Site-specific processing of Ras and Rap1 Switch I by a MARTX toxin effector domain

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 DUF5(Vv) from Vibrio vulnificus to be a site-specific endopeptidase that cleaves within the Switch 1 region of Ras and Rap1. DUF5(Vv) 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 DUF5(Vv) homologues found in other bacteria. In addition, DUF5(Vv )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.

Figure 1. DUF5_Vv-dependent disruption of Ras-ERK-dependent cell proliferation.

(a) Major categories of yeast mutants enabling growth in the presence of DUF5_Vv-C2. (b,d) Representative immunoblots (n=3) of lysates prepared from cells treated for 24 h (b) or time indicated (d) with LF_NDUF5_Vv in the absence (−) or the presence (+) of PA. Trimmed ERK1/2 blots are shown unedited in Supplementary Fig. 2. (c,e) Clonogenic colony-formation assay (n=2) of cells treated for 24 (c) or 1 h (e). Error bars represent the range of the data.

Figure 2. DUF5_Vv is a Ras site-specific endopeptidase.

(a) Coomassie-stained 18% SDS-polyacrylamide gel of anti-HA immunoprecipitated proteins from cells expressing HA-HRas treated for 24 h as indicated. Lower band (HRas*) was excised for peptide sequencing with HRas peptide coverage highlighted in yellow. (b) Same fractions probed by immunoblotting to detect the N terminus (anti-HA) and C terminus (isotype-specific antibody). (c) Lysates from cells expressing HA-tagged KRas, NRas or HRas probed by immunoblotting as indicated. (d) In-vitro cleavage of 10 μM rKRas to KRas* with 10 μM rDUF5_Vv (inset) or concentration indicated. Error bars indicate mean±s.d. (n=3). (e) In-vitro cleavage of 10 μM rKRas, rHRas and rNRas with 10 μM rDUF5_Vv. Identical results of Edman degradation were obtained for all three proteins. (f). Black arrow indicates the cleavage site in the Switch I region (red) of HRas.

Figure 3. DUF5 homologues and other GTPase substrates.

(a) Schematic diagram of DUF5 (orange) within the mosaic architecture of effector domains in MARTX toxins from V. vulnificus (Vv), A. hydrophila (Ah), Vibrio splendidus (Vs), Xenorhabdus nematophila (Xn) and Yersinia kristensii (Yk) or as stand-alone proteins in Photorhabdus luminescens (Pl) and P. asymbiotica (Pa) as previously described. (b) In-vitro cleavage of 10 μM KRas with 10 μM rDUF5 from various species. (c) LF_NDUF5_Ah tested for in-vivo loss of all Ras isoforms after 24 h under the same conditions as in Fig. 1 b. (d) Amino acid identity in Switch I regions of representative GTPases (left) from five major Ras families (right). (e) Bar graph of per cent GFP-fusion protein cleaved after delivery of LF_NDUF5_Vv+PA, quantified from immunoblots (Supplementary Fig. 5). Error bars indicate mean±s.d. (n=3). (f) Representative in-vitro cleavage (n=3) of GST-fusion proteins to release GST*. Negative cleavage reactions for nine other substrates are shown in Supplementary Fig. 6.

Figure 4. DUF5_Vv during bacterial infection and as a potential treatment of malignancies.

(a) MARTX toxin effector domain configuration in V. vulnificus isolates CMCP6 (DUF5_Vv⁺) and M06-24/O (DUF5_Vv⁻). (b) Representative immunoblots (n=2) of lysates from cells incubated with V. vulnificus as indicated and probed for Ras cleavage and ERK1/2 dephosphorylation. (c) Phase-contrast images and immunoblot detection of Ras from HCT116 and MDA-MB-231 cells treated as indicated for 24 h. (d) In-vitro processing of 10 μM rKRas with mutations as indicated.