Improved Stability of Human CGI-58 Induced by Phosphomimetic S237E Mutation.
Miriam Livier Llamas-GarcíaEdgar D Páez-PérezClaudia G Benitez-CardozaGabriela M Montero-MoránSamuel Lara-GonzalezPublished in: ACS omega (2022)
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.
Keyphrases
- wild type
- protein kinase
- fatty acid
- endothelial cells
- adipose tissue
- amino acid
- protein protein
- induced pluripotent stem cells
- helicobacter pylori
- signaling pathway
- helicobacter pylori infection
- insulin resistance
- small molecule
- single molecule
- artificial intelligence
- electronic health record
- machine learning
- quantum dots
- molecular dynamics simulations
- molecularly imprinted