematic representation of ex vivo culture model is shown.
(A) The explants were treated with ERX-11 for 48 hr. Effect of ERX-11 on Ki67 expression in ER-positive tumors with representative sections from three individual tumors and overall trend are shown (B…
Figure 7—figure supplement 1
Effect of ERX-11 treatment on the status of ER.
Effect of ERX-11 treatment on ER + PR + patient-derived explants from three individual patients, as assessed by Ki67, ER and PELP1 immunohistochemistry.
Appendix 1—chemical structure 1
Compound 3a.
https://doi.org/10.7554/eLife.26857.032
Appendix 1—chemical structure 2
Compound 3b.
https://doi.org/10.7554/eLife.26857.033
Appendix 1—chemical structure 3
Compound 3c.
https://doi.org/10.7554/eLife.26857.034
Appendix 1—chemical structure 4
Compound 3d.
https://doi.org/10.7554/eLife.26857.035
Appendix 1—chemical structure 5
Compound 3e.
https://doi.org/10.7554/eLife.26857.036
Appendix 1—chemical structure 6
Compound 3f.
https://doi.org/10.7554/eLife.26857.037
Appendix 1—chemical structure 7
Compound 3g.
https://doi.org/10.7554/eLife.26857.038
Appendix 1—chemical structure 8
Compound 3i.
https://doi.org/10.7554/eLife.26857.039
Appendix 1—chemical structure 9
ERX-7.
https://doi.org/10.7554/eLife.26857.040
Appendix 1—chemical structure 10
ERX-8.
https://doi.org/10.7554/eLife.26857.041
Appendix 1—chemical structure 11
ERX-9.
https://doi.org/10.7554/eLife.26857.042
Appendix 1—chemical structure 12
ERX-5.
https://doi.org/10.7554/eLife.26857.043
Appendix 1—chemical structure 13
ERX-13.
https://doi.org/10.7554/eLife.26857.044
Appendix 1—chemical structure 14
ERX-10.
https://doi.org/10.7554/eLife.26857.045
Appendix 1—chemical structure 15
ERX-12.
https://doi.org/10.7554/eLife.26857.046
Appendix 1—chemical structure 16
Compound 8.
https://doi.org/10.7554/eLife.26857.047
Appendix 1—chemical structure 17
Compound 9.
https://doi.org/10.7554/eLife.26857.048
Appendix 1—chemical structure 18
ERX-11.
https://doi.org/10.7554/eLife.26857.049
Appendix 1—chemical structure 19
Compound 10.
https://doi.org/10.7554/eLife.26857.050
Appendix 1—chemical structure 20
Compound 11.
https://doi.org/10.7554/eLife.26857.051
Appendix 1—chemical structure 21
Compound 12.
https://doi.org/10.7554/eLife.26857.052
Appendix 1—chemical structure 22
ERX-11-biotin.
https://doi.org/10.7554/eLife.26857.053
Appendix 1—figure 1
1H and 13C NMR of compound 3a.
https://doi.org/10.7554/eLife.26857.055
Appendix 1—figure 2
1H and 13C NMR of compound 3b.
https://doi.org/10.7554/eLife.26857.056
Appendix 1—figure 3
1H and 13C NMR of compound 3c.
https://doi.org/10.7554/eLife.26857.057
Appendix 1—figure 4
1H and 13C NMR of compound 3e.
https://doi.org/10.7554/eLife.26857.058
Appendix 1—figure 5
1H and 13C NMR of compound 3 f.
https://doi.org/10.7554/eLife.26857.059
Appendix 1—figure 6
1H and 13C NMR of compound 3 g.
https://doi.org/10.7554/eLife.26857.060
Appendix 1—figure 7
1H and 13C NMR of compound 3i.
https://doi.org/10.7554/eLife.26857.061
Appendix 1—figure 8
1H and 13C NMR of ERX-5.
https://doi.org/10.7554/eLife.26857.062
Appendix 1—figure 9
1H and 13C NMR of ERX-7.
https://doi.org/10.7554/eLife.26857.063
Appendix 1—figure 10
1H and 13C NMR of ERX-8.
https://doi.org/10.7554/eLife.26857.064
Appendix 1—figure 11
1H and 13C NMR of ERX-9.
https://doi.org/10.7554/eLife.26857.065
Appendix 1—figure 12
1H and 13C NMR of ERX-10.
https://doi.org/10.7554/eLife.26857.066
Appendix 1—figure 13
1H and 13C NMR of ERX-12.
https://doi.org/10.7554/eLife.26857.067
Appendix 1—figure 14
1H and 13C NMR of ERX-13.
