Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

Expression, purification and structural analysis of the Pyrococcus abyssi RNA binding protein PAB1135

Abstract Background The gene coding for the uncharacterized protein PAB1135 in the archaeon Pyrococcus abyssi is in the same operon as the ribonuclease P (RNase P) subunit Rpp30. Findings Here we report the expression, purification and structural analysis of PAB1135. We analyzed the interaction of PAB1135 with RNA and show that it binds efficiently double-stranded RNAs in a non-sequence specific manner. We also performed molecular modeling of the PAB1135 structure using the crystal structure of the protein Af2318 from Archaeoglobus fulgidus (2OGK) as the template. Conclusions Comparison of this model has lead to the identification of a region in PAB1135 that could be involved in recognizing double-stranded RNA.

Expression and accumulation of the two-domain odorant-binding protein AaegOBP45 in the ovaries of blood-fed Aedes aegypti

BACKGROUND: Aedes aegypti mosquitoes are the main vectors of dengue viruses. Despite global efforts to reduce the prevalence of dengue using integrated vector management strategies, innovative alternatives are necessary to help prevent virus transmission. Detailed characterizations of Ae. aegypti genes and their products provide information about the biology of mosquitoes and may serve as foundations for the design of new vector control methods. FINDINGS: We studied the Ae. aegypti gene, AAEL010714, that encodes a two-domain odorant-binding protein, AaegOBP45. The predicted gene structure and sequence were validated, although single nucleotide polymorphisms were observed. Transcriptional and translational products accumulate in the ovaries of blood fed females and are not detected or are at low abundance in other tissues. CONCLUSIONS: We validated the Ae. aegypti AAEL010714 gene sequence and characterized the expression profile of a two-domain OBP expressed in ovaries. We propose that AaegOBP45 function as a component of the mosquito eggshell.

Probing the interaction of brain fatty acid binding protein (B-FABP) with model membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called "portal region", formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that BFABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.

Characterization of the Staphylococcus aureus bone sialoprotein-binding protein SdrE and the serine protease EpiP

In an attempt to develop a Staphylococcus aureus vaccine, we have applied reverse vaccinology approach, mainly based on in silico screening and proteomics. By using this approach SdrE, a protein belonging to serine-aspartate repeat protein family was identified as potential vaccine antigen against S. aureus. We have investigated the biochemical properties as well as the vaccine potential of SdrE and its highly conserved CnaBE3 domain. We found the protein SdrE to be resistant to trypsin. Further analysis of the resistant fragment revealed that it comprises a CnaBE3 domain, which also showed partial trypsin resistant behavior. Furthermore, intact mass spectrometry of rCnaBE3 suggested the possible presence of isopeptide bond or some other post-translational modification in the protein.However, this observation needs further investigation. Differential Scanning Fluorimetry study reveals that calcium play role in protein folding and provides stability to SdrE. At the end we have demonstrated that SdrE is immunogenic against clinical strain of S. aureus in murine abscess model. In the second part, I characterized a protein, annotated as epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. The crystal structure of the rEpiP was solved at 2.05 Å resolution by x-ray crystallography . The structure showed that rEpiP was cleaved somewhere between residues 95 and 100 and cleavage occurs through an autocatalytic intra-molecular mechanism. In addition, the protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine if the protein acts as a serine protease, we mutated the catalytic serine 393 residue to alanine, generating rEpiP-S393A and solved its crystal structure at a resolution of 1.95 Å. rEpiP-S393A was impaired in its protease activity, as expected. Protective efficacy of rEpiP and the non-cleaving mutant protein was comparable, implying that the two forms are interchangeable for vaccination purposes.

