Introducing Immunophilins. From Organ Transplantation to Plant Biology

The discovery of the family of highly effective and specific fungal metabolites cyclosporin, FK-506, and rapamycin ranks among one of the most important advancements in the field of organ transplantation: these compounds have been shown to act as potent immunosuppressants and their administration to transplant recipients to significantly cut organ rejection rates. By binding to their intracellular receptor proteins, the immunophilins, they selectively block transcriptional activation of defense-related genes early in the T-cell signal transduction pathway (O'Keefe et al., 1992). During such a response, an antigen detected by the cell surface T-cell receptor triggers a rise in cytosolic calcium, resulting in the activation of calcineurin (CaN). CaN dephosphorylates the nuclear factor of activated T-cell (NF-AT) transcription factors that regulate the expression of a number of genes. These include the growth factors such as interleukin 2 and related proteins required for setting up the autocrine loop for T-cell proliferation. Once applied, the immunosuppressant and its corresponding immunophilin (cyclosporin to cyclophilins, FK-506 and rapamycin to FK506 binding protein or FKBP) form a receptor-ligand complex, which targets distinct components of the T-cell activation signaling pathway. Cyclophilin-CsA and FKBP-FK506 complexes display near identical modes of action; by binding CaN and inhibiting its phosphatase activity, T-cell activation is ultimately blocked by preventing NF-AT induced activation of gene expression (Molkentin et al., 1998). In contrast to cyclosporin and FK-506, rapamycin blocks T-cell proliferation by inhibiting the interleukin signal transduction cascades. The pharmacological properties of these powerful immunosuppressants have stimulated intense research into their therapeutic applications, while acting as a catalyst for the characterization of the biological function of immunophilins in vivo.

A TWIST IN THE TALE: CONVERGENCE OF IMMUNOPHILINS AND PPIASES

Cyclophilin A was initially identified and purified from bovine spleen as the cellular receptor of CsA (Handschumacher et al., 1984). In a separate experiment, a peptidyl-prolyl cis-trans isomerase (PPIase) purified from porcine kidney was found to be identical to cyclophilin A (Fischer et al., 1989). PPIases catalyze the cis-trans isomerization of bonds located between proline and its preceding residue: a rate-limiting step in the folding of newly synthesized proteins (Brandts et al., 1975). FKBPs possess the same enzymatic activity as cyclophilins, although their catalytic domains show no significant sequence homology (Schreiber, 1991). PPIases vary considerably in both form and biological function, and have been identified in a wide variety of organisms, ranging from Escherichia coli to humans (Andreeva et al., 1999; Ivery, 2000). The abundance and diversity of single and multidomain immunophilins identified to date underlines the functional versatility of this family, which is further exemplified by the wide range of cellular processes in which they are involved. Nuclear cyclophilin CypH assembles into spliceosomal complexes where it assists with mRNA splicing (Reidt et al., 2003; Ingelfinger et al., 2003); CypA functionally interacts with the major virion-associated HIV accessory protein viral protein R, but also modulates the catalytic activity of interleukin-2 Tyr kinase (Brazin et al., 2002; Zander et al., 2003); CypB exerts its function in the nucleus as a part of the prolactin transcriptional control machinery (Rycyzyn and Clevenger, 2002), but also triggers the integrin-mediated adhesion of peripheral blood T lymphocytes to the extracellular matrix (Allain et al., 2002). FKBP12 constitutes an integral component of well-documented receptors such as the TGFβ receptor Type II or calcium channels such as the ryanodine receptor or the inositol 1,4,5-triphosphate receptor. When bound to the latter, FKBP12 anchors calcineurin to the complex, promoting a complex negative feedback loop in which calcineurin activity is regulated by calcium flux (Cameron et al., 1997; Marx et al., 2000).

