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

E. Quintero-Troconis*, N. Buelvas, C. Carrasco-López, M. R. Domingo-Sananes, L. González-González, R. Ramírez-Molina, L. Osorio, A. Lobo-Rojas, A. J. Cáceres, P. A. Michels, H. Acosta, W. Quiñones, J. L. Concepción

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)651-660
Number of pages10
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1866
Issue number5-6
DOIs
Publication statusPublished - 10 Mar 2018

Keywords

  • activity inhibition
  • affinity constant
  • co-immunoprecipitation
  • enolase
  • protein-protein interaction

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