https://doi.org/10.7554/eLife.26857.068
Appendix 1—figure 15
1H and 13C NMR of compound 9.
https://doi.org/10.7554/eLife.26857.069
Appendix 1—figure 16
1H and 13C NMR of ERX-11.
https://doi.org/10.7554/eLife.26857.070
Appendix 1—figure 17
1H and 13C NMR of compound 11.
https://doi.org/10.7554/eLife.26857.071
Appendix 1—figure 18
1H NMR of compound 12.
https://doi.org/10.7554/eLife.26857.072
Appendix 1—figure 19
1H and 13C NMR of compound ERX-11-biotin.
https://doi.org/10.7554/eLife.26857.073
Appendix 1—figure 20
Characterization of SRC1-LXXLL peptide.
(a) HPLC chromatogram of SRC1-LXXLL peptide. (b) MALDI-TOF of SRC1-LXXLL peptide.
Appendix 1—figure 21
Characterization of SRC2-LXXLL peptide.
(a) HPLC chromatogram of SRC2-LXXLL peptide. (b) MALDI-TOF of SRC2-LXXLL peptide.
Appendix 1—figure 22
Characterization of AIB1-LXXLL peptide.
(a) HPLC chromatogram of AIB1-LXXLL peptide. (b) MALDI-TOF of AIB1-LXXLL peptide.
Appendix 1—figure 23
Characterization of PELP1-LXXLL peptide.
(a) HPLC chromatogram of PELP1-LXXLL peptide. (b) MALDI-TOF of PELP1-LXXLL peptide.
Tables
Table 1
Top proteins pulled down by biotinylated ERX-11 in MCF-7 cells, as identified by IP-MS. The column marked E2 represents spectral counts for the protein bound to biotinylated control eluted from …
| Protein | Description | Length (AA) | Mw (Da) | PSMs | Peptide seqs | % Coverage | E2 | E2 + ERX-11 | E2 + ERX-11/E2 |
|---|---|---|---|---|---|---|---|---|---|
| P38117 | ETFB_HUMAN Electron transfer flavoprotein subunit beta OS = Homo sapiens GN = ETFB PE = 1 SV = 3 | 255 | 37501.10 | 12 | 7 | 34.10 | 1 | 7.00 | 5.00 |
| Q96PZ0 | PUS7_HUMAN Pseudouridylate synthase seven homolog OS = Homo sapiens GN = PUS7 PE = 1 SV = 2 | 661 | 75186.30 | 20 | 14 | 26.30 | 1.00 | 7.98 | 10.99 |
| O95336 | 6 PGL_HUMAN 6-phosphogluconolactonase OS = Homo sapiens GN = PGLS PE = 1 SV = 2 | 258 | 27601.60 | 13 | 8 | 41.50 | 0.99 | 6.00 | 6.04 |
| Q8TD06 | AGR3_HUMAN Anterior gradient protein three homolog OS = Homo sapiens GN = AGR3 PE = 1 SV = 1 | 166 | 19194.90 | 14 | 9 | 47.00 | 0.99 | 5.96 | 7.03 |
| P18754 | RCC1_HUMAN Regulator of chromosome condensation OS = Homo sapiens GN = RCC1 PE = 1 SV = 1 | 421 | 48241.20 | 27 | 13 | 55.30 | 1.99 | 11.92 | 7.00 |
| O60506 | HNRPQ_HUMAN Heterogeneous nuclear ribonucleoprotein Q OS = Homo sapiens GN = SYNCRIP PE = 1 SV = 2 | 623 | 69739.70 | 20 | 20 | 48.50 | 1.97 | 10.82 | 3.50 |
| P03372 | ESR1_HUMAN Estrogen receptor OS = Homo sapiens GN = ESR1 PE = 1 SV = 2 | 595 | 66335.20 | 36 | 15 | 30.80 | 1.83 | 9.88 | 5.93 |
