The interleukin-like epithelial-mesenchymal transition inducer ILEI exhibits a non-interleukin-like fold and is active as a domain-swapped dimer

Abstract

Production and secretion of pro-metastatic proteins is a feature of many tumor cells. The FAM3C interleukin-like epithelial-to-mesenchymal-transition (EMT) inducer (ILEI) has been shown to be strongly up-regulated in several cancers and to be essential for tumor formation and metastasis in epithelial cells, correlating with a significant decrease in overall survival in colon and breast cancer patients. ILEI has been seen to interact with the γ-secretase presenilin 1 subunit (PS1). However, not much is known about the mechanism-of-action or the detailed ILEI structure. We present here the crystal structures of FAM3C ILEI and show that it exists as monomers but also as covalent dimers. The observed ILEI β-β-α fold confirmed previous indications that the FAM3C proteins do not form classical four-helix-bundle structures as was initially predicted. This provides the first experimental evidence that the interleukin-like EMT inducers are not evolutionarily related to the interleukins. However, more surprisingly, the ILEI dimer structure was found to feature a trans-linked domain swap, converting an intramolecular disulfide to intermolecular. Interestingly, dimeric but not monomeric ILEI was subsequently found to cause a dose-dependent increase in EpRas cell invasiveness comparable with TGF-β, indicating that the dimer might be the active ILEI species. This is in line with a parallel study showing that covalent oligomerization of ILEI is essential for EMT and tumor progression in vivo The structures and the activity data give some first insight into the relationship between dimerization and ILEI function as well as indicate an intriguing link between ILEI, the PS1-protease, TGF-β, and the TGF-β receptor 1.

Keywords: cancer; crystallography; epithelial-mesenchymal transition (EMT); presenilin; transforming growth factor β (TGF-β); γ-secretase.

Figure 1.

Constructs for recombinant expression of ILEI in suspension-adapted HEK cells. A, construct 1 containing a native signal peptide and the sequence of human ILEI followed by a FLAG tag and a C-terminal His-tag. Constructs 2 and 3 were designed based on expression of FAM3B PANDER (13) with the native signal peptide replaced by CD33 signal peptide. Human and mouse variants of C3 were used for crystallization experiments. C4 was designed with an N-terminal Fc fusion preceded by an IgK signal peptide. In the C4 construct the ILEI coding region also contains two mutations, Ala-63 mutated to Gly and Glu-210 mutated to Asp. Mouse variants of C3 and C4 were coexpressed to produce a chimeric ILEI heterodimer. B, Coomassie-stained SDS-PAGE showing yields of human ILEI protein in the supernatant after small-scale expression and Ni-NTA purification of constructs hC1, hC2, and hC3. C, SDS-PAGE of mouse ILEI dimer and monomer, construct mC3, under non-reducing (lanes 1, 3, 5, and 7) and reducing conditions (lanes 2, 4, 6, and 8). Samples in lanes 3, 4, 7, and 8 are concentrated versions of lanes 1, 2, 5, and 6. Note the presence of the dimer and the split monomer bands under non-reducing conditions compared with the single band monomer under reducing conditions.

Figure 2.

The FAM3C ILEI monomer fold. A, the ILEI monomer structure, composed of two layers of β sheets and three short helices. The two cysteine bridges between Cys-58 and Cys-86 as well as between Cys-64 and Cys-221 are indicated in yellow together with a buried cysteine, Cys-185, located on β7. B, a highly conserved SPFE motif breaks the N-terminal end of β8 compared with the FAM3B PANDER structure (supplemental Fig. S1). C, the water-filled ILEI monomer pocket.

Figure 3.

Disulfide topology diagram of FAM3C ILEI monomers and dimers. Both monomer (A) and dimer (B) ILEI contain two disulfide bridges. The Cys-58–Cys-86 disulfide is conserved in both. However, the Cys-64–Cys-221 bridge is converted into a trans-linked disulfide in the dimer, introducing a covalent bond between the N-terminal strand of one chain with the C-terminal strand of the other.

Figure 4.

FAM3C ILEI dimer fold. A, the domain-swapped FAM3C ILEI dimer structure with the two chains colored in blue and red, respectively. B, surface representation of the dimer. C, a highly conserved SPFE motif located in the dimer interface breaks the N-terminal end of β8. D, the water-filled ILEI pocket split by the two dimer chains.

Figure 5.

ILEI dimers increase in vitro invasive capability of EpRas cells in a dose-dependent manner. Trans-well invasion assay of EpRas cells treated with increasing concentrations of recombinant mC3 ILEI dimers and monomers toward a 10% FCS attractant. Invasion was performed for 24 h and is shown as -fold change over non-treated control (ctrl). Recombinant human TGF-β1 (hTGFb1) treatment under the same conditions was used as positive control. Data represent the mean ± S.E. of three independent experiments. Statistical significance (*, p ≤ 0.05, *** p ≤ 0.001) as determined by an unpaired two-sided Student's t test.