Skip to main content
NCBI home page
International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 1999 Dec;80(6):305–315. doi: 10.1046/j.1365-2613.1999.00128.x

Cyclophilins and their possible role in the stress response

Larisa Andreeva *, Richard Heads †, Colin J Green *
PMCID: PMC2517841  PMID: 10632780

Abstract

Cyclophilins are proteins which are remarkably conserved through evolution; moreover they have been found in every possible existing organism, which indicates their fundamental importance. Due to their enzymatic properties, multiplicity, cellular localization and role in protein folding they belong to the group of proteins termed molecular chaperones. All the proteins of the cyclophilin family possess enzymatic peptidyl-prolyl isomerase activity (PPI-ase), which is essential to protein folding in vivo. Recently PPI-ase activity was suggested as playing a role in regulation of transcription and differentiation. However, not all cyclophilin functions are explained by PPI-ase activity. For instance, one of the cyclophilins plays a regulatory role in the heat shock response and the mitochondrial cyclophilin (Cyclophilin D) is an integral part of the mitochondrial permeability transition complex, which is regarded as having a crucial role in mechanisms of cell death. In support of a role in the stress response, the expression of certain cyclophilins has recently been shown to be up-regulated under various stressful conditions. Current evidence of functional involvement of cyclophilins in various intracellular pathways is reviewed along with the indications that cyclophilin D (Cyp D) represents a crucial part of the mitochondrial permeability transition pore, which is detrimental in apoptotic and necrotic cell death. This review does not attempt to cover all the existing information related to cyclophilin family of proteins, but focus on the existing evidence of the involvement of these proteins in the intracellular stress response.

Keywords: cyclophilins, stress response, molecular chaperones, protein folding, mitochondria, calcineurin

Cyclophilins and peptidyl-prolyl isomerases

Cyclophilins were first discovered and described in 1984. Firstly, an 18 kD protein was sequenced as the cellular receptor for the newly discovered potent immunosuppressive drug cyclosporin A (CsA) (Handschumacher et al. 1984). In the same year cyclopilin was described as a protein possessing the specific enzymatic property of catalysing cis-trans isomerization of peptidyl-prolyl bonds and was named peptidyl-prolyl- cis-trans isomerase (PPI-ase) (Fischer et al. 1984). Since then a substantial number of proteins possessing this enzymatic activity have been described. They are now organized into three major groups: cyclophilins, FK-binding proteins (FKBPs)-(proteins, which are receptors for the immunosuppressive drug FK506) and parvulins (Galat & Metcalfe 1995; Kops et al. 1998).

The most abundant cyclophilin is cyclophilin A (CypA), isolated from both yeast and human cytosol, sharing about 80% sequence identity (Galat & Metcalfe 1995). The main sequence of human CypA also has a high degree of sequence homology with human cyclophilin B (CypB), cyclophilin C (CypC) and CypD. CypB (human) and CypC (murine) are endoplasmic reticulum localized cyclophilins and they have the N-terminal sequences which target them to this intracellular localization (Bergsma et al. 1991). CypD is the mitochondrial cyclophilin. Another cyclophilin of 40 kD, termed Cyp40, has been isolated from bovine brain cytosol and also shares sequence similarity with CypA (Kieffer et al. 1992); interestingly, it also has sequence similarity to human FKBP52 (Kieffer et al. 1993), which is a member of the second family of PPI-ases. It is possible that Cyp40, CypA, FKBP52 and FKBP25 have dual localization in the cytoplasm and in the nucleus (Dolinski et al. 1997). For a more detailed classification of the different cyclophilins the reader is urged to refer to other reviews (Galat 1993; Galat & Metcalfe 1995).

Molecular chaperones and cyclophilins

Cyclophilins are remarkably conserved proteins — and this applies equally to the aminoacid sequence and to their three dimensional structure. Due to their enzymatic properties, multiplicity, cellular localization and role in protein folding, cyclophilins belong to a diverse set of proteins which are termed molecular chaperones. Two main types of molecular chaperones have been defined at present: firstly, molecular chaperones which are functionally responsible for the correct folding, assembly and transport of newly synthesized proteins in the cell; and secondly, molecular chaperones with enzymatic properties accountable for acceleration of the rate limiting steps of protein folding (Gething & Sambrook 1992) such as disulphide bond formation and prolyl-isomerization. The remarkable conservation of these proteins throughout evolution is suggestive of an essential functional role for cyclophilins. Currently, our understanding of the process of intracellular acquisition of final protein structure has become much more intricate. The view of molecular chaperones has also changed. The term assembly is used in a broader sense to define not only the folding of newly synthesized polypeptide chains, but also the degree of either folding or association that may occur when proteins cross intracellular membranes and perform their functions (Gething & Sambrook 1992). Since cyclophilins catalyse the cis-trans isomerasation of peptidyl prolyl bonds of certain proteins, they act as acceleration factors in protein folding and assembly. In protein peptide bonds the trans state is favoured at least 100 times over cis, which means that in unfolded proteins the trans- cis isomerization is extremely slow and is considered to be a rate limiting step in the folding of proteins with cis peptide bonds in their final conformation (Herzberg & Moult 1991). The first direct indication for the role of prolyl-isomerization in the protein folding process came from investigations of collagen folding in vitro. Steinmann et al. (1991) demonstrated that addition of the immunosuppressant drug cyclosporin A (CsA), blocked the cis-trans isomerase activity of cyclophilin and significantly delayed maturation of collagen. The role of cyclophilin enzymatic activity in protein folding was subsequently confirmed in a similar study using rabbit reticulocyte lysate (Kruse et al. 1995).