Three-dimensional electron microscopy of myriapod hemocyanin, planorbid snail acetylcholine-binding protein and cyanobacterial Vipp1

Three-dimensional electron microscopy (3-D EM) provides a framework for the analysis of large protein quaternary structures. The advantage over the generally higher resolving meth- od of X-ray crystallography is the embedding of the proteins in their physiological environ- ment. However, results of the two methods can be combined to obtain superior structural information. In this work, three different protein types – (i) Myriapod hemocyanin, (ii) vesi- cle-inducing protein in plastids 1 (Vipp1) and (iii) acetylcholine-binding protein (AChBP) – were structurally analyzed by 2-D and 3-D EM and, where possible, functionally interpreted.rnMyriapod hemocyanins have been previously shown to be 6x6-meric assemblies that, in case of Scutigera coleoptrata hemocyanin (ScoHc), show two 3x6-mer planes whith a stag- gering angle of approximately 60°. Here, previously observed structural differences between oxy- and deoxy-ScoHc could be substantiated. A 4° rotation between hexamers of two dif- ferent 3x6-mer planes was measured, which originates at the most central inter-hexamer in- terface. Further information about allosteric behaviour in myriapod hemocyanin was gained by analyzing Polydesmus angustus hemocyanin (PanHc), which shows a stable 3x6-mer and divergent histidine patterns in the inter-hexamer interfaces when compared to ScoHc. Both findings would conclusively explain the very different oxygen binding properties of chilopod and diplopod hemocyanin.rnVipp1 is a protein found in cyanobacteria and higher plants which is essential for thyla- koid membrane function and forms highly variable ring-shaped structures. In the course of this study, the first 3-D analysis of Vipp1 was conducted and yielded reconstructions of six differently sized Vipp1 rings from negatively stained images at resolutions between 20 to 30 Å. Furthermore, mutational analyses identified specific N-terminal amino acids that are essential for ring formation. On the basis of these analyses and previously published results, a hypothetical model of the Vipp1 tertiary and quaternary structure was generated.rnAChBP is a water-soluble protein in the hemolymph of mollusks. It is a structural and functional homologue of the ligand-binding domain of nicotinic acetylcholine receptors. For the freshwater snail Biomphalaria glabrata, we previously described two types of AChBP (BgAChBP1 and BgAChBP2). In this work, a 6 Å 3-D reconstruction of native BgAChBP is presented, which shows a dodecahedral assembly that is unprecedented for an AChBP. Single particle analysis of recombinantely expressed BgAChBP types led to preliminary results show- ing a dodecahedral assembly of BgAChBP1 and a dipentameric assembly of BgAChBP2. This indicates divergent biological functions of the two types.

Insulin-like growth factor-I treatment in primary growth hormone insensitivity: effect of recombinant human IGF-I (rhIGF-I) and rhIGF-I/rhIGF-binding protein-3 complex

Growth hormone insensitivity syndrome (GHIS) is a rare cause of growth retardation characterized by high serum GH levels, and low serum insulin-like growth factor I (IGF-I) levels associated with a genetic defect of the GH receptor (GHR) as well post-GHR signaling pathway. Based on clinical, as well as biochemical characteristics, GHIS can be genetically classified as classical/Laron's syndrome and nonclassical/atypical GHIS. Recombinant human IGF-I (rhIGF-I) treatment is effective in promoting growth in subjects who have GHIS. Further, pharmacological studies of a IGF-I compound containing a 1:1 molar complex of rhIGF-I and rhIGF-binding protein-3 (BP-3) demonstrated that the complex was effective in increasing levels of circulating total and free IGF-I and that the administration in patients with GHIS should be safe, well-tolerated and more effective than rhIGF-I on its own.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease

Inflammatory bowel diseases (IBDs), Crohn's disease, and ulcerative colitis (UC), are multifactorial disorders, characterized by chronic inflammation of the intestine. A number of genetic components have been proposed to contribute to IBD pathogenesis. In this case-control study, we investigated the association between two common vitamin D-binding protein (DBP) genetic variants and IBD susceptibility. These two single nucleotide polymorphisms (SNPs) in exon 11 of the DBP gene, at codons 416 (GAT>GAG; Asp>Glu) and 420 (ACG>AAG; Thr>Lys), have been previously suggested to play roles in the etiology of other autoimmune diseases.

