Site-Specific Nitrosoproteomic Identification of Endogenously S-Nitrosylated Proteins in Arabidopsis

Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.Nitric oxide (NO), a gaseous signaling molecule, plays important regulatory roles in higher plants, including seed dormancy and germination, root development and hypocotyl elongation, floral transition, senescence and cell death, phytohormone signaling, and responses to abiotic and biotic stresses (He et al., 2004; Besson-Bard et al., 2008; Hong et al., 2008; Neill et al., 2008; Leitner et al., 2009; Feng et al., 2013). S-Nitrosoglutathione (GSNO) is a major biologically active form of reactive nitrogen species (RNS) and functions as a primary NO donor. The endogenous GSNO homeostasis is highly dynamic, and the GSNO level is negatively regulated by GSNO reductase (GSNOR), an evolutionally conserved enzyme catalyzing irreversibly degrading GSNO (Liu et al., 2001). Mutations in the GSNOR gene cause the elevated GSNO level and consequently severe abnormalities under physiological and pathological conditions in various species (Liu et al., 2004; Feechan et al., 2005; Que et al., 2005; Lee et al., 2008; Chen et al., 2009; Moore et al., 2009; Kwon et al., 2012).In Arabidopsis (Arabidopsis thaliana), GSNOR1 is a single-copy gene, and the enzymatic activity of the encoded protein has been biochemically characterized (Sakamoto et al., 2002). Genetic studies revealed that the gsnor1-1 and gsnor1-2 mutants are gain-of-function mutations with increased GSNOR activity and a decreased cellular S-nitrosothiol level. Conversely, gsnor1-3 is a loss-of-function mutant with a significantly increased S-nitrosothiol level (Feechan et al., 2005). The defense responses mediated by distinct resistance (R) genes are significantly impaired in the gsnor1-3 mutant, and GSNOR1 functions as a positive regulator of the salicylic acid-regulated signaling network in the defense response (Feechan et al., 2005). In a genetic screen for thermotolerance-defective mutants, the sensitive to hot temperatures5 (hot5) mutant was characterized as having decreased heat acclimation and was shown to be allelic to gsnor1, indicating the importance of GSNOR1-regulated NO homeostasis in the regulation of the abiotic stress response (Lee et al., 2008). In an independent genetic screen for the oxidative stress-related mutants, the paraquat resistant2 (par2) mutant was also identified to be allelic to gsnor1, which showed an anti-cell death phenotype and multiple developmental defects, revealing the critical role of GSNOR1/HOT5/PAR2 in the regulation of oxidative stress-induced cell death (Chen et al., 2009). Similar to gsnor1-3, the hot5 and par2 allelic mutants also accumulate the significantly increased level of NO. As a result of this defect, these gsnor1/hot5/par2 mutants show a pleiotropic phenotype, with severe developmental abnormalities in both reproductive and vegetative stages (Lee et al., 2008; Chen et al., 2009; Kwon et al., 2012). These studies highlight the critical role of GSNOR1/HOT5/PAR2-modulated NO homeostasis in diverse physiological processes, including plant growth and development as well as in responses to both biotic and abiotic stresses. However, little is known about the underpinning molecular mechanisms of the NO-modulated signaling in various physiological processes.A major physiological effect of NO is executed by protein S-nitrosylation, a reversible posttranslational modification by covalent addition of an NO molecule onto a Cys thiol to form S-nitrosothiol (Jaffrey et al., 2001; Stamler et al., 2001). S-Nitrosothiols are dynamically labile in response to the intracellular redox status, allowing protein S-nitrosylation as a highly sensitive mechanism in the regulation of cellular signaling (Stamler et al., 2001; Hess et al., 2005). Emerging evidence indicates that S-nitrosylation regulates the function of the modified proteins by various mechanisms, including enzymatic activity, stability, subcellular localization, three-dimensional conformation changes, protein-protein interaction, and ligand binding (Hess et al., 2005; Wang et al., 2006; Astier et al., 2011; Gupta, 2011; Hess and Stamler, 2012). In Arabidopsis, S-nitrosylation has been shown as an important mechanism in regulating the stress responses. The activity of Met adenosyltransferase1 (MAT1), which catalyzes S-adenosyl-Met synthesis, was shown to be inhibited by S-nitrosylation (Lindermayr et al., 2006). S-nitrosylation negatively regulates the activity of a peroxynitrite detoxification enzyme, peroxiredoxin II E (PrxII E), and an NADPH oxidase, thereby modulating the oxidative stress in the defense response (Romero-Puertas et al., 2007; Yun et al., 2011). Moreover, S-nitrosylation has also been shown to regulate the conformational changes of NONEXPRESSOR OF PATHOGEN-RELATED1 (NPR1), a master regulator of the defense response, and the activity of SALICYLIC ACID-BINDING PROTEIN3 (SABP3), a key enzyme for salicylic acid biosynthesis (Tada et al., 2008; Wang et al., 2009). In addition, S-nitrosylation of TRANSPORT INHIBITOR RESPONSE1 (TIR1) and Arabidopsis Histidine Phosphotransfer Protein1 (AHP1), two key signaling components of the auxin and cytokinin pathways, respectively, plays an important role in regulating respective phytohormone signaling (Terrile et al., 2012; Feng et al., 2013). These studies illustrate the importance of S-nitrosylation in the regulation of diverse physiological processes in plants.S-Nitrosylation has been considered as one of the most important posttranslational modification mechanisms (Lane et al., 2001; Stamler et al., 2001; Hess et al., 2005). A growing number of S-nitrosylated proteins have been identified using the proteomic approach. To date, the S-nitrosoproteomic studies have identified more than 2,200 S-nitrosylated proteins, covering more than 4,100 S-nitrosylated Cys residues. Of those S-nitrosylated proteins, more than 95% were identified from mammals (Lee et al., 2012). Several proteomic studies in Arabidopsis identified a number of S-nitrosylated proteins (Lindermayr et al., 2005; Romero-Puertas et al., 2008; Palmieri et al., 2010; Fares et al., 2011; Puyaubert et al., 2014). In GSNO-treated cell suspension cultures and NO-treated leaves derived from Arabidopsis, 63 and 52 S-nitrosylated proteins were identified, which are involved in stress response, redox homeostasis, cytoskeleton organization, metabolic processes, and cellular signaling (Lindermayr et al., 2005). In an independent study, 16 S-nitrosylated proteins were identified from Arabidopsis seedlings undergoing the hypersensitive response (Romero-Puertas et al., 2008). In another independent analysis, 46 S-nitrosylated proteins were identified from cultured Arabidopsis suspension cells (Fares et al., 2011). In a more specific analysis, 11 mitochondria proteins were identified to be S-nitrosylated and/or glutathionylated (Palmieri et al., 2010). More recently, 62 endogenously S-nitrosylated proteins were identified from Arabidopsis seedlings (Puyaubert et al., 2014). Notably, a large number of the S-nitrosylated proteins are repeatedly identified in these analyses, thus confirming the validation of each study. Because of the labile nature of S-nitrosylation, most of the S-nitrosoproteomic studies used the protein samples treated with NO donors or the protein extracts prepared from NO donor-treated cells or tissues. The Arabidopsis gsnor1-3 mutants accumulate an excessive amount of NO (Feechan et al., 2005; Lee et al., 2008; Chen et al., 2009), and the identification of S-nitrosylated proteins in gsnor1-3 should depict a more comprehensive map of S-nitrosoproteome in Arabidopsis, and provide important clues on the molecular basis of the pleiotropic phenotype of the mutant.Because of the labile and dynamic nature of protein S-nitrosylation, large-scale identification of endogenously S-nitrosylated proteins remains technically challenging. At present, two major methods for identification of S-nitrosoproteome are shotgun and site-specific nitrosoproteomic analysis, both of which are based on the biotin-switch method and mass spectrometry (Jaffrey et al., 2001; Hao et al., 2006; Torta et al., 2008). In the shotgun analysis, S-nitrosylated proteins were first biotinylated, enriched by affinity-chromatography, and then identified by mass spectrometry. Although the method is relatively simple, the number of S-nitrosylated proteins identified by shotgun proteomics is often few due to various technical limitations (Torta et al., 2008). The identification capacity of nitrosoproteomics was greatly improved by the site-specific strategy, in which biotinylated proteins were first digested by trypsin and the enriched peptides were then characterized by mass spectrometry (Hao et al., 2006; Chen et al., 2010). Moreover, S-nitrosylated Cys residues can also be identified from site-specific nitrosoproteomic analysis.In this study, we performed a large-scale, site-specific proteomic analysis of endogenously S-nitrosylated proteins in Arabidopsis wild-type and gsnor1-3 seedlings, and identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the model plant species, representing the largest data set thus far reported in any organisms and providing important resources for future studies on S-nitrosylation-regulated signaling in plants.     

Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys⁷³ only after the formation of a Cys³²-Cys³⁵ disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1^C32S/C35S (a mutant Trx1 with both Cys³² and Cys³⁵ replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys¹⁷³ and Cys⁸³ and protects it from H₂O₂-induced overoxidation. Functionally, we found that Cys⁷³-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation.Nitric oxide (NO) is an important second messenger for signal transduction in cells. The production of cGMP by guanylyl cyclase, enabled by the binding of NO onto heme, is considered the primary mechanism responsible for the plethora of functions exerted by NO (1). However, S-nitrosylation, the covalent addition of the NO moiety onto cysteine thiols, is increasingly recognized as an important post-translational modification for regulating protein functions (for reviews, see Refs. 2 and 3). S-Nitrosylation is dynamic, reversible, site-specific, and modulated by selected cellular stimuli (4–7). With improved detection sensitivity, an increasing number of S-nitrosylated proteins have been identified by proteomics technologies (5, 8–13). Among the known modified proteins, nitrosylation occurs only on selected cysteines (4, 6, 14–17). Non-enzymatic mechanisms proposed to determine S-nitrosylation specificity include the availability of specific NO donors and protein microenvironments that stabilize the pK_a of acidic target cysteines (18). Furthermore, several enzymes, including hemoglobin (19, 20), superoxide dismutase 1 (21, 22), S-nitrosoglutathione reductase (23–25), and protein-disulfide isomerase (26), have been shown to possess either transnitrosylase or denitrosylase activities. However, an enzymatic system that governs site-specific transnitrosylation and denitrosylation, analogous to the kinase/phosphatase paradigm for regulating protein phosphorylation, has remained largely uncharacterized.Trx1¹ is an important antioxidant protein with protein reductase activity (27, 28). It has been characterized as an antiapoptotic protein because of its ability to suppress proapoptotic proteins, including apoptosis signal-regulating kinase 1 via disulfide reduction and Casp3 via transnitrosylation of Cys¹⁶³ (14, 29). Conversely, Trx1 can denitrosylate Casp3 at Cys¹⁶³, resulting in Casp3 activation (7). Trx1 appears to govern site-specific reversible nitrosylation of selected protein targets (14, 15), but what are the underlying mechanisms that regulate Trx1 transnitrosylation and denitrosylation activities? Are there additional Trx1-mediated transnitrosylation or denitrosylation targets that have not yet been identified? In this study, we used ESI-Q-TOF mass spectrometry (MS) to analyze the nitrosylation of Trx1 and a Casp3 peptide (Casp3p) under different redox conditions. Because of the labile nature of the S–NO bond, direct identification of S-nitrosylated proteins and their specific nitrosylation sites by MS remains challenging (8). A biotin switch method that is based on the derivatization of protein S–NO with a biotinylating agent is typically used for such analyses (8). However, like any indirect method, both false positive and negative identifications have been reported (30). Recently, we developed a method for direct analysis of protein S-nitrosylation by ESI-Q-TOF MS without prior chemical derivatization (31). Here we applied the same technique to determine the regulation of Trx1 by stepwise oxidative and nitrosative modifications of distinct cysteines and its subsequent ability to transnitrosylate target proteins. Nitrosative modification at Cys⁷³ of Trx1 cannot occur without prior attenuation of the Trx1 disulfide reductase and denitrosylase activities via either disulfide bond formation between Cys³² and Cys³⁵ or their mutation to serines. This is a key observation that has never been previously reported. Consequently, we designed a proteomics approach and discovered over 40 putative Trx1 transnitrosylation target proteins. We further characterized the Trx1 transnitrosylation proteome and identified three consensus motifs surrounding the putative Trx1 transnitrosylation sites, suggesting a protein-protein interaction mechanism for determining transnitrosylation specificity.     

