A novel class of dual-family immunophilins

Abstract

Immunophilins are protein chaperones with peptidylprolyl isomerase activity that belong to one of two large families, the cyclosporin-binding cyclophilins (CyPs) and the FK506-binding proteins (FKBPs). Each family displays characteristic and conserved sequence features that differ between the two families. We report a novel group of dual-family immunophilins that contain both CyP and FKBP domains for which we propose the name FCBP (FK506- and cyclosporin-binding protein). The FCBP of Toxoplasma gondii, a protozoan parasite, contained N-terminal FKBP and C-terminal CyP domains joined by tetratricopeptide repeats. Structure-function analysis revealed that both domains were functional and exhibited family-specific drug sensitivity. The individual domains of FCBP inhibited calcineurin (protein phosphatase 2B) in the presence of the appropriate drugs. In binding studies, FCBP recruited calcineurin in the presence of FK506 and a putative target of rapamycin homolog in the presence of rapamycin. Two additional FCBP sequences in Flavobacterium and one in Treponema (spirochete) were also identified in which the CyP and FKBP domains were in the reverse order. T. gondii growth was inhibited by cyclosporin and FK506 in a moderately synergistic manner. The knockdown of FCBP by RNA interference revealed its essentiality for T. gondii growth. Clearly, the FCBPs are novel chaperones and potential targets of multiple immunosuppressant drugs.

Fig 1. Representative domain arrangements of FKBPs, Cyps, and the dual-family FCBPs

The “single-family” enzymes (FKBP, CyP) are shown for comparison only. The proteins are named by the initials of the organism (Dm, Drosophila melanogaster; Hs, Homo sapiens; Td, T. denticola; At, Arabidopsis thaliana) followed by the domain name(s) and the theoretical molecular mass in kDa. The accessory domains, i.e. TPR, WD40, and the putative CaM-binding domains, are also indicated. CyP domains are black rectangles, and FKBDs are white.

Fig. 2. Sequence homology of the dual-family immunophilins of Tg, Fj, and T. denticola (Td)

The nomenclature has been described in Fig. 1. Highlighted residues are important for various functions, including PPIase activity, substrate binding, and interaction with immunosuppressant drugs (CsA for the CyP domain and FK506 for the FKBP domain). A, homology of the FKBP domains. The prototype human FKBP (hFKBP12) and the P. falciparum FKBP35 (21, 22) are included for comparison. B, homology of the CyP domains. The prototype human CyP (hCyP18) is included for comparison. Residues important for CN binding are underlined. The W (for Trp), marked with overhead circle, is especially important for CsA binding but is replaced by H (for His) in Toxoplasma, which explains the relative CsA resistance of the latter (Fig. 4). Note how the residue numbers differ between the Toxoplasma and the bacterial FCBPs because of the reversal of the domain arrangement.

Fig. 3. PPIase (A) and chaperone activity (B) of recombinant full-length T. gondii FCBP and its domains

Assays were carried out as described under “Experimental Procedures.” The following His-tagged recombinants were assayed: 1, full-length TgFCBP57; 2, Δ1TPR (the most N-terminal TPR deleted); 3, Δ2TPR (two N-terminal TPRs deleted); 4, Δ3TPR (all three TPRs deleted); 5, FKBP domain (residues 25−154) only; 6, CyP domain (residue 338−521) only; and 7, no enzyme control. Note that both domains have independent and roughly equal PPIase and folding activity.

Fig. 4. Drug sensitivity of the individual domains of TgFCBP57

A, full-length enzyme. B, FKBP domain only (residues 25−154). C, CyP domain only (residues 338−521). Open symbols, WT; closed symbols, His-474 → Trp mutant (see Fig. 2) in which full CsA sensitivity was restored. Results represent the average of three experiments with mean ± S.E. bars.

Fig. 5. Sensitivity of FCBPs to immunosuppressant drugs

PPI-ase activity of the recombinant enzymes from T. gondii and F. johnsonii was determined in the presence of the indicated concentrations of CsA (gray bars) or ethyl-FK506 (black bars) or a mixture of the two (striped bars). The uninhibited activity of the corresponding enzymes (white bars) was taken as 100. Results represent an average of three experiments with mean ± S.E.

Fig. 6. Inhibition of T. gondii CN activity by recombinant FKBP domain (residues 25−154) or CyP domain (residues 338−521) of TgFCBP57 in the presence of 10 μm CsA (gray bars) or ethyl-FK506 (black bars)

Uninhibited CN activity (white bar) was taken as 100. Results are average of three experiments with mean ± S.E.

Fig. 7. T. gondii drug sensitivity

A, anti-toxoplasma activity of ethyl-FK506 and CsA. The IC₅₀ of the two drugs are 0.65 and 0.76 μm, respectively. B, anti-toxoplasma activity of a combination of ethyl-FK506 and CsA. Note that the experimental line is slightly concave, indicating modest synergism, in contrast to the hypothetical straight line (dotted) that would have been obtained if there were no interaction between the drugs. The numbers on both axes are drug concentrations expressed as fractions of their individual IC₅₀ values. Results represent average of three experimental sets with mean ± S.E.

Fig. 8. RNA interference phenotype of TgFCBP57 knockdown

RNAi knockdown was done with dsRNA as described under “Experimental Procedures.” T. gondii cells, transfected with 1 or 2 μg of dsRNA, were used to infect human foreskin fibroblast cells, and at 18 and 24 h later, the cells were analyzed for TgFCBP57 by immunoblot. Results using antibody A (“Experimental Procedures”) are shown, although antibody B produced essentially the same result. [³H]Uracil incorporation of identical parallel cultures was measured and expressed as percentage of untreated (No RNA) value. Single-stranded sense RNA (sRNA,4 μg, 24 h) was used as another negative control. The numbers are average of three experiments with the mean ± S.E. as shown. Control Hsp90, detected by immunoblot (11), was unaffected (bottom panel).

Fig. 9. Drug-specific association of FCBP with downstream proteins

The experiment was done essentially as described previously (38). Purified His-tagged recombinant TgFCBP57 (10 μg) was immobilized on Ni²⁺-agarose column (60 μl), and 400 μg of T. gondii cell extract was passed through it. Where shown, the extract was pre-mixed with 10 μm drug. Where indicated (last lane), 0.2 μg of competing TOR peptide (FRBD region) was added to the extract. The column was then washed with 1 ml of wash buffer (Novagen) containing 0.2 mm imidazole. The bound proteins were stripped by boiling in 2× SDS sample buffer and analyzed by SDS-PAGE and silver staining. The catalytic subunit of T. gondii CN was further confirmed by immunoblot (data not shown). See “Results” for details.

Fig. 10. A putative T. gondii TOR homolog

The C-terminal end of the deduced primary structures of human (P42345; hTOR) and T. gondii TOR (TgTOR) proteins are aligned. The TgTOR sequence is based on TgTigrScan_2443 in ToxoDB but was conceptually corrected and partially sequenced. The following domains are marked. Part of the FAT (FKBP-rapamycin-associated protein, ATM, and transformation/transcription domain-associated protein (TRRAP)) domain (smaller box), FRBD (larger box), and phosphatidylinositol 3-kinase-like domain (underlined) is shown. Identical residues are denoted by asterisk, and similar ones are represented by dots. We tentatively estimate the full-length TgTOR to be 2845 amino acids long.