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.     

A Second-generation Protein–Protein Interaction Network of Helicobacter pylori

Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein–protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.Helicobacter pylori is a Gram-negative, microaerophilic bacterium that colonizes the stomach, an unusual highly acidic niche for microorganisms. In 1983, Warren and Marshall found it to be associated with gastric inflammation and duodenal ulcer disease (1, 2). A chronic infection with H. pylori can lead to development of stomach carcinoma and MALT lymphoma (reviewed in (3)). Hence, the World Health Organization has classified H. pylori as a class I carcinogen (4). It is estimated that half of the world′s population harbors H. pylori but with large variations in the geographical and socioeconomic distribution while causing annually 700,000 deaths worldwide (reviewed in (5)).The pathogenesis of H. pylori has been extensively studied, including the effector CagA, cytotoxin VacA, its adhesins and urease (reviewed in (3, 5–7)). The latter allows the bacterium to neutralize the stomach acid through ammonia production. However, H. pylori is not a classical model organism and thus many gaps in our knowledge still exist.The genome of H. pylori reference strain 26695 was completely sequenced in 1997 (8) and encodes 1587 proteins of which about 950 (61%) have been assigned functions (excluding “putatives”; Uniprot, CMR (9)). These numbers indicate that a large fraction of the proteins of H. pylori has not been functionally characterized.Protein–protein interactions (PPIs)¹ are required for nearly all biological processes. Unbiased interactomes are helpful to understand proteins or pathways and how they are linking poorly or uncharacterized proteins via their interactions. For instance, our study of the Treponema pallidum interactome (10) has led to the characterization of several previously “unknown” proteins such as YbeB, a ribosomal silencing factor (11), or TP0658, a regulator of flagellar translation and assembly (12, 13). However, only a few other comprehensive bacterial interactome studies have been published to date, including Campylobacter jejuni (14), Synechocystis sp. (15), Mycobacterium tuberculosis (16), Mesorhizobium loti (17), and recently Escherichia coli (18). In addition, partial interactomes are available for Bacillus subtilis (19) and H. pylori (20). Most of them used the yeast two-hybrid (Y2H) screening technology (21) which allows the pairwise detection of PPIs. Furthermore, a few other studies (22–25) systematically identified protein complexes and their compositions in bacteria.In 2001, Rain and colleagues have established a partial interactome of H. pylori, the first published protein interaction network of a bacterium (20). In this study, 261 bait constructs were screened against a random prey pool library resulting in the detection of over 1500 PPIs. Although this network likely represents a small fraction of all PPIs that occur in H. pylori, many downstream studies were motivated by these results (see below).Recent studies have disproved the notion that Y2H data sets are of poor quality (26, 27). Similarly, a high false-negative rate can be avoided by multiple Y2H expression vector systems (28–30) or protein fragments as opposed to full-length constructs (31). The aim of this study was to systematically screen the H. pylori proteome for binary protein interactions using a complementary approach to that of Rain et al. to produce an extended protein–protein interaction map of H. pylori. As a result, we have roughly doubled the number of known binary protein–protein interactions for H. pylori in this study.     

