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

Authors

Chakradhara Rao S. Uppugunduri^1,2, Jayaraman Muthukumaran^3,4, Shannon Robin², Teresa Santos- Silva³ and Marc Ansari^1,2

Organizations

1. Onco-Haematology Unit, Department of Paediatrics, Obstetrics and Gynaecology, Geneva University Hospitals, Geneva, Switzerland
2. Research Platform on Pediatric Onco-Hematology, Department of Paediatrics, Obstetrics and Gynaecology, Faculty of Medicine, University of Geneva, Geneva, Switzerland
3. UCIBIO-Applied Molecular Biosciences Unit, REQUIMTE, Departamento de Qu ?mica, Faculdade de Ci ^ encias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal
4. Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India Communicated by Ramaswamy H. Sarma

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.

CovalX Technology Used

MALDI-ToF
HM4
XL Kits
Complex Tracker

Outcomes

In this publication, CovalX’s technology was used to perform in vitro crosslinking and mass spectrometry experiments to validate in silico results to investigate protein-protein interactions between the seven cytosolic GSTs, MAPK8 and ASK1. To perform controlled crosslinking experiments, a matrix composed of GST-M1, GST-T1 and MAPK8 protein samples were prepared using CovalX’s K200 MALDI Kit. Then the samples were analyzed using MALDI-TOF MS modified with CovalX’s HM4 Interaction module to optimize MS for the detection up to 2 MDa with nano-molar sensitivity. The mass-spectrometer data were analyzed using CovalX’s Complex Tracker analysis software. The CovalX’s technology was useful for in vitro investigations that used pure GST-M1 (23.6 kDa), GST-T1 (31.5 kDa) and MAPK8 (50.5 kDa) recombinant human proteins (PROSPEC).