Lack of Abundance of Cytoplasmic Cyclosporin A-binding Protein Renders Free-living Leishmania donovaniResistant to Cyclosporin A

Dutta, Madhuri; Delhi, P.; Sinha, Krishna M.; Banerjee, Rahul; Datta, Alokmay

doi:10.1074/jbc.m009379200

Cited by 21 publications

(33 citation statements)

References 53 publications

(40 reference statements)

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“…results are consistent with the interpretation that N terminustruncated LdCyP acts as better chaperone than the full-length LdCyP in reactivating AdK. Further, it is to be noted that unlike previous studies, which implicated involvement of the -SH group of CyP in binding of the protein substrate (30), N 22-88 -DEL peptide, despite having no cysteine residue in its sequence (28), is capable of interacting with AdK.…”

Section: Table II Removal Of Ldcyp Renders Activated Adk Inactivesupporting

confidence: 77%

“…In this context, it may be worth pointing out that the cysteine residue of CyP from porcine kidney was implicated in binding of peptide substrate (30). However, our experiments with truncated LdCyP, which does not harbor any cysteine (28), show that this is not applicable in the present case. These results may indicate that the C-terminal region of the full-length LdCyP, responsible for binding with AdK, was probably not fully accessible due to masking.…”

Section: And References Therein)contrasting

confidence: 52%

“…While investigating the possible cause of this inactivation, we discovered that AdK, which is fully active under dilute conditions, tends to form soluble aggregates at higher concentrations, resulting in inactivation. Recently, we reported identification and cloning of a CyPB homologue (LdCyP) from L. donovani and suggested chaperonic function of this protein (28). Using the inactive AdK as the substrate, we herein report a novel finding that this small single-domain CyP from L. donovani, alone, can completely convert this enzyme from inactive oligomers to its active monomeric form.…”

mentioning

confidence: 75%

“…Construction and Expression of Full-length and Deleted LdCyPsFull-length recombinant LdCyP was expressed and purified using procedures described previously (28). To construct the N-terminal deleted clone of LdCyP (N 22-88 -DEL), the coding sequence (amino acids 89 -187) was amplified by PCR and ligated in-frame at the BamHI/SalI sites of the expression vector pQE30 (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

“…Following preincubation (30°C for 30 min) of a fixed concentration of AdK with variable stoichiometric proportions of LdCyP or its truncated form, the mixtures were further incubated for another 30 min in the presence of 0.5 mM DSS. An equal amount of the reaction mixture from each reaction were separated on SDS-PAGE (10 or 13%) and visualized after immunoblotting with polyclonal antisera against both LdCyP and AdK (28,31).…”

Section: Methodsmentioning

confidence: 99%

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A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

Chakraborty¹,

Das²,

Datta³

et al. 2002

Journal of Biological Chemistry

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Disaggregation and reactivation of aggregated proteins by chaperones is well established. However, little is known regarding such kind of function of single-domain small cyclophilins (CyPs). Here we demonstrate that, with increasing concentrations, fully active adenosine kinase (AdK) of Leishmania donovani tends to form soluble aggregates, resulting in inactivation. Using this inactive enzyme as the substrate, it is shown that a CyP from L. donovani (LdCyP) alone can cause complete disaggregation, leading to reactivation of the enzyme. The reactivating ability of LdCyP remains unaffected even in the presence of cyclosporin A and macromolecular crowding agents. The reactivation occurs noncatalytically and is reversible. A truncated LdCyP, devoid of 88 amino acids from the N terminus, is found to be required in near stoichiometric proportion to reactivate AdK, suggesting essentiality of the C-terminal region. Gel filtration and light-scattering experiments together with protein cross-linking studies revealed that both full-length LdCyP and the truncated form directly interact with AdK and convert oligomeric forms of the enzyme to monomeric state. Homology modeling studies suggest that the exposed hydrophobic residues of LdCyP, by interacting with solvent-accessible hydrophobic surface of AdK, pull apart its aggregated inactive oligomers to functional monomers. Clearly, the results are consistent with the interpretation that the higher efficiency of the truncated LdCyP is most likely due to increased exposure of the hydrophobic residues on its surface. These observations, besides establishing L. donovani AdK as one of the model enzymes to study aggregation-disaggregation of proteins, raise the possibility that single-domain small CyPs, under physiological conditions, may regulate the activity of aggregation-prone proteins by ensuring their disaggregation.

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Section: Table II Removal Of Ldcyp Renders Activated Adk Inactivesupporting

confidence: 77%

Section: And References Therein)contrasting

confidence: 52%

mentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

Chakraborty¹,

Das²,

Datta³

et al. 2002

Journal of Biological Chemistry

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Peptidyl‐prolylcis/transIsomerases

Fischer

2008

Protein Science Encyclopedia

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The stereospecificity of peptidyl prolyl cis/trans isomerases (PPIases) was studied using tetrapeptide substrate analogs in which one amino acid residue was replaced by the cognate D-amino acid in various positions of the peptide chain. Reversed stereocenters around proline markedly increased the rate of the spontaneous trans to cis isomerization of the prolyl bond whereas cis to trans isomerizations were less sensitive. PPIases like human cyclophilin18, human FKBP12, Escherichia coli parvulin10 and the PPIase domain of E. coli trigger factor exhibited stereoselectivity demanding at the P I to P P P position of the substrate chain. The discriminating factor for stereoselectiv-ity was the lack of formation of the Michaelis complexes of the diastereomeric substrates. However, D-alanine at the P I position preserved considerable affinity to the active site, and largely prevented activation of the catalytic machinery for all PPIases investigated. z 1998 Federation of European Biochemical Societies.

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Catalysis of Peptidyl‐prolylcis/transIsomerization by Enzymes

Fischer¹

2005

Protein Folding Handbook

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Lack of Abundance of Cytoplasmic Cyclosporin A-binding Protein Renders Free-living Leishmania donovaniResistant to Cyclosporin A

Cited by 21 publications

References 53 publications

A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

Peptidyl‐prolylcis/transIsomerases

Catalysis of Peptidyl‐prolylcis/transIsomerization by Enzymes

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