Heat shock proteins are molecular chaperones involved in folding pathways: their co-operation with cyclophilins

Since cyclophilins have recently been identified as stress-inducible proteins, it is likely that their role in the stress response is an extension of their molecular chaperoning properties under normal conditions and their interaction with heat shock proteins (hsps). All proteins which are synthesized or up-regulated in cells in response to insults such as thermal stress, ultraviolet irradiation, changes in pH of cell environment, treatments with oxidants are classified as hsps. Environmental stresses are generally ‘proteotoxic’ causing unfolding, misfolding or aggregation of intracellular proteins. Hence, damaged proteins within the cells promote a heat shock transcriptional response and increased synthesis of hsps. Increased levels of hsps help cells to overcome the consequences of the stress damage, aid recovery and protect against further stress. The same set of hsps are significantly elevated in disease states such as cancer, fever, ischaemia, and oxidative injury. The protective properties of hsps in the context of the cellular stress response are seen as an increase in molecular chaperone function (Craig et al. 1993; Parsell & Lindquist 1993)

Some molecular chaperones may interact with specific targets in the cell. For instance, the process of maturation of the progesterone receptor complex has been studied in reticulocyte lysate (Jakob et al. 1995). The complex folding pathway of the receptor involves nine different proteins hsp90, hsp70, Hip, p60 and three peptidyl-prolyl cis-trans isomerases-namely FKBP 52, FKBP 51 and cyclophilin 40 (Cyp 40). All these proteins, which dissociate finally from the activated receptor upon hormone binding, play an important role in achieving active conformation of the receptor (Freeman et al. 1996). Examination of the role of Cyp40 in this folding pathway proved a functional similarity and co-operation with hsp90 and hsp70 in their ability to assist folding to a native proteolysis-resistant state, folding competency and solubility (Bose et al. 1996). Cyp40 is capable of maintaining the protein in a folding competent state with an efficiency comparable to that of hsp90. Interestingly, the molecular chaperoning activity of Cyp40 was not suppressed by CsA, which indicates that it may be separate from its peptidyl-prolyl cis-trans isomerase activity (Freeman et al. 1996). Similar results were obtained by Bose and colleagues (Bose et al. 1996), who showed that another immunophilin-(FKBP) prevents citrate synthase aggregation in vitro, and CsA had no effect. Therefore the functional role of cyclophilins is not solely attributable to their PPI-ase activity. Although it is clear that Cyp40 has molecular chaperone properties, its role in different protein maturation pathways and its enzymatic properties require further analysis in vivo. Johnson & Toft (1994) precipitated a complex of hsp90, Cyp40 and p23. Formation of this trimeric complex is Mg2++/ATP dependent and the number of these complexes was not different in tissues with varying numbers of steroid receptors, which indicates that the cyclophilin/hsp90 complex may form part of a uniform intracellular protein folding machinery (Johnson & Toft 1994).

Protein import to the mitochondria

Cyclophilins are essential for the process of protein import to mitochondria. Eukaryotic cells synthesize many proteins on the cytosolic ribosomes and import them into mitochondria. Several major molecular chaperones of the cytosol and mitochondria are involved in the folding, import and processing of the newly synthesized proteins into mitochondria. It has been found that cyclophilin 20 (Cyp 20) in Neurospora crassa is functionally linked with the molecular chaperones hsp70, hsp60 and hsp10 (or cpn10) (Rassow et al. 1995). Hsp 70 is the main component of the chaperone machinery in mammalian cells and associates with nascent polypeptides on ribosomes controlling folding and assembly of newly synthesized proteins. Besides the regulation of polypeptide folding, another important intracellular function of the cytosolic hsp70 is to transport newly synthesized proteins between intracellular compartments (Figure 1).

Figure 1.

Figure 1

Open in a new tab

Schematic representation of the mitochondrial chaperone complex. Cytosolic heat shock protein (c-hsp70); mitochondrial heat shock protein 70 (m-hsp70), mitochondrial heat shock protein 60 (m-hsp 60), mitochondrial chaperone 10 (− cpn 10).

A mitochondrial homologue of hsp70 (mhsp70) is the 75 kD chaperoning ATP-ase, which is encoded by a special heat shock gene in the nucleus (Bhattacharyya et al. 1995). This hsp is localized in the mitochondrial matrix where it is essential for the processes of import folding and assembly of mitochondrial proteins (Langer et al. 1994). The functioning activity of mhsp70 is linked with the activity of two other members of the mitochondrial chaperoning machinery — cytoplasmic hsp70 and mitochondrial hsp60 (mhsp 60) (Langer & Neupert 1994; Kabakov & Gabai 1997). (see Figure 1).

In eukaryotic cells hsp 60 is a nuclear gene product which is synthesized in cytoplasm and then translocated into the mitochondria. In mitochondrial matrix hsp 60 forms a ring-like oligomeric structure of seven subunits and possesses evident chaperoning properties (Langer & Neupert 1994; Kabakov & Gabai 1997). It has been shown that expression of this chaperone is enchanced by thermal shock; moreover ischaemic injury is a potent stimulator of mhsp 60 in cardiomyocytes (Marber et al. 1993). Mestril and colleagues demonstrated that coexpression of hsp 60 with another mitochondrial chaperone, cpn10, protected against simulated ischaemia (Lau et al. 1997), proving that protection was dependent on co-operativity between more than one mitochondrial chaperone protein. The function of the eukaryotic hsp 60 under normal conditions is ATP dependent refolding of proteins imported into the mitochondria and folding of proteins which are synthesized on intramitochondrial ribosomes. These chaperoning actions of hsp60 are only possible in co-operation with mitochondrial hsp70 and hsp10 (Langer & Neupert 1994) as well as with one of the cyclophilins — e.g. Cyp 20 is considered an essential component of the intramitochondrial protein folding machinery. Interestingly, it has been shown that Cyp A is capable of protecting cardiomyocytes against oxidative stress induced by t-butyl-hydroperoxide (Doyle et al. 1999).