PLANT IMMUNOPHILINS: THE STORY SO FAR…

The discovery of plant immunophilins has not only demonstrated conservation of these proteins in a full spectrum of biological systems but has also provided clues as to their potential functions. For example, some plant cyclophilin genes have been shown to be induced by a variety of biotic and abiotic stresses, suggesting that they may play a role in environmental response processes (Chou and Gasser, 1997; Kurek et al., 1999). Early work showing the distribution of immunophilins throughout the plant cell (Breiman et al., 1992; Luan et al., 1994) has been recently confirmed and expanded by genomic and proteomic approaches, which have provided detailed subcellular localization data for these large gene families. Most striking is the finding that the Arabidopsis genome encodes 16 chloroplast immunophilin isoforms: while a single cyclophilin is located in the stroma, 5 cyclophilins and 9 FKBPs are targeted to the thylakoid lumen. Experimental corroboration of the genomic data has been carried out for most of these isoforms (Gupta et al., 2002; Peltier et al, 2002; Schubert et al., 2002). Whilst two of the lumenal immunophilins have been shown to be functionally associated with photosynthetic complex components (Fulgosi et al., 1998; Gupta et al., 2002), the specific function of the majority of chloroplast isoforms remains to be elucidated.

Meanwhile, genetics analysis of developmental mutants identified three multidomain immunophilins containing tetratricopeptide repeats that regulate plant development: plants lacking the FKBP52-like PAS1 protein show severe developmental aberrations including ectopic cell proliferation in cotyledons, extra layers of cells in the hypocotyl, and an abnormal apical meristem (Faure et al., 1998; Vittorioso et al., 1998); severe developmental abnormalities are also observed in twisted dwarf (twd1) mutants, which lack the integral membrane FKBP-like TWD1, an immunophilin which has been shown to interact with the Arabidopsis multidrug-resistance-like ABC transporter AtPGP1 involved in auxin transport (Kamphausen et al., 2002; Geisler et al., 2003; Pérez-Pérez et al., 2004); Arabidopsis Cyp40 has been shown to be required for the vegetative maturation of the shoot (Berardini et al. 2001). Moreover, an insight into the mechanisms underlying immunophilin/PPIase functions has been obtained from two studies that provide a potential link between peptidyl-prolyl isomerization and specific Pro residues of IAA/AUX proteins. Point mutations in the catalytic domain of the tomato cyclophilin LeCYP1 have been shown to be responsible for the pleiotropic, auxin-resistant diageotropica phenotype (Oh et al., 2003). The isomerization state of two conserved Pros located in domain II of the AUX/IAA proteins may also play an important role in auxin signaling. Dharmasiri et al. (2003) showed that auxin-induced BA3::GUS expression in the root elongation zone can be repressed by exogenous addition of juglone, a specific inhibitor of parvulin-type PPIases. Juglone was also shown to specifically inhibit the auxin-induced degradation of the AXR3NT-GST fusion protein. The authors thus speculate that the parvulin catalyzed prolyl-isomerization occurring within domain II of the AUX/IAA protein (or in another step of auxin signaling) is required for recognition and regulatory degradation by the ubiquitin-dependent proteolysis.

In this issue, Patrick Romano and coworkers offer a comprehensive phylogenetic analysis of the Arabidopsis cyclophilin protein family: composed of 29 members, it is the largest cyclophilin family identified in any organism to date (Romano et al. 2004a). He and Luan (2004) expanded the genomics analysis into all PPIases including both immunophilins (CYPs and FKBPs) and parvulins. Of these analysis, the most striking findings are: (1) the structural and potentially functional diversity of these protein foldases; (2) the broad distribution of PPIases in the subcellular compartments; and (3) the large number of PPIases localized to the chloroplasts. With relevance to chloroplast immunophilins, Romano and coworkers further studied the Arabidopsis cyclophilin AtCYP20-2 and show that this cyclophilin may associate with the periphery of PSII supercomplexes (Romano et al., 2004b). Together with earlier reports (Fulgosi et al., 1998; Gupta et al., 2002), these studies suggest that a subset of immunophilins may have specifically evolved to fulfill functions that are unique to photosynthetic organisms. Towards the functional significance of immunophilins in developmental processes, Vespa et al. (2004) show that FIP37, an interacting partner for AtFKBP12, is involved in cell division control. More specifically, the endoreduplication of trichome cells was altered in the transgenic plants that overexpressed FIP37 whereas the null mutant is embryonic lethal.

While the information regarding the function of this large and diverse protein family remains sporadic in plants, evidence obtained so far suggest that they may be involved in a wide variety of functions including, but not limited to, photosynthesis and development. The aim of this focus issue is to introduce the reader to immunophilins and PPIases, underlining their potential importance in plant biology.