Immunophilins, Refsum disease, and lupus nephritis: the peroxisomal enzyme phytanoyl-COA alpha-hydroxylase is a new FKBP-associated protein

Abstract

FKBP52 (FKBP59, FKBP4) is a "macro" immunophilin that, although sharing high structural and functional homologies in its amino-terminal domain with FKBP12 (FKBP1), does not have immunosuppressant activity when complexed with FK506, unlike FKBP12. To investigate the physiological function of FKBP52, we used the yeast two-hybrid system as an approach to find its potential protein partners and, from that, its cellular role. This methodology, which already has allowed us to find the FK506-binding protein (FKBP)-associated protein FAP48, also led to the detection of another FKBP-associated protein. Determination of the sequence of this protein permitted its identification as phytanoyl-CoA alpha-hydroxylase (PAHX), a peroxisomal enzyme that so far was unknown as an FKBP-associated protein. Inactivation of this enzyme is responsible for Refsum disease in humans. The protein also corresponds to the mouse protein LN1, which could be involved in the progress of lupus nephritis. We show here that PAHX has the physical capacity to interact with the FKBP12-like domain of FKBP52, but not with FKBP12, suggesting that it is a particular and specific target of FKBP52. Whereas the binding of calcineurin to FKBP12 is potentiated by FK506, the specific association of PAHX and FKBP52 is maintained in the presence of FK506. This observation suggests that PAHX is a serious candidate for studying the cellular signaling pathway(s) involving FKBP52 in the presence of immunosuppressant drugs.

Figure 1

Effect of FK506 on the FKBP52-I/clone 2 (ΔPAHX) two-hybrid interaction. β-galactosidase activities were quantified by liquid assay in the strain FKBP52-I/clone2 (ΔPAHX), grown in the presence or absence of FK506. The β-galactosidase activity measured in the extract of the untreated strain was taken as 100; the values obtained for the extracts of this strain treated with drug were standardized with respect to the reference sample. Values correspond to the means of triplicates.

Figure 2

Northern blot analysis of PAHX. Poly(A)⁺ RNA (5 μg) isolated from Jurkat cells was electrophoresed in a 1% denaturing gel, blotted onto a Hybond membrane, and hybridized with labeled clone 2. After 1 week of exposure, a band around 1,700 bp was revealed. The position of RNA transcript molecular mass markers (Amersham Pharmacia) is shown on the left.

Figure 3

Interaction of ΔPAHX, or PAHX with FKBP52, and FKBP52-I in the yeast two-hybrid system. β-Galactosidase activities were quantified by liquid assay in the strains FKBP52-I/ΔPAHX, FKBP52-I/PAHX, FKBP52/ΔPAHX, and FKBP52/PAHX.

Figure 4

Analysis of the FKBP52/PAHX complex in vitro using a gel-shift assay. FKBP52 alone or incubated with GST or PAHX, as indicated, was subjected to nondenaturing PAGE followed by Western blotting using the polyclonal antibody 173 directed against FKBP52.

Figure 5

In vitro binding assay of PAHX with immunophilin. (A) Human PAHX specifically binds FKBP52 in vitro. In vitro binding assays were carried out by incubation of 500 pmol GST-FKBP52 resin (lanes 1–3), 500 pmol GST-FKBP12 resin (lanes 4–6), or 500 pmol GST resin as control (lanes 7–9) in the presence of increasing quantities of PAHX. The molar ratio of PAHX added to different resins is indicated under each lane. After processing the resin as described in Material and Methods, the presence of PAHX retained on the GSH beads was analyzed by Western blotting by using a polyclonal antibody directed against PAHX. Recombinant purified PAHX is loaded as control in lane 10. (B) The PAHX/immunophilin complex formation is not modulated by FK506 in vitro. (Upper) GSH beads loaded with 500 pmol of GST-FKBP52 were treated with increasing quantities of FK506 as indicated under each lane, before incubation with 500 pmol of PAHX. Bound proteins were subjected to Western blotting analysis by using a polyclonal antibody directed against PAHX. Lane 1, no PAHX was added to the resin. (Lower) GSH beads loaded with 500 pmol of GST-FKBP52 (lanes 1–3) or 500 pmol of GST-FKBP12 (lanes 4–6) were treated or not with 10 nmol of FK506 before incubation with 500 pmol of PAHX, as indicated in Materials and Methods. Bound proteins were subjected to Western blotting analysis by using a polyclonal antibody directed against PAHX. Lanes 1 and 4, no PAHX was added to the resin. Recombinant purified PAHX was loaded as control in lane 7. Migration of molecular size markers (New England Biolabs) is indicated on the left.