Enolase from Trypanosoma cruzi is inhibited by its interaction with metallocarboxypeptidase-1 and a putative acireductone dioxygenase

Biochim Biophys Acta Proteins Proteom. May-Jun 2018;1866(5-6):651-660. doi: 10.1016/j.bbapap.2018.03.003. Epub 2018 Mar 10.

Abstract

Purification of enolase (ENO) from the cytosol of Trypanosoma cruzi indicated that it may interact with at least five other proteins. Two of them were identified as metallocarboxypeptidase-1 (TcMCP-1) and a putative acireductone dioxygenase (ARDp). Subcellular localization studies confirmed the presence of ARDp in the cytosol, as is the case for ENO and TcMCP-1. Analysis of the ARDp sequence showed that this protein has two domains, an N-terminal ARD and a C-terminal TRP14 (thioredoxin-related protein) domain. The interactions between ENO, TcMCP-1 and ARDp were confirmed for the natural proteins from the trypanosome (using size-exclusion chromatography and co-immunoprecipitation from a cytosolic fraction) and recombinant forms (using ELISA ligand-binding assay and ENO activity assays). The ELISA ligand-binding assays permitted to verify the optimal physicochemical conditions for the interactions (representative for the physiological conditions) and to determine the affinity constants (Kd): ENO/ARDp: 9.54 ± 0.82 nM, ARDp/ENO 10.05 ± 1.11 nM, and ENO/TcMCP-1: 5.66 ± 0.61 nM. The data also show that the interaction between TcMCP-1 and ARDp is mediated by ENO acting as a "bridge". Furthermore, considerable inhibition of the ENO activity, up to 85%, is observed when the enzyme interacts with TcMCP-1 and ARDp simultaneously. All these data confirm that the interaction between ENO, TcMCP-1 and ARDp, occurring in T. cruzi's cytosol, modulates the ENO activity and suggest a possible physiological mechanism for regulation of the ENO activity by the protein-protein interaction.

Keywords: Activity inhibition; Affinity constant; Co-immunoprecipitation; Enolase; Protein-protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Carboxypeptidases / chemistry
  • Carboxypeptidases / genetics
  • Carboxypeptidases / metabolism*
  • Chromatography, Gel
  • Cloning, Molecular
  • Cytosol / enzymology
  • Dioxygenases / chemistry
  • Dioxygenases / genetics
  • Dioxygenases / metabolism*
  • Enzyme-Linked Immunosorbent Assay
  • Immunoprecipitation
  • Kinetics
  • Phosphopyruvate Hydratase / antagonists & inhibitors
  • Phosphopyruvate Hydratase / chemistry
  • Phosphopyruvate Hydratase / genetics
  • Phosphopyruvate Hydratase / metabolism*
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protozoan Proteins / antagonists & inhibitors
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Recombinant Proteins / metabolism
  • Sequence Analysis, Protein
  • Trypanosoma cruzi / enzymology*
  • Trypanosoma cruzi / genetics

Substances

  • Protozoan Proteins
  • Recombinant Proteins
  • Dioxygenases
  • aci-reductone oxidase (CO-forming)
  • Carboxypeptidases
  • Phosphopyruvate Hydratase