Mitochondrial permeability transition

It is now accepted that the lesion responsible for the collapse of mitochondrial membrane potential during cell injury such as ischaemia-reperfusion damage or programmed cell death is a CsA sensitive mitochondrial permeability transition pore (MPTP). Crompton and colleagues using pulse flow techniques determined the mitochondrial transition pore size at about 1.0–1.7 (Crompton & Costi 1988). Molecular components of the MPTP are not fully characterized, but it is now evident that it represents a dynamic multiprotein complex located at the contact site between the inner and outer mitochondrial membranes, which is crucial for coordination between the mitochondrial intermembrane space and cellular matrix (Marzo et al. 1998) (Figure 2).

Figure 2.

Figure 2

Open in a new tab

Representation of the CsA sensitive mitochondrial permeability transition pore (MPTP). CsA binds to the cyclophilin in the mitochondrial membrane in an ADP dependent manner. The exact composition of the MPTP is not fully known, but it is thought to involve proteins from the cytosol including voltage dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), mitochondrial cyclophilin D, the antiapoptotic proteins BCL-2, proapoptotic protein Bax, and hexokinase. Evidence exists for the involvement of these proapoptotic and antiapoptotic proteins in the formation of the MPTP complex in addition to the previously described proteins such as ANT, VDAC, cyclophilin D and hexokinase.

This pore is currently implicated in all types of cell death. The involvement of several proteins in MPTP has been reported including voltage-dependent outer membrane anion channels (VDAC), adenine nucleotide translocator (ANT) and hexokinase (Marzo et al. 1998; Woodfield et al. 1998). CsA has been described as a potent and selective inhibitor of the MPTP (Haworth & Hunter 1980), acting by binding to a unique matrix cyclophilin (Halestrap & Davidson 1990). Exploiting the synergism of the inhibitory action of CsA and ADP on the mitochondrial pore, the main receptor for CsA in heart mitochondria was identified as cyclophilin localized in the inner mitochondrial membrane (Andreeva & Crompton 1994). The aminoacid sequence confirmed significant similarity with the human mitochondrial CypD (Tanveer et al. 1996). Cloning of a rat CypD homologue supported this finding (Woodfield et al. 1998). It has been shown that Cyp D binds to inner mitochondrial membrane in a CsA sensitive manner; but whether the PPI-ase activity is essential for the conformational and structural changes involving the pore components or whether its role in mitochondrial protein folding and transportation is crucial for pore regulation is not yet determined. However, it seems likely that PPI-ase enzymatic activity is necessary for protein conformational changes associated with MPT pore formation. The regulatory role of the antiapoptotic proteins Bcl-2 and Bcl-Xl in stabilizing the membrane potential has been reported (Kajstura et al. 1996; Okuno et al. 1998). The pro-apototic protein Bax (another member of the Bcl-2 family) has been described as a protein disrupting the mitochondrial potential (Xiang et al. 1996). Hence there is some evidence for the involvement of these pro-apoptotic and antiapoptotic proteins in the formation of the MPTP complex in addition to the previously described proteins such as ANT, VDAC, cyclophilin D and hexokinase.

Peptidyl-prolyl-isomerase activity and transcription factor regulation

Regulation of the heat shock response by heat shock transcription factor (HSF) is based on the understanding that the levels of active HSF are proportional to the subsequent synthesis of hsps. In the stressed cell the level of unfolded, partially aggregated proteins increases and the need for hsp70 and other molecular chaperones which are necessary for correct folding or preventing aggregation rises greatly. HSF, which is maintained in the cytosol as an active complex with a number of chaperones, is released from its inactive state and triggers the HSF monomer conversion into a trimer and translocation to the nucleus, where it binds in the promoters of the heat shock genes (Abravaya et al. 1991). As the number of unfolded proteins is reduced as a consequence of increased hsps expression, the HSF reassociates with proteins such as hsp70 which are thought to cause dissociation of the trimer, exit from the nucleus and return to an inactive conformation in the monomeric state in the cytosol (Morimoto et al. 1997). Until recently, the association of hsp70 with HSF was consistent with this model. However, lately hsp90 has been identified as a possible key component regulating the activity of HSF. In addition, both hsp90 and hsp70 were described as specific ligands of the heat inducible PPI-ase FKBP59 (Meshinchi et al. 1990; Czar et al. 1994). Hoffmann and Handshumacher reported that hsp90 is closely associated with Cyp40 (Hoffmann & Handschumacher 1995). Moreover, this complex formation is not dependent upon PPI-ase enzymatic activity, as CsA has no effect on it. Recent genetic studies in vivo using Saccharomyces cerevisiae demonstrated that mutations reducing the level of hsp90 or eliminating Cyp40 caused the activation of the HSF. These experiments indicated that hsp90 and Cpr7 (a yeast analogue of Cyp40) function synergistically to repress gene expression from HSF-dependent promoters. Essentially these proteins are required for negative regulation of the heat shock response in yeast (Duina et al. 1998). Furthermore, it has been shown that DNA binding activity of the c-Myb protooncogene transcription factor is negatively regulated by Cyp40 (Leverson & Ness 1998) and that DNA-binding activity of c-Myb is dependent on its PPI-ase activity, hence implicating this enzymatic activity in the regulation of transcription, transformation and differentiation. Addition of recombinant Cyp40 to nuclear extracts eliminated the DNA binding activity of c-Myb. Addition of CsA, in turn, restores the DNA-binding activity of this protooncogene by neutralizing the N-terminal PPI-ase domain of Cyp40 (Leverson & Ness 1998).

