The Helicobacter pylori GroES Cochaperonin HspA Functions as a Specialized Nickel Chaperone and Sequestration Protein through Its Unique C-Terminal Extension

The transition metal nickel plays a central role in the human gastric pathogen Helicobacter pylori because it is required for two enzymes indispensable for colonization, the nickel metalloenzyme urease and [NiFe] hydrogenase. To sustain nickel availability for these metalloenzymes while providing protection from the metal''s harmful effects, H. pylori is equipped with several specific nickel-binding proteins. Among these, H. pylori possesses a particular chaperone, HspA, that is a homolog of the highly conserved and essential bacterial heat shock protein GroES. HspA contains a unique His-rich C-terminal extension and was demonstrated to bind nickel in vitro. To investigate the function of this extension in H. pylori, we constructed mutants carrying either a complete deletion or point mutations in critical residues of this domain. All mutants presented a decreased intracellular nickel content measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced nickel tolerance. While urease activity was unaffected in the mutants, [NiFe] hydrogenase activity was significantly diminished when the C-terminal extension of HspA was mutated. We conclude that H. pylori HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in nickel-dependent maturation of hydrogenase.Helicobacter pylori is a Gram-negative, microaerophilic bacterium that is the only persistent inhabitant of the human stomach. Its presence in humans is associated with a variety of pathologies, ranging from gastric and duodenal peptic ulcers to the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (1, 39). Indeed, H. pylori is the only formally recognized bacterial carcinogen for humans (17), infecting half of the world''s population (19).In H. pylori, metal ions play a central role, since the transition metal nickel is the cofactor of the urease enzyme and is also required for [NiFe] hydrogenase. Urease catalyzes the hydrolysis of urea into the buffering compounds bicarbonate and ammonia, enabling H. pylori to persist in the acidic environment of the stomach. This enzyme accounts for up to 6% of the soluble cellular proteins and requires 24 nickel ions per active enzymatic complex (16). The uptake-type hydrogenase of H. pylori is a nickel-dependent enzyme containing a binuclear [NiFe] active site. This [NiFe] hydrogenase catalyzes the oxidation of molecular hydrogen and permits the utilization of hydrogen as an energy source during respiration-based energy production in the mucosa (21). Both enzymes are important for host colonization, as shown with several animal models (9, 10, 28, 42, 43). To sustain nickel availability for urease and hydrogenase while providing protection from the metal''s harmful effects, H. pylori possesses an elaborate and strictly controlled nickel metabolism.The incorporation of nickel ions into apohydrogenase requires the participation of the HypAB (HP0869 and HP0900) accessory proteins; for apourease, both the UreEFGH (HP0070-0067) accessory proteins and HypAB are necessary (4, 29). Besides these widely distributed accessory proteins, H. pylori possesses several specific proteins that are present in all H. pylori strains, namely, the histidine-rich proteins Hpn (HP1427) and Hpn-like (HP1432). These cytoplasmic and abundant proteins (Hpn represents 2% of the total protein content) bind nickel ions (five Ni²⁺ ions per monomer; dissociation constant [K_d] for nickel of 7.1 μM) and protect H. pylori against metal overload (15). Furthermore, it has recently been proposed that Hpn and Hpn-like can compete for nickel ions with the urease enzyme and thus regulate its enzymatic activity. In vivo and in vitro experiments indicate that Hpn and Hpn-like sequester nickel ions at neutral pH but donate them for urease activation under acidic pH conditions (14, 35, 44). Hydrogenase activity was unchanged in the Δhpn and Δhpn-like mutants (35).In addition to these proteins, H. pylori possesses a particular chaperone, HspA (HP0011), that is a homolog of the highly conserved and essential bacterial heat shock protein GroES (40). No other gene encoding a GroES homolog is found in the genome of H. pylori. GroES is the cochaperonin of the heptameric GroEL-GroES barrel complex, which mediates the correct folding of a variety of cellular proteins and which is conserved and essential in prokaryotes and eukaryotes (30). In addition to the conserved GroES chaperonin domain (domain A, amino acids 1 to 90) (Fig. ​(Fig.1A),1A), HspA contains a C-terminal extension of 28 amino acids (domain B, amino acids 91 to 118) (Fig. 1A and B) that contains 8 His and 4 Cys residues. Based on this high number of His and Cys residues known to bind transition metal ions, the purified recombinant HspA protein specifically binds two nickel ions per molecule (K_d of 1.1 to 1.8 μM) (7, 18). This domain also contains an HX₄DH motif (boxed in Fig. ​Fig.1B)1B) that is considered to be a nickel-binding signature sequence in the nickel-cobalt (NiCoT) transporter family (11). In addition, Loguercio et al. (20) observed that in vitro, the HspA C-terminal domain is folding into two vicinal disulfide bounds engaging two cysteine pairs that form a unique closed-loop structure. However, since HspA is a cytoplasmic protein, the in vivo relevance of this structure is uncertain.Open in a separate windowFIG. 1.(A) Representation of the HspA protein of H. pylori with the GroES-like domain A and the nickel-binding domain B. (B) Amino acid sequence of domain B of wild-type HspA and of three mutants: HspA-ΔC, with a complete deletion of this domain, and HspA-NB and -CC, each carrying two substitutions that are underlined. Cysteine and histidine residues are in blue and red, respectively. The HX₄DH motif, which in the nickel-cobalt (NiCoT) transporter family is considered to be a nickel-binding signature sequence, is boxed. (C) Immunoblot experiment with whole-cell lysates from the H. pylori wild-type strain and from the three hspA mutants after denaturing SDS-PAGE and using the monoclonal antibody P1-1, which specifically recognizes a conserved epitope of HspA domain A. The predicted molecular mass of the wild-type HspA monomer is 13 kDa, and that of HspA-ΔC is 9.8 kDa. The monomeric (M) and dimeric (D) forms of the HspA wild type (WT) are indicated on the left side of the blot. A cross-reacting unspecific protein band is marked with a star (*) and served as a loading control. Molecular mass standards are indicated at right.The domain B sequence is conserved in and restricted to H. pylori and the closely related Helicobacter acinonychis species but is absent from all other available sequenced Helicobacter species (see Fig. S1 in the supplemental material). When expressed in Escherichia coli, HspA protected bacteria from nickel overload (7) and increased urease activity 4-fold from the coexpressed H. pylori urease gene cluster (18). Therefore, HspA was hypothesized to function in nickel sequestration and as a specialized nickel donor protein for urease (18). However, no functional characterization of the C terminus was carried out for H. pylori due to the essential nature of HspA (40).In this study, we investigated the role of the nickel-binding C terminus of HspA in H. pylori. We found that the unique C terminus of HspA is involved in nickel sequestration and protection against nickel overload. Contrary to previous data from heterologous studies of E. coli, HspA seemed not to provide nickel ions for urease activation. In contrast, we have found an unexpected and specific function of the HspA C-terminal region in the nickel-dependent maturation of the important colonization factor hydrogenase.     

