Isomerase-independent Chaperone Function of Cyclophilin Ensures Aggregation Prevention of Adenosine Kinase Both in Vitro and Under in Vivo Conditions

Biochemistry. 2004 Sep 21;43(37):11862-72. doi: 10.1021/bi049490o.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Kinase / chemistry
  • Adenosine Kinase / genetics
  • Adenosine Kinase / metabolism*
  • Animals
  • Cyclophilins / genetics
  • Cyclophilins / metabolism*
  • Enzyme Activation
  • Isomerases / genetics
  • Isomerases / metabolism*
  • Leishmania donovani / enzymology
  • Models, Biological
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Protein Denaturation
  • Protein Renaturation
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Molecular Chaperones
  • Protein Subunits
  • Protozoan Proteins
  • Recombinant Proteins
  • Adenosine Kinase
  • Isomerases
  • Cyclophilins