Cyclophins and their nuclease activity

Cyclophilins have two different active sites in spite of their relatively small molecular size. One site is responsible for their peptidyl-prolyl cis-trans isomerize activity, as discussed above and the other active site is capable of catalytic degradation of DNA in a calcium/magnesium-dependent manner (Montague et al. 1994). This nuclease activity of cyclophilins is similar to the activity of apoptotic endonucleases. Cyclophilins are capable of degrading single stranded, double stranded and supercoiled DNA. Both Mg++ and of Ca++ stimulate this property of cyclophilins and it has been demonstrated that Mg++ alone is sufficient for CypC nuclease activity. A combination of Mg++ and Ca++ is optimal for CypA and CypB activity in this regard (Montague et al. 1997). This data strongly indicates that cyclophilins might be involved in apoptotic genome degradation, exercising their nuclease properties. Cyclophilin involvement in apoptosis as part of the MPT has been investigated in endothelial cells; the addition of CsA prevented apoptosis by blocking the opening of the MTP, thus stabilizing the mitochondria and preventing the release of mitochondrial cytochrome c into the cytosol (Walter et al. 1998). In turn, cytochrome c release has been shown to be a trigger of apoptotic cysteine protease (caspase) cascade activation (Kroemer et al. 1995; Li et al. 1997). Whether the antiapoptotic effect of CsA is due solely to the prevention of the MTP opening, or CsA is also effective in decreasing the nuclease activity of the cyclophilins should be further explored.

Preconditioning, the heat shock response and a possible role for cyclophilins

Several PPI-ases have recently been identified as heat inducible proteins in widely divergent species (Sykes et al. 1993). As mentioned above, it is likely that the role of the cyclophilins in the stress response is an extension of their role as molecular chaperones, in a similar manner to hsps. It has been reported that two PPI-ases from Saccharomyces cervisae, one located in the cytosol and one in the endoplasmic reticulum (Cyp1 and Cyp2, respectively), are heat inducible and the presence of at least one of them is necessary for maximal survival of the yeast cell after heat shock (Sykes et al. 1993). In our studies cyclophilins were reported to be essential for growth and viability of a bacterium under starvation conditions (Gothel et al. 1998). Cyclophilins were induced by heat shock and hypoxia-reoxygenation in cardiac derived cultured myogenic cells in vitro. (Andreeva et al. 1997). As we described earlier cyclophilins form complexes with and act as coregulatory subunits of various heat shock proteins such as hsp70 and hsp90 (Rassow et al. 1995; Johnson & Craig 1997) suggesting a potential role in protection provided by these proteins during ischaemia-reperfusion injury. The 40 kD cytosolic cyclophilin (Cyp40) has been shown to be a component of inactive steroid receptors along with hsp70 and hsp90 (Zou et al. 1998). Cyclophilins are also induced by nitric oxide (NO) donors such as S-nitroso-N-acetyl penicillamine (SNAP) and this induction is associated with cytoprotection 18–24 h following SNAP treatment (unpublished observation). As discussed above, more specific functions of certain cyclophilins are suggested by associations with macromolecular complexes such as MPTP and steroid receptors.

The phenomenon of ischaemic preconditioning is based on the observation that an adaptive response to a mild, brief ischaemic stress in the heart delays the onset of tissue necrosis following a prolonged episode of myocardial ischaemia. Also, an initial study by Currie's group demonstrated that sublethal, whole body heat stress protects the isolated, Langendorff perfused heart against ischaemia (Knowlton et al. 1998). Since then several studies have described the inverse relationship between the expression of major heat shock proteins in heart and the severity of injury resulting from coronary occlusion. Hsp70 has been described as having a prominent role in this form of adaptation and it was shown to be elevated by brief, intermittent ischaemia and reperfusion which caused delayed (or ‘second window’) preconditioning (Marber et al. 1993; Heads et al. 1995). A cytoprotective function of the hsps in the ischaemic heart has been proposed to result from induction of hsp70 and other stress proteins expression during or following ischaemia and apparent analogy with the thermotolerance induced by prior heat shock. More direct information on the protective mechanism of hsp70 is derived from overexpression studies in transfected cell lines or transgenic models (Marber et al. 1995; Brar et al. 1999). Although it is very likely that heat shock proteins play a role in preconditioning, the response to myocardial ischaemia is rather complex as a result of the number of antioxidant genes and other factors which are transcriptionally activated following ischaemia including immediate early genes and cyclophilins. Nevertheless, heat stress preconditioning has been shown to improve respiratory function in isolated mitochondria from rabbit hearts (Yellon et al. 1992). In addition, mitochondria were proposed as the target for induced thermotolerance (Polla et al. 1996), since hsps synthesis prevented impairment of ATP synthase activity at elevated temperatures (Patriarca & Maresca 1990). Therefore, mitochondria may represent a common target for thermotolerance and ischaemic preconditioning.

Role of cyclophilins, FKBPs and calcineurin in cell death and survival

The calcium-calmodulin dependent phosphatase calcineurin (protein phosphatase 2B or PP2B) also shows interaction with both cyclophilins and FKBPs, since calcineurin is the common target for the immunosuppressant action of both structurally unrelated CsA and FK506 which, in this case, bind to CypA and FKBP12, respectively. However it has been established that cis-trans isomerase activity plays no role in immunosuppresion (Figure 3).