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS),² a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7–9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13–15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18–20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with TopoIIIα and BLAP75 (22–24). BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yeast homolog Sgs1 may play a role at the initial steps of DNA double-strand break repair by participating in exonucleolitic resection of the DNA ends to generate DNA molecules with the 3′-ssDNA tails, a substrate for RAD51 binding (26–29).In vivo, the process of HR is tightly regulated by various mechanisms (30). Whereas some proteins promote HR (14, 31), others inhibit this process, thereby preventing its untimely initiation (32, 33). Disruption of the Rad51-ssDNA nucleoprotein filament appears to be an especially important mechanism of controlling HR. This filament disruption activity was demonstrated for the yeast Srs2 helicase (34, 35) and human RECQ5 helicase (36). Recently, we found that BLM can also catalyze disruption of the RAD51-ssDNA filament (25). This disruption only occurs if the filament is present in an inactive ADP-bound form, e.g. in the presence of Mg²⁺. Conversion of RAD51 into an active ATP-bound form, e.g. in the presence of Ca²⁺ (37), renders the filament resistant to BLM disruption (25). In this study, we analyze the interactions of BLM with an active ATP-bound RAD51-ssDNA filament. Surprisingly, we found that BLM stimulates the DNA strand exchange activity of RAD51. Thus, depending on the conformational state of the RAD51 nucleoprotein filament, BLM may either inhibit or stimulate the DNA strand exchange activity of RAD51. Our analysis demonstrated that, in contrast to several known stimulatory proteins that act by promoting formation of the RAD51-ssDNA filament, BLM stimulates the DNA strand exchange activity of RAD51 at a later stage, during synapsis. Stimulation appears to be independent of the ATPase activity of BLM. We suggest that this stimulation of RAD51 may represent a novel function of BLM in homologous recombination.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Phylogenetic Ubiquity and Shuffling of the Bacterial RecBCD and AddAB Recombination Complexes

RecBCD and AddAB are bacterial enzymes that share similar helicase and nuclease activities and initiate repair of DNA double-strand breaks by homologous recombination. Examination of the phylogenetic distribution of AddAB and RecBCD revealed that one or the other complex is present in most sequenced bacteria. In addition, horizontal gene transfer (HGT) events involving addAB and recBCD appear to be common, with the genes encoding one complex frequently replacing those encoding the other. HGT may also explain the unexpected identification of archaeal addAB genes. More than 85% of addAB and recBCD genes are clustered on the genome, suggesting operon structures. A few organisms, including the Mycobacteria, encode multiple copies of these complexes of either the same or mixed classes. The possibility that the enzymatic activities of the AddAB and RecBCD enzymes promote their horizontal transfer is discussed, and the distribution of AddAB/RecBCD is compared to that of the RecU/RuvC resolvases. Finally, it appears that two sequence motifs, the Walker A box involved in ATP binding and an iron-sulfur-cysteine cluster, are present only in subsets of AddB proteins, suggesting the existence of mechanistically distinct classes of AddB.Homologous recombination is central to the repair of DNA damaged by single-strand (SS) and double-strand (DS) breaks and gaps. Such discontinuities can occur after exposure to exogenous agents such as ionizing radiation but also when DNA replication forks break and as intermediates in DNA repair processes. Homologous recombination can also rearrange genetic information, making it important in processes such as phase variation of bacterial outer membrane proteins (for an example, see reference 2).Homologous recombination is highly conserved among viruses, bacteria, archaea, and eukaryotes. There are three recognizable stages (for reviews, see references 25 and 35). In the first stage, presynapsis, the DNA substrate is processed to give a SS region coated with strand exchange protein(s). In bacteria, this protein is RecA. In the second stage, synapsis, the protein-DNA complex pairs with its complementary homologous DNA target, displacing the other strand at the target site and forming a joint molecule. In the final stage, postsynapsis, DNA replication fills in any gaps in the joint molecule, which is then resolved to give recombinant DNA products.In bacteria, there appear to be two different presynaptic pathways that use either the related AddAB or RecBCD holoenzymes (Fig. ​(Fig.1)1) or, alternatively, the RecFOR proteins. RecFOR proteins appear to operate on SS gaps to ensure RecA is loaded there, whereas RecBCD and AddAB act on DNA DS breaks (DSBs), processing them to yield SS 3′ DNA ends coated with RecA (3, 43).Open in a separate windowFIG. 