Cyclophilin

Historically, an 18-kDa cytosolic protein from pig kidney was the first peptidyl prolyl cis/trans isomerase (abbreviated PPIase) discovered and characterized enzymatically (1). These enzymes catalyze the rotation of a peptidyl prolyl moiety in peptides and proteins. In 1989 it was found that this protein is nearly identical in its primary structure to the already known cytosolic receptor of the immunosuppressive undecapeptide cyclosporin A in human T lymphocytes, previously named cyclophilin [Cyp18; for nomenclature see (2)] (3-5). This enzyme represents the archetype of cyclophilins in that it constitutes the catalytic domain of larger cyclophilin-like proteins. Now two additional families of such isomerases are known, those that bind FK506 (FKBP) and the parvulin family.

 Alignment of amino acid sequences of the cyclophilin family defines residues that are highly conserved among vertebrates, plants, fungi, and bacteria. Nearly perfect conservation is located primarily in the central segment of the protein, including the sequences -Phe-His-Arg-Ile/Val-Ile-(Xaa)₅-Gln-Gly-Gly- at positions 53 to 65 and -Met-Ala-(Xaa)_9–10-Gln-Phe-Phe/Tyr-Ile/Val- at positions 100 to 114, where Xaa is any amino acid and / separates alternatives. These sequences are used as typical motifs to search for other cyclophilins. The three-dimensional structure of the archetypal cyclophilin consists of an eight-stranded antiparallel b-sheet barrel capped by a-helices (6, 7) .

The characterization of 11 distinct cyclophilin genes in the genome of the nematode Caenorhabditis elegans demonstrates that organisms use numerous cyclophilins (8). As many as 12 human homologues have been identified thus far from the initial assessment of gene diversity by expressed sequence tag analysis. The C-terminal and/or N-terminal amino acid extensions of larger cyclophilins consist of additional domains, directing the proteins into specific cellular compartments, mediating protein/protein and DNA/protein interactions, or generating other biochemical functions.

 The cytosolic Cyp18 often represents the major cyclophilin of cells. Kidney tubules and endothelial cells, for example, contain 10 µg of Cyp18 per mg total protein (9). Despite the numerous studies reporting the three-dimensional structures of Cyp18-bound oligopeptide substrates and inhibitors (10-12) , a definite catalytic mechanism for cyclophilins and other PPIases is still lacking. It is currently believed that catalysis arises from a combination of desolvation of the substrate and substrate-assisted catalysis. Electrophilic assistance, for example, by the Arg55 residue of Cyp18, can also be envisaged to account for the more effective catalysis found for cyclophilins, compared to FKBPs. The positively charged side-chain of this amino acid residue is located within hydrogen-bonding distance and perpendicular to the plane of the substrate proline ring in Cyp18/substrate complexes. This environment may cause additional weakening of the reactive C–N linkage by immobilizing the lone pair of electrons of the nitrogen atom. Indeed, a Cyp18 variant in which Arg55 has been replaced retains only 0.1% of the wild-type enzymatic activity (13).

Because of the putative functional redundancy and overlapping of the many PPIases present in most organisms, gene deletion experiments often do not lead to a recognizable phenotype under normal growth conditions. Among the seven cyclophilins found in S. cerevisiae, deletions of only two are associated with any phenotype. Disruption of Cpr3, the gene encoding the mitochondrial isoform of yeast cyclophilin, affects growth on L-lactate medium (14), whereas cpr7D cells are defective in normal cell growth (15). The expression of many PPIase genes seems to be sensitive to heat shock and to the chemical stress response.

Recently, it was shown that host cell cytosolic Cyp18 is required for HIV-1 infection before reverse transcription but subsequent to receptor binding and membrane fusion in T cells (16). A proline-rich segment of the capsid domain of Pr55^gag mediates incorporation of Cyp18 into HIV-1 virions (17).

This conserved segment that contains four proline residues occurs in the sequence -Pro-(Xaa)₄-Pro222-(Xaa)₂-Pro-(Xaa)₅-Pro-. Mutant proteins of HIV-1_HXB2 that have site-directed mutations in which Pro²²² is replaced by Ala or Gly221 by Ala, fail to bind to the fusion protein glutathione-S-transferase/Cyp18. Virions that contain these altered proteins cannot sequester Cyp18 into the released virions, emphasizing the importance of the Gly-Pro²²² bond for Pr55^gag /Cyp18 complex formation. The ability of many cyclosporin derivatives to dissociate the Pr55^gag/Cyp18 complex reveals a quantitative relationship between Cyp18 inhibition and complex decomposition. Obviously, the antiviral effect of cyclosporin A must be caused by a pathway distinctive from immunosuppression because cyclosporin A derivatives with negligible immunosuppressive activity, but high affinities for the active site of Cyp18, retain potent anti-HIV activity (18-20.(

 A genetic cDNA screen was used to identify the bovine homologue of the retina-specific cyclophilin NinaA of Drosophila. The membrane-localized PPIase of the secretory pathway of the fly is required for proper folding and trafficking of Rh1-6 opsin in photoreceptor cells (21). The bovine RanBP2 cyclophilin has a domain that binds to the GTPase Ran, as well as to red/green opsin. A still unknown modification of opsin, possibly a prolyl bond isomerization catalyzed by the Cyp-domain of RanBP2, augments and stabilizes the interaction between the Ran-binding domain and opsin. This modification is important in membrane trafficking of long-wavelength opsin in photoreceptors (22).

References

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