Association of the (CA)n repeat polymorphism of insulin-like growth factor-I and -202 A/C IGF-binding protein-3 promoter polymorphism with adult height in patients with severe growth hormone deficiency

A number of mathematical models for predicting growth and final height outcome have been proposed to enable the clinician to 'individualize' growth-promoting treatment. However, despite optimizing these models, many patients with isolated growth hormone deficiency (IGHD) do not reach their target height. The aim of this study was to analyse the impact of polymorphic genotypes [CA repeat promoter polymorphism of insulin-like growth factor-I (IGF-I) and the -202 A/C promoter polymorphism of IGF-Binding Protein-3 (IGFBP-3)] on variable growth factors as well as final height in severe IGHD following GH treatment. DESIGN, PATIENTS AND CONTROLS: One hundred seventy eight (IGF-I) and 167 (IGFBP-3) subjects with severe growth retardation because of IGHD were studied. In addition, the various genotypes were also studied in a healthy control group of 211 subjects.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Absence of the calcium-binding protein calretinin, not of calbindin D-28k, causes a permanent impairment of murine adult hippocampal neurogenesis

Calretinin (CR) and calbindin D-28k (CB) are cytosolic EF-hand Ca(2+)-binding proteins and function as Ca(2+) buffers affecting the spatiotemporal aspects of Ca(2+) transients and possibly also as Ca(2+) sensors modulating signaling cascades. In the adult hippocampal circuitry, CR and CB are expressed in specific principal neurons and subsets of interneurons. In addition, CR is transiently expressed within the neurogenic dentate gyrus (DG) niche. CR and CB expression during adult neurogenesis mark critical transition stages, onset of differentiation for CR, and the switch to adult-like connectivity for CB. Absence of either protein during these stages in null-mutant mice may have functional consequences and contribute to some aspects of the identified phenotypes. We report the impact of CR- and CB-deficiency on the proliferation and differentiation of progenitor cells within the subgranular zone (SGZ) neurogenic niche of the DG. Effects were evaluated (1) two and four weeks postnatally, during the transition period of the proliferative matrix to the adult state, and (2) in adult animals (3 months) to trace possible permanent changes in adult neurogenesis. The absence of CB from differentiated DG granule cells has no retrograde effect on the proliferative activity of progenitor cells, nor affects survival or migration/differentiation of newborn neurons in the adult DG including the SGZ. On the contrary, lack of CR from immature early postmitotic granule cells causes an early loss in proliferative capacity of the SGZ that is maintained into adult age, when it has a further impact on the migration/survival of newborn granule cells. The transient CR expression at the onset of adult neurogenesis differentiation may thus have two functions: (1) to serve as a self-maintenance signal for the pool of cells at the same stage of neurogenesis contributing to their survival/differentiation, and (2) it may contribute to retrograde signaling required for maintenance of the progenitor pool.

Lactococcus lactis HemW (HemN) is a haem-binding protein with a putative role in haem trafficking

Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 μM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.

Characterisation of transthyretin and retinol-binding protein in plasma and cerebrospinal fluid of dogs

The aim of this study was to investigate differences in concentrations of vitamin A, transthyretin (TTR) and retinol-binding protein (RBP) between plasma and cerebrospinal fluid (CSF) in dogs. RBP was detected using ELISA, and both RBP and TTR by Western blot analysis after separation on SDS-PAGE. Vitamin A was determined by high performance liquid chromatography. RBP and TTR as well as vitamin A were detected in all samples but at substantially lower concentrations in CSF compared to plasma. RBP in dog plasma showed a similar molecular mass to that of humans, whereas canine TTR had a lower molecular mass. Comparison between plasma and CSF showed that both RBP and TTR were of lower molecular mass in CSF. In CSF, RBP and retinol were present at 10-100-fold lower concentrations compared to plasma. Retinyl esters were present only in minute amounts in 5/17 samples. In conclusion, the CSF of dogs compared to humans is significantly different in terms of both quality and quantity of transport proteins for vitamin A.