Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ

Abstract

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

Figure 1. Shigella IpaJ and VirA disrupt Golgi morphology

a, Fluorescence microscopy HeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red). The cis-Golgi (green) was detected by α-GM130 antibodies. Scale bar, 10 μm. b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 μm. c, Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains. Scale bar, 10 μm. d, Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild-type Shigella (M90T) or the indicated mutants. Error bars, means ± s.e.m. calculated from three independent experiments. e, Number of recoverable c.f.u. per mouse lung 24 h after intranasal inoculation with 1 ×10⁶ of wild-type M90T or each mutant Shigella strain as indicated. Limit of detection (LOD) for this assay is indicated by the dotted line. Error bars, geometrical means ± s.e.m. **P < 0.001.

Figure 2. IpaJ belongs to the C39-like family of cysteine proteases and targets ARF-family GTPases

a, Sequence–structure alignment of S. flexneri IpaJ with members of C39-peptidase-like family identified by HHPred. IpaJ possesses invariant catalytic triad residues Cys 64, His 206 and Asp 218 (red). The conserved Gln 58, which helps form the oxyanion hole found in many proteases, is shown in green. Invariant residues and core hydrophobic residues found in the C39-family are shown in blue and magenta, respectively. Characterized C39-family member (GCN5-acyetyl transferase) and a C1-family member (Papain) are shown. b, Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 μm. c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source. d, Illustration of the yeast genomic clones isolated from the IpaJ suppressor screen. ARF1p, ARF2p and VPS15p are highlighted. e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.

Figure 3. IpaJ cleaves the N-myristoylated glycine of lipidated substrates

a–c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7). d, Structure of N-myristoylated ARF1 N terminus in untreated (left) and IpaJ-treated (right) samples. R group following Asn 3 denotes ARF1-strep protein residues 4–240. e, Reconstitution of ARF1-His N-myristoylation (N-myr) by NMTp in bacterial cells and IpaJ cleavage reaction in vitro (see Supplementary Information and Methods). f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain. g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel). h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.

Figure 4. Conformational-dependent cleavage of lipidated substrates by IpaJ

a, Multiple-sequence alignment of N-myristoylated glycine and downstream sequences from the indicated proteins (Hs, Homo sapiens; Sc, S. cerevisiae). b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide–eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS–polyacrylamide gel electrophoresis caused by proteolytic reaction. c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis. d, Fluorescence microscopy showing ARF1–mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 μm. e, Fluorescence microscopy showing ARF1–eGFP (green) and Golgi membranes (blue, GM130) after infection with Shigella M90T and the indicated mutants (red; mCherry-expressing bacteria). Scale bar, 10μm.