Isomerase-Independent Chaperone Function of Cyclophilin Ensures Aggregation Prevention of Adenosine Kinase Both in vitro and under in vivo Conditions

Abstract

Using inactive aggregates of adenosine kinase (AdK) from Leishmania donovani as the model substrate, we recently demonstrated that a cyclophilin (LdCyP) from the same source in an isomerase-independent fashion reactivated the enzyme in vitro by disaggregating its inactive oligomers [Chakraborty et al. (2002) J. Biol. Chem. 277, 47451-47460]. Besides disrupting preformed aggregates, LdCyP also prevents reaggregation of the newly formed active protein that is generated after productive refolding from its urea-denatured state. To investigate possible physiological implications of such phenomena, a unique expression system that simultaneously induces both AdK and LdCyP in naturally AdK-deficient Escherichia coli, was developed. Both in vitro and in vivo experiments revealed that oligomerization is an inherent property of this particular enzyme. In vivo protein cross-linking studies, activity determination analysis and Ado phosphorylation experiments carried out in cells coexpressing both the proteins unequivocally demonstrated that, similar to the phenomena observed in vitro, aggregates of the enzyme formed in vivo are able to interact with both LdCyP and its N-terminal truncated form (N(22-88)DEL LdCyP) in a crowded intracellular environment, resulting in aggregation prevention and reactivation of the enzyme. Our results indicate that the isomerase-independent chaperone function of LdCyP, detected in vitro, participates in vivo as well to keep aggregation-prone proteins in a monomeric state. Furthermore, analogous to yeast/bacterial two-hybrid systems, development of this simple coexpression system may help in the confirmation of interaction of two proteins under simulated in vivo conditions.

Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability

“…Based on previously published observations, Leishmania CyPs may have important amastigote-specific chaperone functions and participate in protein disaggregation [20]. We tested if CsA treatment affects thermotolerance of promastigotes and amastigotes following the number of propidium iodide stained, dead parasites as a read out.…”

“…Although these findings define members of the Leishmania CyP protein family as potential important drug targets, only little is known on this protein family in trypanosomatids and the mechanisms of the anti-parasitic effects of CsA on intracellular Leishmania remain elusive. A potential role of Leishmania CyPs in amastigote differentiation and virulence can be postulated based on the role of Leishmania donovani LdCyP in disaggregation of adenosine kinase aggregates [20], an important enzyme in the Leishmania purine salvage pathway, whose activity substantially increases during the pro- to amastigote differentiation [21]. Furthermore, the amastigote-specific phosphorylation of cyclophilin 40 [22], [23] suggests that activity, localization, and interaction of this protein may be regulated in a stage-specific manner by post-translational modification.…”

Adenosine Kinase: Exploitation for Therapeutic Gain

“…The reactivation occurred noncatalytically and was reversible (Chakraborty et al, 2002). The prevention of ADK aggregation by cyclophilin was shown to be mediated by an isomeraseindependent chaperone function of cyclophilin (Chakraborty et al, 2004). It was further shown that ADP stabilized the aggregated form of ADK and that cyclophilin was able to disaggregate and activate ADK (Sen et al, 2006).…”

A Legionella pneumophila Peptidyl-Prolyl cis - trans Isomerase Present in Culture Supernatants Is Necessary for Optimal Growth at Low Temperatures

“…Given its PPIase activity (37), PpiB may catalyze the isomerization of secreted proteins, such as type II substrates, that promote survival at low temperatures. Alternatively, PpiB might assist cold-adapted exoenzymes as a chaperone (5,19,36,38). Finally, PpiB, whether as an isomerase or as a chaperone, might promote the functioning of a secretion apparatus.…”