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose

Post-translational modification of nucleocytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRα and LXRβ are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRα O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRα O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.     

Maintenance of segment and appendage primordia by the Tribolium gene knödel

For homeotic and segment-polarity genes in Drosophila, a switch in gene regulation has been described that distinguishes patterning and maintenance phases. Maintenance of segment and organ primordia involves secondary patterning and differentiation steps, as well as survival factors regulating proliferation and organ size. In a screen for embryonic lethal mutations in the flour beetle Tribolium castaneum, we have recovered two alleles of the kn?del gene, which result in short, bag-like embryos. These embryos have severely reduced appendages and differentiate a cuticle that lacks most overt signs of segmentation. In addition, they lack bristles and display defects in the nervous system. Early patterning in kn?del mutant embryos is normal up to the extended germ band stage, as indicated by the formation of regular even-skipped (Tc'eve) and wingless (Tc'wg) stripes. Afterwards, however, these patterns degenerate. Similarly, proximo-distal growth and patterning of limbs are nearly normal initially, but limb primordia shrink, and proximo-distal patterns degenerate, during subsequent stages. kn?del could be a segment polarity gene required for segment border maintenance in both trunk and appendages. Alternatively, it may have a more general role in tissue or organ maintenance.     

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-beta was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-kappaB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.     

Functional dichotomy between OX40 and 4-1BB in modulating effector CD8 T cell responses

Members of the TNFR family are thought to deliver costimulatory signals to T cells and modulate their function and survival. In this study, we compare the role of two closely related TNFR family molecules, OX40 and 4-1BB, in generating effector CD8 T cells to Ag delivered by adenovirus. OX40 and 4-1BB were both induced on responding naive CD8 T cells, but 4-1BB exhibited faster and more sustained kinetics than OX40. OX40-deficient CD8 T cells initially expanded normally; however, their accumulation and survival at late times in the primary response was significantly impaired. In contrast, 4-1BB-deficient CD8 T cells displayed hyperresponsiveness, expanding more than wild-type cells. The 4-1BB-deficient CD8 T cells also showed enhanced maturation attributes, whereas OX40-deficient CD8 T cells had multiple defects in the expression of effector cell surface markers, the synthesis of cytokines, and in cytotoxic activity. These results suggest that, in contrast to current ideas, OX40 and 4-1BB can have a clear functional dichotomy in modulating effector CD8 T cell responses. OX40 can positively regulate effector function and late accumulation/survival, whereas 4-1BB can initially operate in a negative manner to limit primary CD8 responses.     

Natural genetic transformation: A novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus

Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately 40 billion dollars per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering. Due to the recent publication of three complete genome sequences, it is now possible to use a rational approach for designing S. thermophilus starter strains with improved properties. Progress in this field, however, is hampered by a lack of genetic tools. Therefore, we developed a system, based on natural transformation, which makes genetic manipulations in S. thermophilus easy, rapid, and highly efficient. The efficiency of this novel tool should make it possible to construct food-grade mutants of S. thermophilus, opening up exciting new possibilities that should benefit consumers as well as the dairy industry.     

Abrogation of the CLK-2 checkpoint leads to tolerance to base-excision repair intermediates

Incorporation of uracil during DNA synthesis is among the most common types of endogenously generated DNA damage. Depletion of Caenorhabditis elegans dUTPase by RNA interference allowed us to study the role of DNA damage response (DDR) pathways when responding to high levels of uracil in DNA. dUTPase depletion compromised development, caused embryonic lethality and led to activation of cell-cycle arrest and apoptosis. These phenotypes manifested as a result of processing misincorporated uracil by the uracil-DNA glycosylase UNG-1. Strikingly, abrogation of the clk-2 checkpoint gene rescued lethality and developmental defects, and eliminated cell-cycle arrest and apoptosis after dUTPase depletion. These data show a genetic interaction between UNG-1 and activation of the CLK-2 DDR pathway after uracil incorporation into DNA. Our results indicate that persistent repair intermediates and/or single-stranded DNA formed during repair of misincorporated uracil are tolerated in the absence of the CLK-2 checkpoint in C. elegans.     

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.