Figure 3.

Figure 3

Open in a new tab

The immunosuppressive effects of CsA and FK 506 originate from the binding of the drug-immunophilin complex to molecules of calcineurin (CN) and subsequent interaction with calmodulin (CAM). De-phosphorylation of activated T-cells class of transcription factors (NFAT) inhibits their translocation to the cell nucleus and subsequent activation of interleukin-2 (IL-2) expression. Activation of calcineurin leads to induction of nitric oxide synthase (NOS) by a dephosphorylation mechanism, overproduction of nitric oxide (NO) and cell death.

The basis of the immunosuppressive effects originates from the binding of the drug-immunophilin complex to calcineurin and inhibition of its phosphatase activity. This inhibits activation of the nuclear factor of activated T-cells (NF-AT) class of trancription factors by de-phosphorylation, thus inhibiting their translocation to the nucleus and subsequent activation of interleukin-2 expression (Molkentin et al. 1998). Interestingly, NF-AT3 has recently been implicated in the development of cardiac hypertrophy, which is also blocked by FK506 (Molkentin et al. 1998) (Figure 4).

Figure 4.

Figure 4

Open in a new tab

Representation of the IP3 receptor which regulates calcium release from endoplasmic reticulum. FKBP anchors calcineurin to the IP3 receptor complexes and calcium flux is regulated by a mechanism involving calcineurin phosphatase activity which is disrupted by FK 506.

FKBP12 has been described as a component of the striated muscle ryanodine receptor (RyR) and the IP3 receptor which regulate calcium release from the sarcoplasmic reticulum and endoplasmic reticulum, respectively. FK506 is thus able to modulate calcium flux. Whilst this was originally thought to be independent of calcineurin, it has recently been shown that, FKBP12 anchors calcineurin to the RyR and IP3-R complexes (Cameron et al. 1997) and that calcium flux may be regulated by a mechanism involving calcineurin phosphatase activity which is disrupted by FK506 or rapamycin (which also binds and dissociates FKBP12 from the channels) (Brillantes et al. 1994; Cameron et al. 1995). Calcineurin has recently been found to play an important role in cell death and survival. These functions, whilst probably not dependent on PPI-ase activity per se, are still dependent on its interaction with CypA and FKBP12. For instance, activation of calcineurin and changes in mitochondrial function play a role in N-methyl-d-aspartate (NMDA) receptor mediated neurotoxicity (Ankarcrona et al. 1996). Both CsA and FK506 prevented both acute and delayed NMDA-induced cell death and CsA also prevented collapse of the mitochondrial transmembrane potential (ψm). Activation of calcineurin by elevated [Ca++]i leads to the induction of nitric oxide synthase (NOS) by a dephosphorylation mechanism (Dawson et al. 1993), overproduction of NO and cell death. This is also blocked by FK506. It has been shown that FK506 protects isolated cardiac myocytes against simulated ischaemia (Cumming et al. 1996). Whilst most evidence suggests that PPI-ase activity is not involved in regulation of calcineurin function, it is important to point out that there is also evidence to suggest that PPI-ase activity may itself play a role in cell survival as discussed above. In addition, nonimmunosuppressive FK506 analogues which retain their ability to inhibit PPI-ase activity but do not inhibit calcineurin have been shown to be both neuroprotective and neurotrophic (Snyder et al. 1998). However, the cellular mechanism of these neuroprotective effects remains unclear, since the levels of FKBP12 and CypA in cells are higher (micromolar) than the low (picomolar) effective concentrations of the drugs; it is should also be noted that CsA undergoes cis-trans isomerization about particular bonds upon binding its receptors -cyclophilins and this action results in significant conformational and structural changes at the surfaces of these complexes. It is conceivable that the drug-immunophilin complexes bind another, as yet unidentified protein target within the cell. This possibility should be explored alongside other possibilities, which might account for the intracellular role of cyclophilins, their functional diversity and remarkable conservation.

Concluding remarks

Cyclophilins are responsible for a crucial step in protein folding pathways. In vivo these versatile and ubiquitous expressed proteins act in concert with major chaperones to regulate interactions with peptide substrates and hence ATP activity. The overall abundance of these constitutively expressed proteins and their enzymatic properties allow us to propose the involvement of PPI-ase activity in the refolding of cellular proteins denatured as a result of ATP depletion or oxidative stress. Another possible explanation for the functional association between cyclophilins and major hsps under conditions of stress is their involvement in the structural transformation leading to the opening of mitochondrial permeability transition pore, with the subsequent dissipation of mitochondrial potential. The highlighted studies suggest multiple possible functions for cyclophilins in conditions of stress and indicate the necessity for further molecular and biochemical investigations for full comprehension of their intracellular role.

Acknowledgments

We would like to thank Dr Su Metcalfe for her constructive criticism and extremely useful comments. We wish also to express gratitude to all our colleagues who are involved in this and associated work carried out at Northwick Park Institute for Medical Research including Eleanor Benton, Najeem Folarin, Rekha Bassi, Padmini Sarathchandra, Roberta Foresti, Harprakash Kaur, Roberto Motterlini. This work was supported by grants from the British Heart Foundation and from the National Heart Research Fund.