Transnitrosylating Nitric Oxide Species Directly Activate Type I Protein Kinase A, Providing a Novel Adenylate Cyclase-independent Cross-talk to ��-Adrenergic-like Signaling

The transnitrosylating nitric oxide (NO) donor nitrocysteine (CysNO) induced a disulfide bond between the two regulatory RI subunits of protein kinase A (PKA). The conventional NO donor S-nitroso-N-acetylpenicillamine failed to do this, consistent with our observation that it also did not promote protein S-nitrosylation. This disulfide oxidation event activated PKA and induced vasorelaxation independently of the classical β-adrenergic or NO signaling pathway. Activation of PKA had also been anticipated to exert a positive inotropic effect on the myocardium but did not. The lack of positive inotropy was explained by CysNO concomitantly activating protein kinase G (PKG) Iα. PKG was found to exert a partial negative inotropic influence regardless of whether PKA was activated by classical β-receptor stimulation or by disulfide bond formation. This work demonstrates that NO molecules that can induce S-nitrosylation directly activate type I PKA, providing a novel cross-talk to β-adrenergic-like signaling without receptor or adenylate cyclase stimulation. However, the expected positive inotropic consequences of PKA activation by this novel mechanism are countermanded by the simultaneous dual activation of PKGIα, which is also activated by CysNO.Nitric oxide (NO) initiates cell signaling by binding and activating soluble guanylate cyclase (sGC)² to produce the second messenger cGMP. cGMP primarily allosterically activates protein kinase G (PKG) but can also regulate other proteins. Although this NO-sGC-cGMP-PKG pathway is well defined (1), a second major mechanism of NO-dependent regulation has subsequently emerged. This involves NO covalently adducting to protein thiols, a process known as S-nitrosylation or S-nitrosation (2).Significant evidence continues to accumulate supporting protein S-nitrosylation as a fundamental regulator of protein and thus cell function (3). NO is produced in a regulated way (4), with a defined structural basis for selectivity in the proteins it covalently modifies (5, 6). Additional regulatory control can be achieved by the localization of NO synthase enzymes proximal to target proteins (6) and by reverse denitrosylation being enzymatically controlled (7). Indeed, many proteins appear to be basally S-nitrosylated, offering the potential for attenuation (8) as well as potentiation of signaling.Although stable regulatory S-nitrosylation occurs in some proteins, in others, it serves as an intermediate prior to transition to other redox states, especially disulfides (9). Previously, we searched for proteins that form interprotein disulfides in response to hydrogen peroxide (H₂O₂), identifying the regulatory RI subunit of protein kinase A (PKA) as such a protein (10, 11). This appears to activate the kinase (11), although the mechanism is not yet precisely defined. There is a rational structural basis for interprotein disulfide formation in PKA RI in response to H₂O₂. The RI dimer is held together by an N-terminal amphipathic leucine zipper in which the monomers are aligned antiparallel to each other with both Cys¹⁷ residues directly facing the corresponding Cys³⁸ residues on the opposite chains (12). H₂O₂-mediated RI disulfide formation is likely via protein sulfenic acid formation by one thiol in the Cys¹⁷ and Cys³⁸ disulfide-forming pair, prior to reduction by the other cysteine to yield the covalently conjugated dimer. Intriguingly, this pair of thiol-disulfide switches in RI is located directly on either side of the protein kinase A anchor protein-binding domain (13). This provides a rational structural basis for the PKA RI-protein kinase A anchor protein interaction being redox-modulated, as the interaction is strongly anticipated to change depending on the oxidation state of the cysteine switches, which flank the interaction locus (11).We hypothesized that NO may also be able to drive RI disulfide formation via an S-nitrosylated catalytic redox intermediate in a mechanism analogous to transient sulfenation formation during H₂O₂-induced covalent conjugation. This conceptual link between NO and PKA was investigated by comparing the biochemical and functional responses of cardiovascular tissue to the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and nitrocysteine (CysNO). The authentic NO donor SNAP did not promote RI disulfide formation, whereas CysNO did so efficiently, consistent with its established thiol-oxidizing transnitrosylating ability. We show that disulfide-mediated activation of PKA significantly contributes to vasorelaxation induced by CysNO. However, disulfide activation of PKA failed to exert a positive inotropic influence in isolated hearts exposed to CysNO, which was difficult to reconcile with the kinase being truly activated by oxidation. Further investigations showed that this lack of positive inotropy following CysNO-induced oxidation is explained by the co-activation of PKGIα, which we demonstrated previously can be disulfide-activated (15). PKGIα serves as a master regulator of cardiac inotropy, dominating the system to prevent increases in cardiac contractility. Thus, thiol-oxidizing derivatives of NO can activate PKA and so exert β-adrenergic-like signaling, although dual activation of PKG prevents the anticipated positive inotropy.     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Delineation of a Carcinogenic Helicobacter pylori Proteome

Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet only a fraction of infected persons ever develop cancer. The extensive genetic diversity inherent to this pathogen has precluded comprehensive analyses of constituents that mediate carcinogenesis. We previously reported that in vivo adaptation of a non-carcinogenic H. pylori strain endowed the output derivative with the ability to induce adenocarcinoma, providing a unique opportunity to identify proteins selectively expressed by an oncogenic H. pylori strain. Using a global proteomics DIGE/MS approach, a novel missense mutation of the flagellar protein FlaA was identified that affects structure and function of this virulence-related organelle. Among 25 additional differentially abundant proteins, this approach also identified new proteins previously unassociated with gastric cancer, generating a profile of H. pylori proteins to use in vaccine development and for screening persons infected with strains most likely to induce severe disease.Helicobacter pylori is a Gram-negative bacterial species that selectively colonizes gastric epithelium and induces an inflammatory response within the stomach that persists for decades (1, 2). Biological costs incurred by the long term relationship between H. pylori and humans include an increased risk for distal gastric adenocarcinoma (3–8), and eradication of this pathogen significantly decreases cancer risk among infected individuals without premalignant lesions (9). However, only a fraction of colonized persons ever develop neoplasia, and enhanced cancer risk is related to H. pylori strain differences, inflammatory responses governed by host genetic diversity, and/or specific interactions between host and microbial determinants (10).H. pylori strains are remarkably diverse (11–15), and the genetic composition of strains can change over time within an individual colonized stomach (16, 17). Despite this diversity, several genetic loci have been identified that augment disease risk. The cag pathogenicity island encodes a type IV bacterial secretion system, and the product of the terminal gene in this island, CagA, is translocated into host epithelial cells by the cag secretion system following adherence (18–20). Within the host cell, CagA undergoes Src- and Abl-dependent tyrosine phosphorylation (21) and activates the eukaryotic phosphatase SHP-2, leading to dephosphorylation of host cell proteins and cellular morphological changes (19–21). CagA also dysregulates β-catenin signaling (22, 23) and apical-junctional complexes (24), events linked to increased cell motility and oncogenic transformation in several models (25, 26). Another H. pylori constituent linked to gastric cancer is the cytotoxin VacA, encoded by the gene vacA, which is present in virtually all H. pylori strains (27). In vitro, VacA induces the formation of intracellular vacuoles (27) and can induce apoptosis (28), and vacuolating activity is significantly associated with the presence of the cag pathogenicity island (3).Approximately 20% of H. pylori bind to gastric epithelial cells in vivo (29), and sequence analysis has revealed that the H. pylori genome contains an unusually high number of ORFs relative to its genome size that are predicted to encode outer membrane proteins (15). BabA, a member of a family of highly conserved outer membrane proteins and encoded by the strain-specific gene babA2, binds the Lewis^b histo-blood group antigen on gastric epithelial cells (30, 31), and H. pylori babA2⁺ strains are associated with an increased risk for gastric cancer (30). However, not all persons infected with cag⁺ babA2⁺ toxigenic strains develop gastric cancer, indicating that additional H. pylori constituents are important in carcinogenesis.We recently identified a strain of H. pylori, 7.13, that reproducibly induces gastric cancer in two rodent models of gastritis, Mongolian gerbils and hypergastrinemic INS-GAS mice (22). This strain was derived via in vivo adaptation of a clinical H. pylori strain, B128, which induces inflammation, but not cancer, in rodent gastric mucosa. The oncogenic 7.13 phenotype is not due to an enhanced ability of strain 7.13 to colonize as there were no significant differences in gastric colonization density or efficiency between strains B128 and 7.13 as assessed by either quantitative culture or histology. However, carcinogenic strain 7.13 binds more avidly to gastric epithelial cells in vitro than does strain B128, suggesting that the two strains may variably express different outer membrane proteins.To define proteins that may mediate the development of H. pylori-induced gastric cancer, we performed two-dimensional (2D)¹ DIGE coupled with MS to identify differentially abundant membrane-associated and cytosolic proteins from non-carcinogenic H. pylori strain B128 and its carcinogenic derivative, strain 7.13 (22). DIGE/MS is a well established proteomics technology based on conventional 2D gel protein separations whereby prelabeling samples with spectrally resolvable fluorescent dyes and multiplexing samples onto a series of gels that contain a mixture of all experimental samples (internal standard) provide quantitative data on abundance changes for thousands of intact proteins from multiple experimental conditions, each measured in replicate for statistical confidence (32–36). Techniques including DIGE/MS have recently been utilized to robustly define differences in protein abundance profiles between bacterial strains and to compare expression patterns of proteins harvested from bacteria maintained under different growth conditions (37, 38).Utilizing DIGE/MS, we detected and identified 26 proteins with statistically significant differences between strains B128 and 7.13, including a novel cysteine-to-arginine mutation in the H. pylori flagellar protein FlaA. We demonstrate that this FlaA mutation results in structural and functional aberrations. Application of this technique to two genetically related bacterial strains that induce distinct phenotypes also identified several novel candidate H. pylori virulence factors, providing a framework for studies targeting the pathogenesis of microbially induced cancer.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).