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Cyclophilins Are Widely Spread

Cyclophilins are widely spread in all studied organisms, can be found in both prokaryotes and eukaryotes and are ubiquitous proteins highly conserved throughout evolution. All known cyclophylin share a commom domain around 109 aminoacids, the Cyp-like domain which is the surrounded by other domains that are unique to each member of the family enabling a specific location and activity in the cell1.

Cyclophylins belong to a group of proteins that have peptidyl-prolyl isomerase activity (PPIases) and are responsible for the cis-trans interconversion of proline isomers, considered important not only in the protein folding but also in the assembly of multi-domain proteins (Figure 1).

Peptide bonds can be connected either in the cis (side chains adjacent to each other) or trans (side chains are 180 degrees apart) configuration during biosynthesis in the ribosome but the later tends to be more favorable due to be more sterically stable with less clashes.

Proline is unique among the natural amino acids and as a relatively small difference in the free energy between the cis and the trans configuration and the X-prolyl peptide will not adopt the intended configuration spontaneously. This isomerization property is common to all proteins in the immunophilins family such as the parvulins and the FK-506-binding proteins.

First mention of PPIases was in 1984 when Fisher G. and colleagues reported and characterized the first protein with such activity2. It was later shown that this isomerization process is an intrinsically slow reaction, typically occurring on the time scale of several minutes under physiological conditions3. No surprise that this crucial step was considered to be a rate-limiting step in protein folding and cyclophilins accelerate the isomerization raising its interest in the cell biology4. This also identifies this protein as a chaperone or foldase in many processes1. In the same report authors describe an assay to measure prolyl isomerase activity based on the activity of chymotrypsin. This proteolytic enzyme has high substrate specificity for the four-residue peptide Ala-Ala-Pro-Phe only when the proline peptide bond is in the trans state. By addition of chymotrypsin to a solution that contains a peptide with this sequence results in the rapid cleavage of about 90% of the peptides, while peptides with cis proline bonds (normally in lower levels, around 10%) are cleaved at a rate limited by uncatalyzed proline isomerization. The addition of a potential prolyl isomerase will accelerate this latter reaction phase if it has true prolyl isomerase activity2. This assay was developed at 10 °C and and the activity was measured in a spectrophotometer at 390nm wavelength.

In the same year Handschumacher and colleagues purified and described the first cyclophylin A (CypA) from bovine thymocytes as an intracellular protein with a high affinity for the immunosuppressive drug cyclosporine A5 (CsA) (Figure 2). Cyclosporins were launched by Sandoz (now Novartis) in 1983 and they are a group of cyclic undecapeptides isolated from several fungi species which includes the most common strain Tolypocladium inflatum. CsA is the most studied form and it is highly used to prevent immune response and organ rejection after a transplant6.

CsA binding to cyclophilin in the PPIase active site inhibits its ability to perform cis- trans- isomerization which is not related to the immunosuppression effect. In fact, this complex when formed binds to calcienurin, Ca2+/calmodulin dependent serine/threonine phosphatase (PP2B), and restrains its phosphatase activity. This event turns off the transcription activity of nuclear factor of activated T cell, leading to T cell activation suppression and cardiac hypertrophy 7.

It was only five years later that it was understood that these two proteins were in fact the same and this immunosuppressant effect was determined to be unrelated to the intrinsic isomerase activity8,9. Besides this immunosuppression and their function in protein evolution, they also stabilize or destabilize protein-protein complexes (calcium channels, steroid receptors and tyrosine kinase receptors)10. They have also been shown to be involved in a variety of pathophysiological processes including inflammation and vascular dysfunction, wound healing, innate immunity to HIV, hepatitis C infection, host-parasite interactions and tumor biology and diverse signaling pathways including mitochondrial apoptosis, RNA splicing and adaptive immunity11,12.

Overall, there are seven major cyclophilins in humans: hsCypA, hsCypB, hsCypC, hsCypD, hsCypE, hsCyp40 and hsCypNK; and a total of 16 unique proteins with hsCypA representing the prototype of the family. Furthermore, Drosophila has at least 9 cyclophilins and the plant Arabidopsis thaliana has 29 putative cyclophilins while 8 could be found in Saccharomyces cerevisae1,12. These numbers elucidate the extended diversity that can be found in cyclophilins.

Although over the years scientific researches pointed and proved the involvement of cyclophilins in several critical process that may lead to the development of numerous diseases, the function and role in each specific disease remains obscure and more research is needed to develop more therapies.

In particular, cyclophillin D is the mitochondrial isoform of the enzyme and it is a key regulator of the mitochondrial permeability transition pore (MPTP). Mitochondria are small organelles essential to cell metabolism and survival and play a vital role in cellular bioenergetics, calcium homeostasis among several other metabolic activities. They have been implicated to play an essential role in neuronal cell survival or death after central nervous system injury because they are regulators of both energy metabolism and apoptotic pathways13. Electron microscopic studies by George Palade revealed that mitochondria have two membrane systems: an outer membrane and an extensive, highly folded inner membrane14 (Figure 2).