| E9PCR7 | E9PCR7_HUMAN 2-oxoglutarate dehydrogenase, mitochondrial OS = Homo sapiens GN = OGDH PE = 2 SV = 1 | 1038 | 115728.00 | 39 | 25 | 32.80 | 3.98 | 18.88 | 4.00 |
| O43488 | ARK72_HUMAN Aflatoxin B1 aldehyde reductase member 2 OS = Homo sapiens GN = AKR7A2 PE = 1 SV = 3 | 359 | 39653.80 | 22 | 11 | 39.80 | 1.99 | 8.96 | 5.51 |
| O95994 | AGR2_HUMAN Anterior gradient protein two homolog OS = Homo sapiens GN = AGR2 PE = 1 SV = 1 | 175 | 22277.70 | 29 | 12 | 65.70 | 2.97 | 11.92 | 4.68 |
| P19338 | NUCL_HUMAN Nucleolin OS = Homo sapiens GN = NCL PE = 1 SV = 3 | 710 | 76766.50 | 99 | 35 | 48.00 | 14.00 | 50.98 | 2.43 |
| O43148 | MCES_HUMAN mRNA cap guanine-N7 methyltransferase OS = Homo sapiens GN = RNMT PE = 1 SV = 1 | 476 | 57831.90 | 16 | 9 | 29.40 | 1.99 | 6.97 | 3.50 |
| Q562R1 | ACTBL_HUMAN Beta-actin-like protein 2 OS = Homo sapiens GN = ACTBL2 PE = 1 SV = 2 | 376 | 42084.00 | 14 | 14 | 39.10 | 2.00 | 7.00 | 3.00 |
| Q9Y5A9 | YTHD2_HUMAN YTH domain family protein 2 OS = Homo sapiens GN = YTHDF2 PE = 1 SV = 2 | 579 | 62457.80 | 15 | 12 | 23.10 | 2.00 | 6.99 | 3.00 |
| P16152 | CBR1_HUMAN Carbonyl reductase [NADPH] 1 OS = Homo sapiens GN = CBR1 PE = 1 SV = 3 | 277 | 30427.90 | 20 | 11 | 56.70 | 2.99 | 9.98 | 2.33 |
| Q9UBS4 | DJB11_HUMAN DnaJ homolog subfamily B member 11 OS = Homo sapiens GN = DNAJB11 PE = 1 SV = 1 | 358 | 40578.70 | 21 | 12 | 35.20 | 3.00 | 10.00 | 2.67 |
Table 2
Top biological processes of coregulators, whose interactions with ER are disrupted by ERX-11 in MCF-7 cells.
| Biology processes | Genes |
|---|---|
| RNA processing | CD2BP2 CHERP CPSF1 CPSF2 CPSF3L CSTF3 DDX17 DDX20DDX23 DHX15 DHX9 DKC1 GEMIN5 HNRNPA3 HNRNPK HNRNPLL HNRNPLL HNRNPR INTS2 INTS4 INTS5 NCBP1 PCF11 POLR2A PPP2R1A PRPF31 PRPF40a PUF60 RBM10 RBM14 SART1 SF1 SF3A3 SF3B1 SF3B3 Sfrs15 SKIV2L2 SMC1A SRRM1 SSB SYNCRIP THOC2 TRNT1 U2AF2 XRN2 ZCCHC8 |
| Transcription | ADNP CCNL1 CSDA CTNND2 DIDO1 DMAP1 EIF2S2 ERCC2 FOXA1 GTF2I GTF3C1 GTF3C KDM3B KDM5B LRPPRC MCM2 MED1 MED24 NCOA3 PELP1 POLR2A POLR3C PSIP1 PUF60 RBM14 RFX1 SAP130 SF1 SMARCA2 SMARCA4 SMARCC2 SMARCD2 THRAP3 Th1l TRIM33 UHRF1 XRN2 ZBTB7A ZMYM2 ZNF217 ZNF512B |
| Protein transport, protein localization | AP2A2 CLTC COG1 COG3 COG5 COG8 COPB1 COPB2 COPG2 CSE1L EXOC2 EXOC3 EXOC4 EXOC5 EXOC8 IPO4 KPNA4 KPNB1 NUP153 NUP155 NUP93 RANBP2 SEC16A SEC23A SEC24B SEC24C SRP72 STXBP2 TNPO1 TRAM1 TRNT1 VCP VPS11 VPS18 VPS39 |
| RNA splicing | CD2BP2 CPSF1 CPSF2 CSTF3 DDX20 DDX23 DHX15 DHX9 GEMIN5 HNRNPA3 HNRNPL HNRNPR LUC7L3 NCBP1 PCF11 POLR2A PPP2R1A PRPF31 PRPF40A PUF60 RBM10 SART1 SF1 SF3A3 SF3B1 SF3B3 SKIV2L2 SMC1A SRRM1 SYNCRIP THOC2 U2AF2 ZCCHC8 |
