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

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

Author information

1
Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela. Electronic address: ejqtroconis@ula.ve.
2
Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela.
3
Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela; Centre for Immunity, Infection and Evolution, Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, United Kingdom.
4
Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela. Electronic address: concepci@ula.ve.

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

PMID:
29530564
DOI:
10.1016/j.bbapap.2018.03.003
[Indexed for MEDLINE]

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