TY - JOUR
T1 - Improved Stability of Human CGI-58 Induced by Phosphomimetic S237E Mutation
AU - Llamas-García, Miriam Livier
AU - Páez-Pérez, Edgar D.
AU - Benitez-Cardoza, Claudia G.
AU - Montero-Morán, Gabriela M.
AU - Lara-González, Samuel
N1 - Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.
PY - 2022/4/19
Y1 - 2022/4/19
N2 - In lipolysis, the activating function of CGI-58 is regulated by its interaction with perilipin 1 (PLIN1) localized on the lipid droplet (LD), and its release is controlled by phosphorylation. Once lipolysis is stimulated by catecholamines, protein kinase A (PKA)-mediated phosphorylation enables the dissociation of the CGI-58/PLIN1 complex, thereby recruiting adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) to initiate fatty acid release. It has been shown that mouse CGI-58 mutant S239E, which mimics the phosphorylation of this residue, is able to dissociate from the CGI-58/PLIN1 complex and activate ATGL. Here, we analyze the stabilizing effect on human CGI-58 of a triple tryptophan to alanine mutant (3WA) on the LD-binding motif, as well as a quadruple mutant in which the phosphomimetic S237E substitution was introduced to the 3WA construct (3WA/S237E). We found that tryptophan residues promote wild-type (WT) protein aggregation in solution since their substitution for alanine residues favors the presence of the monomer. Our experimental data showed increased thermal stability and solubility of 3WA/S237E protein compared to the 3WA mutant. Moreover, the 3WA/S237E protein showed proper folding and a functional binding site for oleoyl-CoA. The analysis of a bioinformatic three-dimensional (3D) model suggests an intramolecular interaction between the phosphomimetic glutamic acid and a residue of the α/β hydrolase core. This could explain the increased solubility and stability observed in the 3WA/S237E mutant and evidences the possible role of serine 237 phosphorylation.
AB - In lipolysis, the activating function of CGI-58 is regulated by its interaction with perilipin 1 (PLIN1) localized on the lipid droplet (LD), and its release is controlled by phosphorylation. Once lipolysis is stimulated by catecholamines, protein kinase A (PKA)-mediated phosphorylation enables the dissociation of the CGI-58/PLIN1 complex, thereby recruiting adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) to initiate fatty acid release. It has been shown that mouse CGI-58 mutant S239E, which mimics the phosphorylation of this residue, is able to dissociate from the CGI-58/PLIN1 complex and activate ATGL. Here, we analyze the stabilizing effect on human CGI-58 of a triple tryptophan to alanine mutant (3WA) on the LD-binding motif, as well as a quadruple mutant in which the phosphomimetic S237E substitution was introduced to the 3WA construct (3WA/S237E). We found that tryptophan residues promote wild-type (WT) protein aggregation in solution since their substitution for alanine residues favors the presence of the monomer. Our experimental data showed increased thermal stability and solubility of 3WA/S237E protein compared to the 3WA mutant. Moreover, the 3WA/S237E protein showed proper folding and a functional binding site for oleoyl-CoA. The analysis of a bioinformatic three-dimensional (3D) model suggests an intramolecular interaction between the phosphomimetic glutamic acid and a residue of the α/β hydrolase core. This could explain the increased solubility and stability observed in the 3WA/S237E mutant and evidences the possible role of serine 237 phosphorylation.
UR - http://www.scopus.com/inward/record.url?scp=85128516384&partnerID=8YFLogxK
U2 - 10.1021/acsomega.1c06872
DO - 10.1021/acsomega.1c06872
M3 - Artículo
C2 - 35474805
AN - SCOPUS:85128516384
SN - 2470-1343
VL - 7
SP - 12643
EP - 12653
JO - ACS Omega
JF - ACS Omega
IS - 15
ER -