TY - JOUR
T1 - Enolase from Trypanosoma cruzi is inhibited by its interaction with metallocarboxypeptidase-1 and a putative acireductone dioxygenase
AU - Quintero-Troconis, E.
AU - Buelvas, N.
AU - Carrasco-López, C.
AU - Domingo-Sananes, M. R.
AU - González-González, L.
AU - Ramírez-Molina, R.
AU - Osorio, L.
AU - Lobo-Rojas, A.
AU - Cáceres, A. J.
AU - Michels, P. A.
AU - Acosta, H.
AU - Quiñones, W.
AU - Concepción, J. L.
PY - 2018/3/10
Y1 - 2018/3/10
N2 - 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.
AB - 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.
KW - activity inhibition
KW - affinity constant
KW - co-immunoprecipitation
KW - enolase
KW - protein-protein interaction
U2 - 10.1016/j.bbapap.2018.03.003
DO - 10.1016/j.bbapap.2018.03.003
M3 - Article
C2 - 29530564
AN - SCOPUS:85044528183
VL - 1866
SP - 651
EP - 660
JO - BBA - Proteins and Proteomics
JF - BBA - Proteins and Proteomics
SN - 1570-9639
IS - 5-6
ER -