HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain-containing proteins is a poorly understood, yet highly conserved post-translational modification. Here, we describe the generation, evaluation, and application of four HypE-specific nanobodies: three that inhibit HypE-mediated target AMPylation in vitro and one that acts as an activator. All heavy chain-only antibody variable domains bind HypE when expressed as GFP fusions in intact cells. We observed localization of HypE at the nuclear envelope and further identified histones H2–H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression.     

An experimental strategy for the identification of AMPylation targets from complex protein samples

AMPylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete Fic proteins that AMPylate Rho GTPases and thus interfere with host cell signaling processes. Although Fic proteins are widespread and found in all kingdoms of life, only a small number of AMPylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMPylation events. Here, we present an unbiased MS‐based approach utilizing stable isotope‐labeled ATP. The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMPylation introducing specific reporter ion clusters that allow detection of AMPylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted Fic protein Bep2 of Bartonella rochalimae, we identified the filamenting protein vimentin as an AMPylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMPylation targets. Furthermore, the approach can be generically adapted to study targets of other PTMs that allow incorporation of isotopically labeled substrates.     

Fic蛋白家族的研究进展

过去的研究发现了一个具有相同保守结构域的Fic(filamentation induced by cAMP)蛋白家族。虽然在原核生物中发现超过3 000种以上含有Fic结构域的不同蛋白质,但到目前为止,在包括人在内的真核生物中仅发现一种Fic蛋白。Fic结构域主要通过含有磷酸基团的化合物在转录中起作用,目前被人类关注的功能是单酰磷酸化(AMPylation, AMP化),一种类似磷酸化的蛋白质调控机制,是以ATP为底物将一磷酸腺苷(AMP)特异地转移至靶标氨基酸残基。该机制主要在细菌侵袭宿主、侵袭后增殖、致病的过程中发挥作用。真核细胞中的单磷酸腺苷化可以调控内质网的稳定性。本文主要对Fic蛋白的特征性结构和作用以及几种主要的Fic蛋白的结构、作用和研究方法进行综述。     

Fido,a Novel AMPylation Domain Common to Fic,Doc, and AvrB

Background

The Vibrio parahaemolyticus type III secreted effector VopS contains a fic domain that covalently modifies Rho GTPase threonine with AMP to inhibit downstream signaling events in host cells. The VopS fic domain includes a conserved sequence motif (HPFx[D/E]GN[G/K]R) that contributes to AMPylation. Fic domains are found in a variety of species, including bacteria, a few archaea, and metazoan eukaryotes.

Methodology/Principal Findings

We show that the AMPylation activity extends to a eukaryotic fic domain in Drosophila melanogaster CG9523, and use sequence and structure based computational methods to identify related domains in doc toxins and the type III effector AvrB. The conserved sequence motif that contributes to AMPylation unites fic with doc. Although AvrB lacks this motif, its structure reveals a similar topology to the fic and doc folds. AvrB binds to a peptide fragment of its host virulence target in a similar manner as fic binds peptide substrate. AvrB also orients a phosphate group from a bound ADP ligand near the peptide-binding site and in a similar position as a bound fic phosphate.

Conclusions/Significance

The demonstrated eukaryotic fic domain AMPylation activity suggests that the VopS effector has exploited a novel host posttranslational modification. Fic domain-related structures give insight to the AMPylation active site and to the VopS fic domain interaction with its host GTPase target. These results suggest that fic, doc, and AvrB stem from a common ancestor that has evolved to AMPylate protein substrates.     

Fic and non-Fic AMPylases: protein AMPylation in metazoans

Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.     

Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu

The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.     

Alpha-Synuclein Is a Target of Fic-Mediated Adenylylation/AMPylation: Possible Implications for Parkinson's Disease

During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic-mediatedadenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE—alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation invitro, suggesting a possible mechanism by which cells cope with αSyn toxicity.     

Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone,BiP/GRP78

Fic (filamentation induced by cAMP) proteins regulate diverse cell signaling events by post-translationally modifying their protein targets, predominantly by the addition of an AMP (adenosine monophosphate). This modification is called Fic-mediated adenylylation or AMPylation. We previously reported that the human Fic protein, HYPE/FicD, is a novel regulator of the unfolded protein response (UPR) that maintains homeostasis in the endoplasmic reticulum (ER) in response to stress from misfolded proteins. Specifically, HYPE regulates UPR by adenylylating the ER chaperone, BiP/GRP78, which serves as a sentinel for UPR activation. Maintaining ER homeostasis is critical for determining cell fate, thus highlighting the importance of the HYPE-BiP interaction. Here, we study the kinetic and structural parameters that determine the HYPE-BiP interaction. By measuring the binding and kinetic efficiencies of HYPE in its activated (Adenylylation-competent) and wild type (de-AMPylation-competent) forms for BiP in its wild type and ATP-bound conformations, we determine that HYPE displays a nearly identical preference for the wild type and ATP-bound forms of BiP in vitro and preferentially de-AMPylates the wild type form of adenylylated BiP. We also show that AMPylation at BiP’s Thr366 versus Thr518 sites differentially affect its ATPase activity, and that HYPE does not adenylylate UPR accessory proteins like J-protein ERdJ6. Using molecular docking models, we explain how HYPE is able to adenylylate Thr366 and Thr518 sites in vitro. While a physiological role for AMPylation at both the Thr366 and Thr518 sites has been reported, our molecular docking model supports Thr518 as the structurally preferred modification site. This is the first such analysis of the HYPE-BiP interaction and offers critical insights into substrate specificity and target recognition.     