References

  1. Abravaya K, Philips B, Morimoto RI. Heat shock–induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting. Mol. Cell Biol. 1991;11:586–592. doi: 10.1128/mcb.11.1.586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andreeva L, Crompton M. An ADP-sensitive cyclosporin-A-binding protein in rat liver mitochondria. Eur. J. Biochem. 1994;221:261–268. doi: 10.1111/j.1432-1033.1994.tb18737.x. [DOI] [PubMed] [Google Scholar]
  3. Andreeva L, Motterlini R, Green CJ. Cyclophilins are induced by hypoxia and heat stress in myogenic cells. Biochem. Biophys. Res. Com. 1997;237:6–9. doi: 10.1006/bbrc.1997.7067. [DOI] [PubMed] [Google Scholar]
  4. Ankarcrona M, Dypbukt JM, Orrenius S, Nicotera P. Calcineurin and mitochondrial function in glutamate-induced neuronal cell death. FEBS Lett. 1996;394:321–324. doi: 10.1016/0014-5793(96)00959-3. [DOI] [PubMed] [Google Scholar]
  5. Bergsma DJ, Eder C, Gross M, et al. The cyclophilin multigene family of peptidyl-prolyl isomerases. Characterization of three separate human isoforms. J. Biol. Chem. 1991;266:23204–23214. [PubMed] [Google Scholar]
  6. Bhattacharyya T, Karnezis AN, Murphy SP, et al. Cloning and subcellular localization of human mitochondrial hsp70. J. Biol. Chem. 1995;270:1705–1710. doi: 10.1074/jbc.270.4.1705. [DOI] [PubMed] [Google Scholar]
  7. Bose S, Weikl T, Bugl H, Buchner J. Chaperone function of Hsp90-associated proteins. Science. 1996;274:1715–1717. doi: 10.1126/science.274.5293.1715. [DOI] [PubMed] [Google Scholar]
  8. Brar BK, Stephanou A, Wagstaff MJ, et al. Heat shock proteins delivered with a virus vector can protect cardiac cells against apoptosis as well as against thermal or hypoxic stress. J. Mol. Cell Cardiol. 1999;31:135–146. doi: 10.1006/jmcc.1998.0857. [DOI] [PubMed] [Google Scholar]
  9. Brillantes AB, Ondrias K, Scott A, et al. Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell. 1994;77:513–523. doi: 10.1016/0092-8674(94)90214-3. [DOI] [PubMed] [Google Scholar]
  10. Cameron AM, Nucifora FCJ, Fung ET, et al. FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine- proline (1400–1401) and anchors calcineurin to this FK506-like domain. J. Biol. Chem. 1997;272:27582–27588. doi: 10.1074/jbc.272.44.27582. [DOI] [PubMed] [Google Scholar]
  11. Cameron AM, Steiner JP, Roskams AJ, Ali SM, Ronnett GV, Snyder SH. Calcineurin associated with the inositol 1,4,5-trisphosphate receptor- FKBP12 complex modulates Ca2+ flux. Cell. 1995;83:463–472. doi: 10.1016/0092-8674(95)90124-8. [DOI] [PubMed] [Google Scholar]
  12. Craig EA, Gambill BD, Nelson RJ. Heat shock proteins: molecular chaperones of protein biogenesis. Microbiol. Rev. 1993;57:402–414. doi: 10.1128/mr.57.2.402-414.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crompton M, Costi A. Kinetic evidence for a heart mitochondrial pore activated by Ca2+, inorganic phosphate and oxidative stress. Europ. J. Biochem. 1988;178:489–501. doi: 10.1111/j.1432-1033.1988.tb14475.x. [DOI] [PubMed] [Google Scholar]
  14. Cumming DV, Heads RJ, Coffin RS, Yellon DM, Latchman DS. Pharmacological preconditioning of primary rat cardiac myocytes by FK506. Basic Res. Cardiol. 1996;91:367–373. doi: 10.1007/BF00788716. [DOI] [PubMed] [Google Scholar]
  15. Czar MJ, Owens-Grillo JK, Dittmar KD, et al. Characterization of the protein–protein interactions determining the heat shock protein (hsp90.hsp70.hsp56) heterocomplex. J. Biol. Chem. 1994;269:11155–11161. [PubMed] [Google Scholar]
  16. Dawson TM, Steiner JP, Dawson VL, Dinerman JL, Uhl GR, Snyder SH. Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc. Natl. Acad. Sci. USA. 1993;90:9808–9812. doi: 10.1073/pnas.90.21.9808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dolinski K, Scholz C, Muir RS, et al. Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae. Mol. Biol. Cell. 1997;8:2267–2280. doi: 10.1091/mbc.8.11.2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Doyle V, Virji S, Crompton M. Evidence that cyclophilin-A protects cells against oxidative stress. Biochem. J. 1999;341:127–132. [PMC free article] [PubMed] [Google Scholar]
  19. Duina AA, Kalton HM, Gaber RF. Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response. J. Biol. Chem. 1998;273:18974–18978. doi: 10.1074/jbc.273.30.18974. [DOI] [PubMed] [Google Scholar]
  20. Fischer G, Bang H, Mech C. Determination of enzymatic catalysis for the cis-trans-isomerization of peptide binding in proline-containing peptides] Biomed. Biochim. Acta. 1984;43:1101–1111. [PubMed] [Google Scholar]
  21. Freeman BC, Toft DO, Morimoto RI. Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23. Science. 1996;274:1718–1720. doi: 10.1126/science.274.5293.1718. [DOI] [PubMed] [Google Scholar]
  22. Galat A. Peptidylproline cis-trans isomerases — immunophilins. Eur. J. Biochem. 1993;216:689–707. doi: 10.1111/j.1432-1033.1993.tb18189.x. [DOI] [PubMed] [Google Scholar]
  23. Galat A, Metcalfe SM. Peptidylproline cis/trans isomerases. Prog. Biophys. Mol. Biol. 1995;63:67–118. doi: 10.1016/0079-6107(94)00009-x. [DOI] [PubMed] [Google Scholar]
