The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypical mechanism to disrupt Ras-ERK signaling

Abstract

The Ras-ERK (extracellular signal–regulated kinase) pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors for treating cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends upon multifunctional-autoprocessing repeats-in-toxin (MARTX), a toxin that releases bacterial effector domains into host cells. One of the effector domains delivered by this toxin is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance (NMR) analysis and nucleotide exchange assays, we determined that RRSP processing did not release any fragments of KRAS or cause KRAS to dissociate from its bound nucleotide, but instead only locally impacted the structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor (GEF)-mediated nucleotide exchange and RAF binding. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance to its target.

Inhibition of tumor growth by a novel engineered chimeric toxin that cleaves activated mutant and wild-type RAS

“…Notably, depletion of RAS and reduction in pERK were also observed in spheroids treated with the catalytically-inactive mutant RRSP*-DTB, but at a 10-fold higher dose. Previous binding studies showed that RRSP* retains its ability to bind recombinant KRAS, especially at high KRAS:RRSP* molar ratios (Biancucci et al, 2018). Therefore, although RRSP*-DTB does not cleave RAS, it might impact its activity when used at high doses and after longer exposure both in vitro and in vivo.…”

“…Imaging System (LI-COR Biosciences) and quantified by densitometry using NIH ImageJ software. Percentage of uncleaved RAS was calculated as previously described (Biancucci et al, 2018).…”

“…Protein concentration was quantified using Bio-Rad protein assay dye reagent concentrate (#5000006). Equal amounts of proteins were separated by SDS-PAGE followed by western blot analysis as previously described (Biancucci et al, 2018). in phosphate buffered saline (PBS)) was then added to each well and the plate incubated at room temperature for 20 min.…”

“…Recently, we discovered a RAS/RAP1 specific endopeptidase (RRSP) from Vibrio vulnificus that site-specifically cleaves RAS and its close homolog RAP1 between residues Y32 and D33 within the Switch I, a region crucial to RAS-mediated signal transduction. RRSP is highly specific for RAS and RAP1 and does not cleave other closely related GTPases (Antic et al, 2015;Biancucci et al, 2018;Biancucci et al, 2017). Importantly, RRSP cleaves all three major RAS isoforms (H, N, and K), as well as oncogenic RAS with mutations at position 12, 13 and 61.…”

“…RRSP also targets both active (GTP-bound) and inactive (GDP-bound) RAS, resulting in destruction of the entire cellular RAS pool. By proteolytically cleaving the Switch I loop, RRSP prevents RAS from undergoing GDP-GTP exchange and binding the downstream effector kinase RAF, ultimately terminating ERK signaling in cells (Antic et al, 2015;Biancucci et al, 2018).…”