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.Protein AMPylation (adenylylation) was recently discovered in bacteria-host interactions where virulence factors catalyze AMPylation using either a conserved Fic domain (e.g., VopS, Vibrio parahaemolyticus (V. para) and IbpA, Histophilus somni) or an adenylyl transferase domain (e.g., DrrA, Legionella pneumophila). These bacterial AMPylation enzymes, or AMPylators, are secreted into the host cells by bacterial secretion systems and transfer AMP from ATP to Tyr or Thr residues of their respective substrates (1–3). In the case of VopS and IbpA, several Rho family GTPases (Rac1, RhoA, and Cdc42) are known substrates and AMPylation disrupts the binding of the GTPase to its downstream effectors, for example, PAK1 (2–6). Considering the conservation of AMPylation domains in both prokaryotic and eukaryotic organisms, we expect that AMPylation plays an important role in a wide range of cellular processes (2, 4, 5, 7–9). Nevertheless, our understanding of this post-translational modification (PTM) is still limited to only a handful of known eukaryotic AMPylation substrates, exclusively belonging to the Rho and Rab GTPase families(10–14). Determining the repertoire of substrates modified by AMPylators will help illuminate both the functional consequences of AMPylation and the mechanistic strategies of pathogens that employ them (6).Significant effort has been devoted to identifying AMPylation substrates. Li et al. systematically investigated the fragmentation patterns of chemically synthesized peptides with Thr, Ser, and Tyr AMPylation using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). They detected AMPylation sites with high confidence and selectively scanned AMPylated peptides in protein mixtures (10). Hao et al. produced a polyclonal antibody that specifically recognized proteins with AMPylation at threonine residues (11). Grammel et al. synthesized an ATP analog, N⁶pATP (N⁶-propargyl adenosine-5′-triphophate), which allows the labeling of AMPylated proteins with azide-functionalized fluorescein or a cleavable biotin enrichment tag (ortho-hydroxy-azidoethoxy-azobiotin) based on copper-catalyzed azide-alkyne cycloaddition (CuAAC)¹. The identification of new substrates for VopS in HeLa cell lysates was explored by a combination of AMP-specific pull-down and LC-MS (12). Using the same approach, Lewallen et al. tried to identify the substrates of VopS in MCF7 cell extracts by employing a commercial N⁶-(6-amino)hexyl-ATP-5-carboxyl-fluorescein (F1-ATP) and anti-fluorescein antibody(13). With these efforts combined, four potential new VopS substrates have been identified (SCCA2, NAGK, NME1, and PFKP), though not yet confirmed. These approaches might miss substrates because of temporal and spatial expression or low abundance in cell lysate, poor recognition by the capture molecules or loss during pull-down procedures (12, 14).Protein microarrays offer a promising approach to identify candidate substrates because they display thousands of unique proteins in a high-throughput and reproducible format (15–17). However, producing arrays with consistent levels of well-folded proteins is challenging because of limitations of protein production, purification, and storage, particularly for mammalian proteins (18).To circumvent these limitations, cell-free protein arrays, which do not require protein purification, have been developed over the past decade (19–22). These methods provide rapid and economical approaches of fabricating protein arrays in terms of cost, shelf life, and storage (23, 24). In cell-free protein arrays, a nucleotide template is printed on the slide and used to produce proteins in vitro with cell-free expression systems from several organisms such as E. coli, wheat germ, and rabbit reticulocyte lysate, etc. (24, 25). These proteins can be engineered to contain fusion tags that enable their capture to the array surface with an appropriate agent. Of these cell-free protein array methods, the Nucleic Acid Programmable Protein Array (NAPPA) is the most advanced, having achieved both high-density and high content containing ∼2300–8000 proteins per slide (20, 26, 27). In NAPPA, a plasmid-based cDNA configured to include an epitope tag is printed on a microscope slide along with the corresponding tag-specific binding reagent, such as an anti-tag antibody, and stored. At the time of experimentation, the cDNA is transcribed/translated into recombinant protein and captured/displayed in situ by the binding reagent. Using a rabbit reticulocyte lysate-based cell-free expression system, NAPPA has been applied toward the identification of novel protein-protein interactions and disease-related antibody biomarkers (20, 26, 28, 29). However, cell-free protein arrays have yet to be employed in the study of PTMs.In this work, we established a novel, nonradioactive unbiased AMPylation screening platform by developing a novel click chemistry-based detection assay for use on high-density cell-free protein microarrays displaying human proteins. Labeling AMP-modified substrates covalently with a fluorophore coupled with the use of human ribosomal machinery and chaperones to produce proteins achieved much higher sensitivity and signal to noise (S/N) ratio compared with previous studies. We screened 10,000 human proteins with two bacterial pathogen AMPylators, VopS and IbpAFic2, identifying more than twenty new substrates each. Two novel Rho GTPases (Rac2 and Rac3) were validated in vivo as substrates of the virulence factor VopS in HEK293T cells during V. para infection. Using mass spectrometry, we verified that a non-GTPase protein, ARHGDIB/LyGDI, was AMPylated by VopS on its threonine 51, which is located in a highly regulated part of this protein. This modification inhibited phosphorylation of LyGDI by Src kinase in vitro. Finally, the identification of these new targets allowed us to build the first bacteria-host interaction AMPylation network and may reveal signaling interactions that could potentially be important for bacterial pathogenesis in the future functional studies.     

Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP.     

A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response

The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP''s ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling.     

Highly conserved amino acids in Pax and Ets proteins are required for DNA binding and ternary complex assembly

Combinatorial association of DNA-binding proteins on composite binding sites enhances their nucleotide sequence specificity and functional synergy. As a paradigm for these interactions, Pax-5 (BSAP) assembles ternary complexes with Ets proteins on the B cell-specific mb-1 promoter through interactions between their respective DNA-binding domains. Pax-5 recruits Ets-1 to bind the promoter, but not the closely related Ets protein SAP1a. Here we show that, while several different mutations increase binding of SAP1a to an optimized Ets binding site, only conversion of Val68 to an acidic amino acid facilitates ternary complex assembly with Pax-5 on the mb-1 promoter. This suggests that enhanced DNA binding by SAP1a is not sufficient for recruitment by Pax-5, but instead involves protein–protein interactions mediated by the acidic side chain. Recruitment of Ets proteins by Pax-5 requires Gln22 within the N-terminal β-hairpin motif of its paired domain. The β-hairpin also participates in recognition of a subset of Pax-5-binding sites. Thus, Pax-5 incorporates protein–protein interaction and DNA recognition functions in a single motif. The Caenorhabditis elegans Pax protein EGL-38 also binds specifically to the mb-1 promoter and recruits murine Ets-1 or the C.elegans Ets protein T08H4.3, but not the related LIN-1 protein. Together, our results define specific amino acid requirements for Pax–Ets ternary complex assembly and show that the mechanism is conserved between evolutionarily related proteins of diverse animal species. Moreover, the data suggest that interactions between Pax and Ets proteins are an important mechanism that regulates fundamental biological processes in worms and humans.     

The SH3 domains of two PCH family members cooperate in assembly of the Schizosaccharomyces pombe contractile ring

Schizosaccharomyces pombe cdc15 homology (PCH) family members participate in many cellular processes by bridging the plasma membrane and cytoskeleton. Their F-BAR domains bind and curve membranes, whereas other domains, typically SH3 domains, are expected to provide cytoskeletal links. We tested this prevailing model of functional division in the founding member of the family, Cdc15, which is essential for cytokinesis in S. pombe, and in the related PCH protein, Imp2. We find that the distinct functions of Imp2 and Cdc15 are SH3 domain independent. However, the Cdc15 and Imp2 SH3 domains share an essential role in recruiting proteins to the contractile ring, including Pxl1 and Fic1. Together, Pxl1 and Fic1, a previously uncharacterized C2 domain protein, add structural integrity to the contractile ring and prevent it from fragmenting during division. Our data indicate that the F-BAR proteins Cdc15 and Imp2 contribute to a single biological process with both distinct and overlapping functions.     

Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases

Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.KEY WORDS: Caenorhabditis elegans, Cell non-autonomous proteotoxicity, Prion-like spreading     

CPNA-1, a copine domain protein,is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans

We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1–null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans.     

Identification and characterization of Crumbs polarity complex proteins in Caenorhabditis elegans

Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting the formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including the scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here, we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1 and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on the physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues and, therefore, not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.     

AMPylation of Rho GTPases Subverts Multiple Host Signaling Processes

Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules. Herein, we demonstrate that AMPylation of Rho GTPases by VopS is a multifaceted virulence mechanism that counters several host immunity strategies. Activation of NFκB, Erk, and JNK kinase signaling pathways were inhibited in a VopS-dependent manner during infection with Vibrio parahaemolyticus. Phosphorylation and degradation of IKBα were inhibited in the presence of VopS as was nuclear translocation of the NFκB subunit p65. AMPylation also prevented the generation of superoxide by the phagocytic NADPH oxidase complex, potentially by inhibiting the interaction of Rac and p67. Furthermore, the interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a nucleic acid programmable protein array and discovered that Rac1 binds to the protein C1QA, a protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted by AMPylation. We conclude that AMPylation of Rho Family GTPases by VopS results in diverse inhibitory consequences during infection beyond the most obvious phenotype, the collapse of the actin cytoskeleton.