Active site mutants of human cyclophilin A separate peptidyl‐prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition

Zydowsky, Lynne D.; Etzkorn, Felicia A.; Chang, Howard Y.; Ferguson, Stephen B.; Stolz, Lesley A.; Ho, Susanna I.; Walsh, Christopher T.

doi:10.1002/pro.5560010903

Cited by 288 publications

(356 citation statements)

References 37 publications

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“…However, for G89 cis, the bound form is only observed at higher CyPA R55A ͞CA N ratios in agreement with a higher affinity of the CA N cis isomer for CypA. activity (40). As expected, the bound cis and trans conformations of CA N G89-P90 can now be separately detected in the complex with CypA R55A (Fig.…”

Section: Catalysis Of Cis͞trans Isomerization Of the G89-p90 Peptide supporting

confidence: 81%

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

Bosco

Eisenmesser

Pochapsky

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

204

211

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Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89 -Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CA N ). Despite the fact that CypA is a peptidyl-prolyl cis͞trans isomerase, catalytic activity on CA N has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CA N but also catalyzes efficiently the cis͞trans isomerization of the Gly-89 -Pro-90 peptide bond. In addition, conformational changes in CA N distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate.

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Section: Catalysis Of Cis͞trans Isomerization Of the G89-p90 Peptide supporting

confidence: 81%

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

Bosco

Eisenmesser

Pochapsky

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

204

211

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“…The conserved binding site is therefore used as the signature for FKBP protein identification. In a similar manner, the residues necessary for cyclophilin activity are highly conserved (Kallen et al, 1991;Zydowsky et al, 1992) and serve as CYP signature for identifying putative CYP members. The sequence alignment for AtFKBP and AtCYP family is shown in Figure 1 and Figure 2, respectively.…”

Section: Resultsmentioning

confidence: 99%

Immunophilins and Parvulins. Superfamily of Peptidyl Prolyl Isomerases in Arabidopsis

2004

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Immunophilins are defined as receptors for immunosuppressive drugs including cyclosporin A, FK506, and rapamycin. The cyclosporin A receptors are referred to as cyclophilins (CYPs) and FK506-and rapamycin-binding proteins are abbreviated as FKBPs. These two groups of proteins (collectively called immunophilins) share little sequence homology, but both have peptidyl prolyl cis/trans isomerase (PPIase) activity that is involved in protein folding processes. Studies have identified immunophilins in all organisms examined including bacteria, fungi, animals, and plants. Nevertheless, the physiological function of immunophilins is poorly understood in any organism. In this study, we have surveyed the genes encoding immunophilins in Arabidopsis genome. A total of 52 genes have been found to encode putative immunophilins, among which 23 are putative FKBPs and 29 are putative CYPs. This is by far the largest immunophilin family identified in any organism. Both FKBPs and CYPs can be classified into single domain and multiple domain members. The single domain members contain a basic catalytic domain and some of them have signal sequences for targeting to a specific organelle. The multiple domain members contain not only the catalytic domain but also defined modules that are involved in protein-protein interaction or other functions. A striking feature of immunophilins in Arabidopsis is that a large fraction of FKBPs and CYPs are localized in the chloroplast, a possible explanation for why plants have a larger immunophilin family than animals. Parvulins represent another family of PPIases that are unrelated to immunophilins in protein sequences and drug binding properties. Three parvulin genes were found in Arabidopsis genome. The expression of many immunophilin and parvulin genes is ubiquitous except for those encoding chloroplast members that are often detected only in the green tissues. The large number of genes and diversity of structure domains and cellular localization make PPIases a versatile superfamily of proteins that clearly function in many cellular processes in plants.Immunosuppressive drugs cyclosporin A (CsA), FK506, and rapamycin are used clinically in transplantation to prevent graft rejection. During the course to understand the molecular mechanisms of immunosuppression by CsA, FK506, and rapamycin, the cellular receptors of these drugs have been purified and characterized (Schreiber, 1991;Fruman et al., 1994). CsA binds to a family of receptors named cyclophilins (CyPs), and FK506 and rapamycin bind to a distinct set of receptors called FKBPs (FK506 and rapamycin-binding proteins). Cyclophilins and FKBPs are collectively referred to as immunophilins (Schreiber, 1991).The complexes formed by immunophilins and their cognate ligands are the functional modules for immunosuppression. The FKBP12-FK506 and CyPCsA complexes, but not their separate components, bind to and inhibit the activity of calcineurin, a Ca 21 , calmodulin-dependent protein phosphatase (Liu et al., 1991). Studies have demonstrated that inhib...

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“…To restore CypA expression in Huh7 CypA knockdown cells, a CypA cDNA bearing silent mutations that render it nontargetable by the CypA shRNA was subcloned into pcDNA3 with HindIII and NotI sites to generate pcDNA3-resistant wild-type CypA, which contains an N-terminal HA tag. Using pcDNA3-resistant wild-type CypA as template, we engineered two plasmids carrying either the H126Q (pcDNA3-resistant H126Q CypA) or the R55A (pcDNA3-resistant R55A CypA) mutation in the hydrophobic pocket of CypA that disallows its isomerase activity (25).…”

Section: Methodsmentioning

confidence: 99%

“…Specifically, we replaced the histidine located at position 126 in the hydrophobic pocket of CypA by a glutamine, creating the H126Q CypA mutant. Importantly, this mutation diminishes CypA isomerase activity by more than 99% compared with wild-type CypA (25). To determine whether HCV requires the isomerase activity of CypA to replicate, we had to transfect the H126Q CypA mutant into the CypA-KD cells and asked if HCV replication can be rescued.…”

Section: Analysis Of the Respective Contribution Of Cyp Members To Hcmentioning

confidence: 99%

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Chatterji

Bobardt

Selvarajah

et al. 2009

Journal of Biological Chemistry

176

220

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Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies. Hepatitis C virus (HCV)2 is the main contributing agent of acute and chronic liver diseases worldwide (1). Primary infection is often asymptomatic or associated with mild symptoms. However, persistently infected individuals develop high risks for chronic liver diseases such as hepatocellular carcinoma and liver cirrhosis (1). The combination of IFN␣ and ribavirin that serves as current therapy for chronically HCV-infected patients not only has a low success rate (about 50%) (2) but is often associated with serious side effects (2). There is thus an urgent need for the development of novel anti-HCV treatments (2).The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV (3). Controlled trials showed that a combination of CsA with IFN␣ is more effective than IFN␣ alone, especially in patients with a high viral load (4, 5). Moreover, recent in vitro studies provided evidence that CsA prevents both HCV RNA replication and HCV protein production in an IFN␣-independent manner (6 -10). CsA exerts this anti-HCV activity independently of its immunosuppressive activity because the nonimmunosuppressive Cyp inhibitors such as Debio 025, NIM811, and SCY-635 also block HCV RNA and...

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Active site mutants of human cyclophilin A separate peptidyl‐prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition

Cited by 288 publications

References 37 publications

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

Immunophilins and Parvulins. Superfamily of Peptidyl Prolyl Isomerases in Arabidopsis

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

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