Abstract
Ras (Rat sarcoma) protein is a central regulator of cell growth and proliferation. Mutations in the RAS gene are known to occur in human cancers and have been shown to contribute to carcinogenesis. In this study, we show that the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin-effector domain DUF5Vv from Vibrio vulnificus to be a site-specific endopeptidase that cleaves within the Switch 1 region of Ras and Rap1. DUF5Vv processing of Ras, which occurs both biochemically and in mammalian cell culture, inactivates ERK1/2, thereby inhibiting cell proliferation. The ability to cleave Ras and Rap1 is shared by DUF5Vv homologues found in other bacteria. In addition, DUF5Vv can cleave all Ras isoforms and KRas with mutations commonly implicated in malignancies. Therefore, we speculate that this new family of Ras/Rap1-specific endopeptidases (RRSPs) has potential to inactivate both wild-type and mutant Ras proteins expressed in malignancies.
Highlights
Ras (Rat sarcoma) protein is a central regulator of cell growth and proliferation
As a strategy to identify molecular targets accounting for this cytotoxicity[20], a genome-wide, arrayed, nonessential gene deletion library was screened for yeast strains that survived enforced expression of C2 (Supplementary Fig. 1)
We have previously demonstrated that the cytotoxic activity of DUF5Vv can be isolated away from the large multifunctional-autoprocessing repeats-intoxin (MARTX) by fusing DUF5Vv to the N terminus of anthrax toxin lethal factor (LFNDUF5Vv) and subsequently delivering the fusion protein to cells in culture using anthrax toxin protective antigen (PA)[20]
Summary
Ras (Rat sarcoma) protein is a central regulator of cell growth and proliferation. We show that the multifunctional-autoprocessing repeats-intoxin (MARTX) toxin-effector domain DUF5Vv from Vibrio vulnificus to be a site-specific endopeptidase that cleaves within the Switch 1 region of Ras and Rap[1]. Rat sarcoma (Ras) oncoprotein is a small GTPase ubiquitous in eukaryotic cells and is a critical node that coordinates incoming signals and subsequently activates downstream target proteins. These targets include rapidly accelerated fibrosarcoma kinase (Raf), phosphatidylinositol-4,5-bisphosphate 3-kinase and mitogen-activated protein kinase (MAPK), which induces expression of genes directing cell proliferation, differentiation and survival. DUF5Vv-C2 was found to inhibit growth when conditionally overexpressed in Saccharomyces cerevisiae[20]
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