  24. Gething MJ, Sambrook J. Protein folding in the cell. Nature. 1992;355:33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  25. Gothel SF, Scholz C, Schmid FX, Marahiel MA. Cyclophilin and trigger factor from Bacillus subtilis catalyze in vitro protein folding and are necessary for viability under starvation conditions. Biochemistry. 1998;37:13392–13399. doi: 10.1021/bi981253w. [DOI] [PubMed] [Google Scholar]
  26. Halestrap AP, Davidson AM. Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem. J. 1990;268:153–160. doi: 10.1042/bj2680153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science. 1984;226:544–547. doi: 10.1126/science.6238408. [DOI] [PubMed] [Google Scholar]
  28. Haworth RA, Hunter DR. Allosteric inhibition of the Ca2+-activated hydrophilic channel of the mitochondrial inner membrane by nucleotides. J. Membr. Biol. 1980;54:231–236. doi: 10.1007/BF01870239. [DOI] [PubMed] [Google Scholar]
  29. Heads RJ, Latchman DS, Yellon DM. Differential stress protein mRNA expression during early ischaemic preconditioning in the rabbit heart and its relationship to adenosine receptor function. J. Mol. Cell Cardiol. 1995;27:2133–2148. doi: 10.1016/s0022-2828(95)91299-1. [DOI] [PubMed] [Google Scholar]
  30. Herzberg O, Moult J. Analysis of the steric strain in the polypeptide backbone of protein molecules. Proteins. 1991;11:223–229. doi: 10.1002/prot.340110307. [DOI] [PubMed] [Google Scholar]
  31. Hoffmann K, Handschumacher RE. Cyclophilin-40: evidence for a dimeric complex with hsp90. Biochem. J. 1995;307(1):5–8. doi: 10.1042/bj3070005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jakob U, Lilie H, Meyer I, Buchner J. Transient interaction of hsp90 with early unfolding intermediates of citrate synthase — inplications for heat shock in vivo. J. Biol. Chem. 1995;270:7288–7294. doi: 10.1074/jbc.270.13.7288. [DOI] [PubMed] [Google Scholar]
  33. Johnson JL, Craig EA. Protein folding in vivo: Unraveling complex pathways. Cell. 1997;90:201–204. doi: 10.1016/s0092-8674(00)80327-x. [DOI] [PubMed] [Google Scholar]
  34. Johnson JL, Toft DO. A novel chaperone complex for steroid-receptors involving heat shock proteins, immunophilins and p23. J. Biol. Chem. 1994;269:24989–24993. [PubMed] [Google Scholar]
  35. Kabakov AE, Gabai V. Heat Shock Proteins and Cytoprotection: ATP-deprived Mammalian Cells, Landes Bioscience. Heidelberg: Springer-Verlag; 1997. Heat shock proteins and the regulation of heat shock gene expression in eukaryotes; pp. 1–13. [Google Scholar]
  36. Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necrotic cell deaths are independent contributing variables of infarct size in rats. Lab. Invest. 1996;74:86–107. [PubMed] [Google Scholar]
  37. Kieffer LJ, Thalhammer T, Handschumacher RE. Isolation and characterization of a 40-kDa cyclophilin-related protein. J. Biol. Chem. 1992;267:5503–5507. [PubMed] [Google Scholar]
  38. Kieffer LJ, Seng TW, Li W, Osterman DG, Handschumacher RE, Bayney RM. Cyclophilin-40, a protein with homology to the P59 component of the steroid receptor complex. Cloning of the cDNA and further characterization. J. Biol. Chem. 1993;268:12303–12310. [PubMed] [Google Scholar]
  39. Knowlton AA, Kapadia S, Torre-Amione G, et al. Differential expression of heat shock proteins in normal and failing human hearts. J. Mol. Cell Cardiol. 1998;30:811–818. doi: 10.1006/jmcc.1998.0646. [DOI] [PubMed] [Google Scholar]
  40. Kops O, Eckerskorn C, Hottenrott S, Fischer G, Mi H, Tropschug M. Ssp1, a site-specific parvulin homolog from Neurospora crassa active in protein folding. J. Biol. Chem. 1998;273:31971–31976. doi: 10.1074/jbc.273.48.31971. [DOI] [PubMed] [Google Scholar]
  41. Kroemer G, Petit P, Zamzami N, Vayssiere JL, Mignotte B. The biochemistry of programmed cell death. FASEB J. 1995;9:1277–1287. doi: 10.1096/fasebj.9.13.7557017. [DOI] [PubMed] [Google Scholar]
  42. Kruse M, Brunke M, Escher A, Szalay AA, Tropschug M, Zimmermann R. Enzyme assembly after de novo synthesis in rabbit reticulocyte lysate involves molecular chaperones and immunophilins. J. Biol. Chem. 1995;270:2588–2594. doi: 10.1074/jbc.270.6.2588. [DOI] [PubMed] [Google Scholar]
  43. Langer T, Neupert W. Chaperoning mitochondrial biogenesis. In: Morimoto R, Tissieres A, Georgopoulos C, editors. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1994. pp. 53–83. [Google Scholar]
  44. Lau S, Patnaik N, Sayen MR, Mestril R. Simultaneous overexpression of two stress proteins in rat cardiomyocytes and myogenic cells confers protection against ischemia- induced injury. Circulation. 1997;96:2287–2294. doi: 10.1161/01.cir.96.7.2287. [DOI] [PubMed] [Google Scholar]
  45. Leverson JD, Ness SA. Point mutations in v-Myb disrupt a cyclophilin-catalyzed negative regulatory mechanism. Mol. Cell. 1998;1:203–211. doi: 10.1016/s1097-2765(00)80021-0. [DOI] [PubMed] [Google Scholar]
  46. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997;91:479–489. doi: 10.1016/s0092-8674(00)80434-1. [DOI] [PubMed] [Google Scholar]
