In silico and in vitro investigations on the protein–protein interactions of glutathione S-transferases with mitogen-activated protein kinase 8 and apoptosis signal-regulating kinase 1

Chakradhara Rao S Uppugunduri,Jayaraman Muthukumaran,Shannon Robin,Teresa Santos-Silva,Marc Ansari

doi:10.1080/07391102.2020.1827036

Abstract

Cytosolic glutathione S-transferase (GST) enzymes participate in several cellular processes in addition to facilitating glutathione conjugation reactions that eliminate endogenous and exogenous toxic compounds, especially electrophiles. GSTs are thought to interact with various kinases, resulting in the modulation of apoptotic processes and cellular proliferation. The present research used a combination of in silico and in vitro studies to investigate protein–protein interactions between the seven most abundant cytosolic GSTs—GST alpha-1 (GST-A1), GST alpha-2 (GST-A2), GST mu-1 (GST-M1), GST mu-2 (GST-M2), GST mu-5 (GST-M5), GST theta-1 (GST-T1) and GST pi-1 (GST-P1)—and Mitogen-activated protein kinase 8 (MAPK8) and Apoptosis signal-regulating kinase 1 (ASK1). MAPK8 and ASK1 were chosen as this study’s protein interaction partners because of their predominant role in electrophile or cytokine-induced stress-mediated apoptosis, inflammation and fibrosis. The highest degree of sequence homology or sequence similarity was observed in two GST subgroups: the GST-A1, GST-A2 and GST-P1 isoforms constituted subgroup1; the GST-M1, GST-M2 and GST-M5 isoforms constituted subgroup 2. The GST-T1 isoform diverged from these isoforms. In silico investigations revealed that GST-M1 showed a significantly higher binding affinity to MAPK8, and its complex was more structurally stable than the other isoforms, in the order GST-M1 > GST-M5 > GST-P1 > GST-A2 > GST-A1 > GST-M2 > GST-T1. Similarly, GST-A1, GST-P1 and GST-T1 actively interacted with ASK1, and their structural stability was also better, in the order GST-T1 > GST-A1 > GST-P1 > GST-A2 > GST-M5 > GST-M1 > GST-M2. To validate in silico results, we performed in vitro crosslinking and mass spectroscopy experiments. Results indicated that GST-M1 interacted with GST-T1 to form heterodimers and confirmed the predicted interaction between GST-M1 and MAPK8. Communicated by Ramaswamy H. Sarma

Highlights

Cytosolic glutathione S-transferase (GST) enzymes are involved in multiple cellular processes, in addition to catalysing glutathione conjugation reactions that eliminate endogenous and exogenous toxic compounds, especially electrophiles (Adler et al, 1999; Gu€lcin, Scozzafava, Supuran, Akıncıoglu, et al, 2016; Gu€lcin, Scozzafava, Supuran, Koksal, et al, 2016; Hayes & Pulford, 1995; Hayes et al, 2005; Taslimi et al, 2020; Temel et al, 2018)
Phylogenetic analyses indicated that it is most likely that the activities of GST pi-1 (GST-P1) would be taken care by GST mu-1 (GST-M1), GST mu-5 (GST-M5), GST mu-2 (GST-M2) followed by GST alpha-1 (GST-A1) or GST alpha-2 (GST-A2) isoforms, whenever there is less abundance or dysfunctional GST-P1 and vice versa
Results indicated that MAPK9 and MAPK10 are closer to Mitogen-activated protein kinase 8 (MAPK8), whereas MAPK6 and MAPK4 are closer to Apoptosis signal-regulating kinase 1 (ASK1). We suggest that both MAPK8 and ASK1 interact with GST isoforms and, based on the results obtained from the phylogenetic analysis, that MAPK4, MAPK6, MAPK9 and MAPK10 (Supporting Information, Figures S2 and S3) may interact with GST isoforms

Summary

Introduction

Cytosolic glutathione S-transferase (GST) enzymes are involved in multiple cellular processes, in addition to catalysing glutathione conjugation reactions that eliminate endogenous and exogenous toxic compounds, especially electrophiles (Adler et al, 1999; Gu€lcin, Scozzafava, Supuran, Akıncıoglu, et al, 2016; Gu€lcin, Scozzafava, Supuran, Koksal, et al, 2016; Hayes & Pulford, 1995; Hayes et al, 2005; Taslimi et al, 2020; Temel et al, 2018). Members of the cytosolic GST family share sequence similarity among themselves and with members of the mitochondrial GST family (Gulcin et al, 2018; Nebert & Vasiliou, 2004; Tu€rkan et al, 2020). Structurewise, Cytosolic GST enzymes comprise two important domains and two critical binding sites: the N-terminal thioredoxin-like domain is responsible for glutathione binding, i.e. at G-sites, and the C-terminal alpha-helical domain and the N-terminal domain both harbour the hydrophobic ligand-

Methods

Results

Conclusion