Molecular interaction between small nuclear ribonucleoprotein polypeptide G and heat shock protein 70.14: a microscale thermophoresis exposition towards developing anti-cancer drugs

Abstract

Background: Targeting protein-protein interactions (PPIs) linked to protein quality control (PQC) pathways as potential anti-cancer drug targets have unanimously widened biological insights and the therapeutic potential of PPIs as smart-drug discovery tools in cancer. PPIs between disease-relevant proteins associated with protein homeostasis in PQC pathways have been linked to improved mechanistic understanding associated with conformational abnormalities and impairment, cellular proteotoxicity, induced apoptosis, and pathogenesis in different types of cancers. In this context, PPIs between small nuclear ribonucleoprotein polypeptide G (SNRPG) and heat shock protein 70.14 (Hsp70.14) have attracted attention as potential smart drug discovery tools in cancer diagnostics and therapeutics. Validated evidence of high-quality biological data has shown the presence of the two proteins in different types of cancers including breast cancer. The links between SNRPG and Hsp70.14 in cancer-cell networks remain elusive, overlooked, and uncharacterized.

Methodology: In this study, we explored the interaction between the two oncogenic proteins using the MST-based assays.

Results: The results revealed a low K_D in the nanomolar concentration range of 2.4673 × 10^-7 demonstrating a great affinity for SNRPG binding to Hsp70.14.

Conclusions: The results suggest a possible involvement between the two proteins in hostile tumour microenvironments. Furthermore, these findings offer a different therapeutic perspective that could pave the way for the creation of novel small molecule inhibitors as drugs for the treatment of cancer.

Keywords: Anti-cancer drug discovery; Hsp70.14; SNRPG; dissociation constant; microscale thermophoresis; protein-protein interactions; proteostasis.

Figure 1

Schematic representation of Sm proteins’ regulation in protein quality control (PQC) during U snRNP assembly. Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, as well as type 2 diabetes and cancer and are just a few diseases examples of the efficient homeostasis that prevent cellular proteotoxicity, apoptosis and disease progression. Figure taken from Prusty et al [5].

Figure 2

A diagram depicting the regulatory functions of house-keeping chaperones in PQC machineries. Hsp70 is actively involved in protein folding thereby dictating protein homeostasis in cell environments. Dysregulation of proteins and their failure to attain final conformation leads to ubiquitylation and degradation by the proteasome. Figure taken from Sannino and Brodsky [4].

Figure 3

Setup for MicroScale Thermophoresis (MST) (A) MST tests are carried out in microscopic glass capillaries. The MST effect is triggered by infrared and fluorescence lasers, which generate sample tracking (B) The time-dependent change in fluorescence after infrared heating of the sample capillaries is explained by temperature-related intensity change (TRIC) and thermophoresis (C) For varied combination ratios of target and ligand molecules, several MST traces are acquired (D) The steady-state affinity of the target-ligand interaction can be determined through dose-response analysis of MST traces. (Figure extracted from Mabonga et al [20]).

Figure 4

MST scans for the 6XHis-tag SNRPG protein in capillaries. The capillary scan graph was perfectly overlayed, indicating that there was no SNRPG protein adsorption onto the capillaries (A). As expected, the capillary scans revealed no changes in protein fluorescence, indicating that the SNRPG protein was successfully labelled (B).

Figure 5

Thermograph of SNRPG binding to the Hsp70.14 at 24.5°C. For varying combination ratios of the SNRPG and Hsp70.14, several MST traces were recorded. To determine the K_D of the contact and avoid potential convection phenomena, the cold zone is set to 0 seconds (blue) and the hot region to 20 seconds (red).

Figure 6

The binding interaction between SNRPG and Hsp70.14 has a dose-response curve. SNRPG protein concentration was held constant at 50 nM, whereas ligand concentrations ranged from 510 nM to 3.11 × 10^-5 M. The interaction’s binding affinity measurement yielded a K_D of 0.0247 nM. The studies were carried out at 24.5°C for 30 minutes at medium MST and 40 percent LED power.