Investigating the activity and interactions of oncogenic KRas proteins

Abstract

Mutations in the Ras protein family are responsible for one of the highest prevalence in cancer. Most commonly affecting the pancreas, colon and lung, these lesions remain challenging for medical science today. The KRas protein, encoded by the Ras gene, plays a central role in regulating signaling pathways. This enzyme can control signaling as a binary switch through its GTPase activity, thereby affecting cell proliferation and division. The function of the KRas protein is facilitated by GAP (GTPase activating protein) proteins, which can increase the rate of GTP hydrolysis by several magnitudes. However, this finely tuned system can easily be disrupted by mutations in the active site of the KRas protein. A single point mutation reduces the ability of GAP to activate KRas proteins, therefore the rate of hydrolysis is significantly reduced, resulting that the protein being unable to fulfil its role in the signaling network. This leds to inproper functioning of cellular processes and as a consequence uncontrolled cell division and tumor development. Several approaches have been developed to restore the signaling, the most common one is drug treatment with inhibitor molecules. However, the restoration of the activation of mutant KRas porteins by GAP proteins holds promise too. Despite being an important target, the mechanism of GAP-catalysed hydrolysis has not been precisely defined yet. To understand this, our collaborators have set up a QM/MM model to systematically design GAP mutants that can activate the KRas G12D mutant, which is the most abundant KRas mutant in the human cancers. In addition, researchers have recently discovered a GAP-like protein, RGS3, which can also cause a catalytic effect on mutant KRas proteins. Our aim is to test these alternative GAP proteins in practice. For this, we investigated the interaction between the mutant KRas and RGS3 by biolayer interferometry and measured the ability of GAP variants to activate KRas mutants by using MESG-PNP enzyme activity assay. These studies may help to understand the interaction of these proteins and thus improve the development of tumor therapeutics.