1.Structure of the RecBCD and AddAB proteins. The RecB, RecD, and AddA proteins include a helicase domain (solid green) with the canonical six-helicase motifs of helicase superfamily I. The most N-terminal of these motifs is the Walker A box (orange). The RecC and AddB proteins include an inactivated helicase domain (striped green). The RecB, AddA, and AddB proteins additionally possess similar short nuclease domains toward their C termini (red). Some, but not all, AddB proteins possess an N-terminal Walker A and/or a mostly C-terminal iron-sulfur motif made up of four cysteines (blue).The RecBCD complex has most thoroughly been studied in Escherichia coli, a member of the Gamma class of the phylum Proteobacteria. RecB and RecD are superfamily I helicases, and RecB is also a nuclease. After the RecBCD complex loads onto a DS DNA end, the RecB and RecD helicases separate the strands, with the slower RecB helicase traveling on one strand in the 3′-to-5′ direction and the faster RecD helicase traveling on the other strand from 5′ to 3′ (38). During translocation, E. coli RecBCD recognizes a specific DNA sequence, Chi (5′-GCTGGTGG-3′), perhaps via the RecC protein, which appears to be an inactivated helicase (Fig. ​(Fig.1)1) (32, 34). Chi is a recombination hot spot that switches the complex to a recombinogenic mode, producing a 3′ SS end at the Chi site via the nuclease activity of RecB (for reviews, see references 25 and 35). In addition to producing a 3′ SS “tail” needed for recombination, the RecBCD complex actively loads the RecA strand exchange protein onto this DNA (5). In E. coli, the RecBCD complex is responsible for essentially all recombinational repair of DSBs.Bacillus subtilis and some other bacteria lack RecBCD but possess a different complex with many of the same activities. This complex is composed of the AddA and AddB proteins (22). Like RecB, AddA is a superfamily I helicase and a nuclease (18, 21), organized with the same domain structure as RecB (Fig. ​(Fig.1).1). Similar to RecC, AddB appears to be an inactivated helicase, but it also possesses a nuclease domain similar to those of RecB and AddA (Fig. ​(Fig.1)1) (2, 11, 43). Like RecBCD, AddAB is an ATP-dependent helicase that acts as a nuclease in conjunction with its helicase activity, with the AddA nuclease acting on the 3′-end strand and the AddB nuclease on the 5′-end strand (43). Also like RecBCD, AddAB recognizes Chi-like control sequences, for instance, 5′-GCGCGTG-3′ in Lactococcus lactis and 5′-AGCGG-3′ in B. subtilis (7, 12). The AddAB complex is required for recombinational repair of DSBs (1, 2, 44), but it is not known if, like RecBCD, it actively loads RecA.Both RecBCD and AddAB are important for bacterial pathogenicity. addAB mutations reduce the ability of Helicobacter pylori to colonize mouse stomachs, and recBC mutants of Salmonella enterica serovar Typhimurium are severely compromised for infection and killing (2, 8).Using a strict set of criteria, Rocha et al. (30) examined the distribution across bacteria of the RecBCD and AddAB enzymes, along with other recombination and DNA repair proteins. They determined that the AddAB proteins are ubiquitous in some taxa and the RecBCD proteins in others, while yet other taxa have examples of both complexes in different species. In addition, they classified several species as lacking both the AddAB and RecBCD complexes. Recently, addAB genes from Epsilonproteobacteria were identified (2, 27, 40). Previously, all examined members of this proteobacterial class had been classified by Rocha et al. as lacking every component of the RecBCD and AddAB systems. This finding suggests that the stringent criteria used by Rocha et al. (30) might have led them to miss other examples of AddAB and RecBCD from sequenced bacteria. On this basis, and to gain a fuller understanding of the importance of the AddAB and RecBCD enzymes, their phylogenetic distribution was reexamined.     

Helicobacter pylori CagA Causes Mitotic Impairment and Induces Chromosomal Instability

Infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric carcinoma. The cagA gene product CagA, which is delivered into gastric epithelial cells, specifically binds to and aberrantly activates SHP-2 oncoprotein. CagA also interacts with and inhibits partitioning-defective 1 (PAR1)/MARK kinase, which phosphorylates microtubule-associated proteins to destabilize microtubules and thereby causes epithelial polarity defects. In light of the notion that microtubules are not only required for polarity regulation but also essential for the formation of mitotic spindles, we hypothesized that CagA-mediated PAR1 inhibition also influences mitosis. Here, we investigated the effect of CagA on the progression of mitosis. In the presence of CagA, cells displayed a delay in the transition from prophase to metaphase. Furthermore, a fraction of the CagA-expressing cells showed spindle misorientation at the onset of anaphase, followed by chromosomal segregation with abnormal division axis. The effect of CagA on mitosis was abolished by elevated PAR1 expression. Conversely, inhibition of PAR1 kinase elicited mitotic delay similar to