| Macromolecular complex subunit organization | CSE1L DARS DDX20 DDX23 EPRS ERCC2 FKBP4 GEMIN5 GTF2I GTF3C4 HSP90AA1 IPO4 KPNB1 LONP1 MCM2 MED1 MED24 NCBP1 PFKL PFKM PFKP POLR2A PPP2R1A PREX1 PRPF31 SF1 SF3A3 SF3B3 THRAP3 TNPO1 TUBA1B TUBB VCP XRN2 |
| Cell cycle | CUL1 CUL2 CUL3 CUL4B Dmc1 DNM2 DYNC1H1 EIF4G2 LIG3 MCM2 MRE11A NUMA1 PDS5B PHGDH PPP3CA PSMC1 PSMC4 PSMD3 PSMD5 RAD50 SART1 SF1 SMC1A SMC3 SMC4 TUBB UHRF1 |
| Chromosome organization | PDS5B KDM5B RBM14 RAD50 MRE11A CHD1L DMAP1 DKC1 EP400 KDM3B MCM2 SAP130 SMC1A SMC3 SMC4 SMCHD1 SMARCA2 SMARCA4 SMARCC2 SMARCD2 |
| Regulation of cell death | ACTN1 ADNP CSDA CUL1 CUL2 CUL3 DDX20 DNM2 ERCC2 PPP2R1A PREX1 SART1 SCRIB TUBB UACA VCP |
Table 3
Selected pathways modulated by ERX-11 treatment. Differentially expressed genes were subjected to pathway analysis using IPA software and the selected top canonical pathways modulated by ERX-11 are …
| Pathway | p-Value | Ratio | Genes |
|---|---|---|---|
| Retinoic-acid-Mediated Apoptosis Signaling | 2.44E + 00 | 1.25E-01 | PARP12,ZC3HAV1,TNFSF10,PARP9,PARP14,CRABP2,RARG,CRABP1 |
| ERK/MAPK Signaling | 1.77E + 00 | 7.49E-02 | SRC,MKNK2,PLA2G4F,DUSP2,BAD,ELF5,PPM1J,PPP1R14B,STAT1,RAC3,ELF4,PPP2R1A,RRAS,RPS6KA4 |
| Cyclins and Cell Cycle Regulation | 1.46E + 00 | 8.97E-02 | HDAC5,TGFB1,PPM1J,PPP2R1A,E2F1,HDAC11,HDAC7 |
| Death Receptor Signaling | 1.53E + 00 | 8.70E-02 | PARP12,ACTG1,ZC3HAV1,LIMK1,TNFSF10,PARP9,PARP14,BIRC3 |
| Inhibition of Matrix Metalloproteases | 2.40E + 00 | 1.54E-01 | HSPG2,MMP10,TIMP1,MMP13,MMP15,SDC1 |
| Estrogen Receptor Signaling | 1.19E + 00 | 7.09E-02 | KAT2B,ERCC2,SRC,G6PC3,TAF6,MED24,TAF6L,RRAS,MED15 |
| Breast Cancer Regulation by Stathmin1 | 4.63E-01 | 4.71E-02 | ADCY1,ARHGEF19,PPM1J,PPP1R14B,LIMK1,PPP2R1A,E2F1,TUBA4A,RRAS |
Table 4
Clinicopathologic characteristics of the 12 patients, whose ER+, PR + status in breast tumors were analyzed by Ki67 and ER staining. This data is related to Figure 7B,C.
| Case # | Tumor | ER% | PR% | HER2 |
|---|---|---|---|---|
| 1 | IDC | 100 | 80 | Negative |
| 2 | IDC | 90 | 90 | Negative |
| 3 | Papillary | 100 | 70 | Non-amplified |
| 4 | IDC | 90 | 90 | Negative |
| 5 | IDC | 100 | 95 | Non-amplified |
| 6 | IDC | 100 | 30 | Negative |
| 7 | IDC | 100 | 10–40 | Negative |
| 8 | IDC | 80 | 50 | Negative |
| 9 | IDC | 50–60 | 80–90 | Negative |
| 10 | IDC | 100 | 100 | Non-amplified |
| 11 | IDC | 100 | 95 | Non-amplified |
| 12 | IDC | 95 | 95 | Negative |
Table 5
Primer sequences used for RTqPCR
| Gene name | Primer sequence |
|---|---|
| SRC-325F | GAGCGGCTCCAGATTGTCAA |
| SRC-410R | CTGGGGATGTAGCCTGTCTGT |
| E2F1-378F | ACGCTATGAGACCTCACTGAA |
| E2F1-626R | TCCTGGGTCAACCCCTCAAG |