  47. Marber MS, Latchman DS, Walker JM, Yellon DM. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation. 1993;88:1264–1272. doi: 10.1161/01.cir.88.3.1264. [DOI] [PubMed] [Google Scholar]
  48. Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J. Clin. Invest. 1995;95:1446–1456. doi: 10.1172/JCI117815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Marzo I, Brenner C, Zamzami N, et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J. Exp Med. 1998;187:1261–1271. doi: 10.1084/jem.187.8.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Meshinchi S, Sanchez ER, Martell KJ, Pratt WB. Elimination and reconstitution of the requirement for hormone in promoting temperature-dependent transformation of cytosolic glucocorticoid receptors to the DNA-binding state. J. Biol. Chem. 1990;265:4863–4870. [PubMed] [Google Scholar]
  51. Molkentin JD, Lu JR, Antos CL, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998;93:215–228. doi: 10.1016/s0092-8674(00)81573-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Montague JW, Gaido ML, Frye C, Cidlowski JA. A calcium dependent nuclease from apoptotic rat thymocytes is homologous with cyclophilin.-Recombinant cyclophilin-A, cyclophilin-B, and cyclophilin-C have nuclease activity. J. Biol. Chem. 1994;269:18877–18880. [PubMed] [Google Scholar]
  53. Montague JW, Hughes FM, Cidlowski JA. Native recombinant cyclophilins A, B, and C degrade DNA independently of peptidylprolyl cis-trans-isomerase activity — Potential roles of cyclophilins in apoptosis. J. Biol. Chem. 1997;272:6677–6684. doi: 10.1074/jbc.272.10.6677. [DOI] [PubMed] [Google Scholar]
  54. Morimoto RI, Kline MP, Bimston DN, Cotto JJ. The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem. 1997;32:17–29. [PubMed] [Google Scholar]
  55. Okuno S, Shimizu S, Ito T, et al. Bcl-2 Prevents Caspase-independent Cell Death. J. Biol. Chem. 1998;273:34272–34277. doi: 10.1074/jbc.273.51.34272. [DOI] [PubMed] [Google Scholar]
  56. Parsell DA, Lindquist S. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 1993;27:437–496. doi: 10.1146/annurev.ge.27.120193.002253. [DOI] [PubMed] [Google Scholar]
  57. Patriarca EJ, Maresca B. Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae. Exp. Cell Res. 1990;190:57–64. doi: 10.1016/0014-4827(90)90143-x. [DOI] [PubMed] [Google Scholar]
  58. Polla BS, Kantengwa S, Francois D, et al. Mitochondria are selective targets for the protective effect of heat-shock against oxidative injury. Proc. Natl. Acad. Sci. USA. 1996;93:6458–6463. doi: 10.1073/pnas.93.13.6458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Rassow J, Mohrs K, Koidl S, Barthelmess IB, Pfanner N, Tropschug M. Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones hsp70 and hsp60. Mol. Cell Biol. 1995;15:2654–2662. doi: 10.1128/mcb.15.5.2654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Snyder SH, Sabatini DM, Lai MM, Steiner JP, Hamilton GS, Suzdak PD. Neural actions of immunophilin ligands. Trends Pharmacol. Sci. 1998;19:21–26. doi: 10.1016/s0165-6147(97)01146-2. [DOI] [PubMed] [Google Scholar]
  61. Steinmann B, Bruckner P, Superti-Furga A. Cyclosporin A slows collagen triple-helix formation in vivo: indirect evidence for a physiologic role of peptidyl-prolyl cis-trans-isomerase. J. Biol. Chem. 1991;266:1299–1303. [PubMed] [Google Scholar]
  62. Sykes K, Gething MJ, Sambrook J. Proline isomerases function during heat shock. Proc. Natl. Acad. Sci. USA. 1993;90:5853–5857. doi: 10.1073/pnas.90.12.5853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tanveer A, Virji S, Andreeva L, et al. Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress. Eur. J. Biochem. 1996;238:166–172. doi: 10.1111/j.1432-1033.1996.0166q.x. [DOI] [PubMed] [Google Scholar]
  64. Walter DH, Haendeler J, Galle J, Zeiher AM, Dimmeler S. Cyclosporin A inhibits apoptosis of human endothelial cells by preventing release of cytochrome C from mitochondria. Circulation. 1998;98:1153–1157. doi: 10.1161/01.cir.98.12.1153. [DOI] [PubMed] [Google Scholar]
  65. Woodfield K, Ck A, Brdiczka D, Halestrap AP. Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition. Biochem. J. 1998;336:287–290. doi: 10.1042/bj3360287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Xiang J, Chao DT, Korsmeyer SJ. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA. 1996;93:14559–14563. doi: 10.1073/pnas.93.25.14559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Yellon DM, Pasini E, Cargnoni A, Marber MS, Latchman DS, Ferrari R. The protective role of heat stress in the ischaemic and reperfused rabbit myocardium. J. Mol. Cell Cardiol. 1992;24:895–907. doi: 10.1016/0022-2828(92)91102-b. [DOI] [PubMed] [Google Scholar]
  68. Zou J, Guo Y, Guettouche T, Smith DF, Voellmy R. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell. 1998;94:471–480. doi: 10.1016/s0092-8674(00)81588-3. [DOI] [PubMed] [Google Scholar]

Articles from International Journal of Experimental Pathology are provided here courtesy of Wiley

RESOURCES