Peptidyl‐Prolyl Isomerase Activity of Immunophilins Could Be the Mere Consequence of Protein Complex Organization

Immunophilins comprise a family of proteins characterized by the presence of a specific sequence that usually shows peptidyl-prolyl-(cis/trans)-isomerase (PPIase) activity, i.e., the reversible cis/trans interconversion of Xaa─Pro bonds. The “PPIase domain” also binds immunosuppressive drugs, a property used to group immunophilins into two main subfamilies: FKBPs (FK506-binding proteins) and CyPs (cyclophilins).[1] Immunophilins elicited the early attention of protein folding researchers since it seemed extremely likely that they should play a cardinal role as folding catalysts.[2] Nonetheless, the demonstration that peptidyl-prolyl bonds isomerization is indeed a rate-limiting step for protein folding is still an unresolved and challenging matter. In this issue, Theo Rein addressed this conundrum by analyzing various controversial proteins such as the ribosome-bound chaperone belonging to the FKBP subfamily, TF (trigger factor), whose PPIase activity is dispensable for nascent-chain binding.[3] CyPA, a cyclophilin that induces leukocyte chemotaxis by binding to the cell surface receptor CD147, shows the same properties for PPIase-inactive mutants. Pin1, an immunophilin upregulated in several types of cancers and downregulated in Alzheimer's disease, binds client-proteins in a PPIase-dependent manner. Nonetheless, it has not been demonstrated yet that cis/trans isomerization is the rate-limiting step for this binding. An interesting case is the role of FKBP51 and FKBP52 on the glucocorticoid receptor (GR). The FD67DV mutation within the PPIase domain of FKBP51 cannot prevent the inhibitory action of this immunophilin on GR, whereas the same mutation turned FKBP52 into a GR inhibitor. This led to the conclusion that the enzymatic activity of FKBP52, but not that of FKBP51, is essential for GR regulation. However, that mutant also showed inefficient binding to client-proteins. Consequently, other mutants were generated (Y57A, F67Y, W90L, and F130Y) showing no effect on the FKBP52-dependent potentiation of GR. Therefore, the original concept related to the essentiality of the PPIase activity of FKBP52 on GR regulation had to be revised. Another remarkable case is the transcription factor NF-kB, FKBP52 being a strong PPIase-dependent enhancer and FKBP51 a PPIase-independent inhibitory regulator in fibroblasts, but in melanoma cells, FKBP51 is a PPIase-dependent activator. Thus, the biological action of FKBPs on NF-kB seems to be unpredictable and cell-type dependent. Inasmuch as immunophilins work associated to other chaperones, it is entirely possible that the final effect is dictated by different heterocomplex rearrangements or by specific protein-protein induced conformations. An interesting speculation of Rein's hypothesis to explain these cases is that substrates could disturb protein–protein interactions due to steric interferences and/or favoring the stabilization of a given protein conformation that affects protein complex interactions. In summary, his challenging proposal asserts that the enzymatic activity of an immunophilin could be the consequence of interactions with certain protein motifs of the associated substrate, more likely through binding to peptidyl-prolyl motifs. This is compatible with the variety of effects observed for the same client-protein in different cell contexts discussed above and transforms the prerequisite of enzymatic activity of the immunophilin into a relative or unnecessary requirement. Therefore, the PPIase activity could be the consequence of protein interactions and not essential in most cases. An exciting possibility is that PPIase activity may have a relevant role in protein function and signal transduction rather than in protein folding per se, as it has always been thought. Of course, this hypothesis needs experimental corroboration. The use of artificial proline analogues able to impact the cis/trans preference and the energy threshold for their interconversion could be an interesting option. Also, the need of time-resolved experiments will elucidate the relevance for accelerating prolyl-isomerization in heterocomplexes. This is a thrilling field to develop, especially for the FKBP subfamily in view of its property to regulate signalling cascade factors, perhaps acting as transient scaffolders rather than accelerating prolyl isomerizations. The author declares no conflict of interest.