that induced by CagA. Thus, CagA-mediated inhibition of PAR1, which perturbs microtubule stability and thereby causes microtubule-based spindle dysfunction, is involved in the prophase/metaphase delay and subsequent spindle misorientation. Consequently, chronic exposure of cells to CagA induces chromosomal instability. Our findings reveal a bifunctional role of CagA as an oncoprotein: CagA elicits uncontrolled cell proliferation by aberrantly activating SHP-2 and at the same time induces chromosomal instability by perturbing the microtubule-based mitotic spindle. The dual function of CagA may cooperatively contribute to the progression of multistep gastric carcinogenesis.Helicobacter pylori is a spiral-shaped bacterium first described in 1984 by Marshall and Warren (1). H. pylori inhabits at least half of the world''s human population. Clinically isolated H. pylori strains can be divided into two major subtypes based on their ability to produce a 120- to 145-kDa protein called cytotoxin-associated gene A antigen (CagA)² (2–5). More than 90–95% of H. pylori strains isolated in East Asian countries such as Japan, Korea, and China are cagA-positive, whereas 40–50% of those isolated in Western countries are cagA-negative. Infection with a cagA-positive H. pylori strain is associated with severe atrophic gastritis, peptic ulcerations, and gastric adenocarcinoma (6–12).H. pylori cagA-positive strains deliver the CagA protein into host cells via the cag pathogenicity island-encoded type IV secretion system (4, 5, 13, 14). Translocated CagA then localizes to the inner surface of the plasma membrane, where it undergoes tyrosine phosphorylation by Src family kinases or Abl kinase at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs present in the C-terminal region of CagA (15–17). Tyrosine-phosphorylated CagA then binds specifically to SHP-2 tyrosine phosphatase and deregulates its phosphatase activity (18–21). Recent studies have revealed that gain-of-function mutations of SHP-2 are associated with a variety of human malignancies, indicating that SHP-2 is a bona fide human oncoprotein. Furthermore, transgenic expression of CagA in mice induces gastrointestinal and hematological malignancies in a manner that is dependent on CagA tyrosine phosphorylation (22). These findings suggest a critical role of CagA-SHP-2 interaction in the oncogenic potential of CagA.A polarized epithelial monolayer is characterized by the presence of well developed cell-cell interaction apparatuses such as tight junctions and adherens junctions. The tight junctions act as a paracellular barrier in polarized epithelial cells and play an essential role in the establishment and maintenance of epithelial cell polarity by delimiting the apical and basolateral membrane domains. CagA disrupts the tight junctions and causes loss of epithelial apical-basal polarity (23, 24). The disruption of tight junctions by CagA is mediated by the specific interaction of CagA with partitioning-defective 1 (PAR1) (25, 26). PAR1 is a serine/threonine kinase originally isolated in Caenorhabditis elegans and highly conserved from yeast to humans (27, 28). In mammals, there are four PAR1 isoforms, which may have redundant roles in polarity regulation. PAR1 acts as a master regulator for the regulation of cell polarity in various cell systems. During epithelial polarization, PAR1 specifically localizes to the basolateral membrane, whereas atypical PKC complexed with PAR3 and PAR6 (aPKC complex) specifically localizes to the apical membrane as well as the tight junctions (29–31). This asymmetric distribution of the two kinases, PAR1 and aPKC complex, ensures formation and maintenance of epithelial apical-basal polarity. Notably, mammalian PAR1 kinases were originally identified as microtubule affinity-regulating kinases (MARKs), which phosphorylate microtubule-associated proteins (MAPs) such as Tau, MAP2, and MAP4 on their tubulin-binding repeats. The PAR1/MARK-dependent phosphorylation causes MAPs to detach from and thereby destabilize microtubules (32, 33). Importantly, microtubules form a mitotic spindle, which plays an indispensable role in chromosomal alignment and separation during mitosis, raising the possibility that PAR1 regulates mitosis through controlling stability of the mitotic spindle. Indeed, during mitosis, MAPs undergo a severalfold higher level of phosphorylation (34, 35), and microtubule dynamics increase ∼20-fold (36). This in turn raises the intriguing possibility that CagA influences chromosomal stability by subverting MAP phosphorylation through systemic inhibition of PAR1.In this study, the effects of CagA on microtubule-dependent cellular events, especially dynamics of the mitotic spindle and chromosomal segregation during mitosis, were examined. The results of this work provide evidence that CagA perturbs mitotic spindle checkpoint and thereby causes chromosomal instability. Given the role of chromosomal instability in cell transformation, the newly identified CagA activity may play a crucial role in the development of gastric carcinoma.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]