| ERCC2-68F | GGAAGACAGTATCCCTGTTGGC |
| ERCC2-169R | CAATCTCTGGCACAGTTCTTGA |
| LIMK1-276F | CAAGGGACTGGTTATGGTGGC |
| LIMK1-367R | CCCCGTCACCGATAAAGGTC |
| MMP15-149F | AGGTCCATGCCGAGAACTG |
| MMP15-305R | GTCTCTTCGTCGAGCACACC |
| DUSP2-491F | GGGCTCCTGTCTACGACCA |
| DUSP2-574R | GCAGGTCTGACGAGTGACTG |
| RCOR2-118F | CACTCGCACGACAGCATGAT |
| RCOR2-285R | CATCGCAATGTACTTGTCAAGC |
| DKK1-95F | CCTTGAACTCGGTTCTCAATTCC |
| DKK1-232R | CAATGGTCTGGTACTTATTCCCG |
| PDGFB-63F | CTCGATCCGCTCCTTTGATGA |
| PDGFB-301R | CGTTGGTGCGGTCTATGAG |
| PGLYRP2-23F | TCCTACTCGGATTGCTACTGTG |
| PGLYRP2-206R | AAGTGGTAGAGGCGATTGTGG |
| ELF5-357F | TAGGGAACAAGGAATTTTTCGGG |
| ELF5-519R | GTACACTAACCTTCGGTCAACC |
| TNFSF10-46F | TGCGTGCTGATCGTGATCTTC |
| TNFSF10-126R | GCTCGTTGGTAAAGTACACGTA |
| STAT1-368F | ATCAGGCTCAGTCGGGGAATA |
| STAT1-553R | TGGTCTCGTGTTCTCTGTTCT |
| XAF1-297F | GCTCCACGAGTCCTACTGTG |
| XAF1-403R | GTTCACTGCGACAGACATCTC |
| IFI6-257F | GGTCTGCGATCCTGAATGGG |
| IFI6-401R | TCACTATCGAGATACTTGTGGGT |
Table 6
Analyses of the amino acids at the flanking sequences of top ER binders whose interactions are blocked by ERX-11 in MCF-7 and ZR-75 cells, as determined by unbiased IP-MS.
| Protein/GENE ID | # LXXLL motifs | LXXLL sequences | Serine at i ± 3/4 |
|---|---|---|---|
| Plectin Q15149-3 | 6 | 210 GHNLISLLEVL 220 213 LISLLEVLSGDS 224 421 YRELVLLLLQWM 431 659 LRYLQDLLAWV 669 1102 YQQLLQSLEQG 1112 4006 TGQLLLPLSDA 4016 | yes |
| FAM83H Q6ZRV2 | 2 | 816 AAQLLDTLGRS 826 966 SLRLRQLLSPK 976 | yes |
| AHNAK Q09666 | 0 | ||
| CLTC: cQ00610 | 5 | 563 TAFLLDALKNN 573 854 RNRLKLLLPWL 864 1001 PNELIELLEKIV 1011 1021 HRNLQNLLILT 1031 1418 PLLLNDLLMVLS 1429 | yes |
| FAS P49327 | 10 | 76 DPQLRLLLEVT 86 418 HATLPRLLRAS 428 560 QIGLIDLLSCM 570 691 APPLLQELKKV 701 1175 QQELPRLLSAA 1185 1211 EDPLLSGLLDSP 1221 1346 GFLLLHTLLRGH 1358 1470 RCVLLSNLSST 1480 2216 QLNLRSLLVNP 2226 2381 NRVLEALLPLKG 2391 | yes |
| TRG P27635 | 1 | 116 VNRLLDSLEPP 126 | no |
| ACTN4 O43707 | 1 | 81 GLKLMLLLEVIS 92 | yes |
| TFG Q92734 | 0 | ||
| Coatomer subunit alpha P53621 | 1 | 83 RRCLFTLLGHLDYI 96 | no |
| Q9NVI7-2 | 1 | 99 ALSLLHTLVWA 109 | no |
Appendix 1—Table 1
Characterization of the peptides
| LXXLL Peptide | Sequence | molecular mass (MALDI-TOF-MS) | |
| calculated | observed | ||
| SRC1 | Ac-LTARHKILHRLLQEGSPSD-NH2 | [M + H]+ for C96H162N32O28: 2212.2 | 2212.4 |
| SRC2 | Ac-DSKGQTKLLQLLTTKSDQM-NH2 | [M + H]+ for C92H162N26O32: 2176.2 | 2176.6 |
| AIB1 | Ac-ESKGHKKLLQLLTCSSDDR-NH2 | [M + H]+ for C92H159N29O31: 2199.2 | 2199.8 |
| PELP1 | Ac-SIKTRFEGLCLLSLLVGESPT-NH2 | [M + H]+ for C103H174N26O31: 2304.3 | 2304.6 |
