Towards data warehousing and mining of protein unfolding simulation data

OBJECTIVES: The prediction of protein structure and the precise understanding of protein folding and unfolding processes remains one of the greatest challenges in structural biology and bioinformatics. Computer simulations based on molecular dynamics (MD) are at the forefront of the effort to gain a deeper understanding of these complex processes. Currently, these MD simulations are usually on the order of tens of nanoseconds, generate a large amount of conformational data and are computationally expensive. More and more groups run such simulations and generate a myriad of data, which raises new challenges in managing and analyzing these data. Because the vast range of proteins researchers want to study and simulate, the computational effort needed to generate data, the large data volumes involved, and the different types of analyses scientists need to perform, it is desirable to provide a public repository allowing researchers to pool and share protein unfolding data. METHODS: To adequately organize, manage, and analyze the data generated by unfolding simulation studies, we designed a data warehouse system that is embedded in a grid environment to facilitate the seamless sharing of available computer resources and thus enable many groups to share complex molecular dynamics simulations on a more regular basis. RESULTS: To gain insight into the conformational fluctuations and stability of the monomeric forms of the amyloidogenic protein transthyretin (TTR), molecular dynamics unfolding simulations of the monomer of human TTR have been conducted. Trajectory data and meta-data of the wild-type (WT) protein and the highly amyloidogenic variant L55P-TTR represent the test case for the data warehouse. CONCLUSIONS: Web and grid services, especially pre-defined data mining services that can run on or 'near' the data repository of the data warehouse, are likely to play a pivotal role in the analysis of molecular dynamics unfolding data.

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Role of sigma(54) in the regulation of genes involved in type I and type IV pili biogenesis in Xylella fastidiosa

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.

Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet

Type II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule. © 2006 International Society of Nephrology.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50%), Cryptococcus gattii VGII (25/56, 44.64%) and C. gattii VGI (3/56, 5.26%). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95% 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.

INFLUENCE OF TYPE 2 BOVINE VIRAL DIARRHEA VIRUS NPRO ON ENHANCEMENT OF BOVINE RESPIRATORY SYNCYTIAL VIRUS REPLICATION MEDIATED BY ANTAGONISM OF HOST CELL INTERFERON TYPE I RESPONSES

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, Family Flaviviridae. The virus can infect many species of animals of the order Artiodactyla. The BVDV genome encodes an auto protease, Npro, that degrades interferon regulatory factor-3 (IRF-3) reducing type I interferon (IFN-I) production from host cells. Bovine respiratory syncytial virus (BRSV) is a member of the genus Pneumovirus, Family Paramyxoviridae. Concurrent infection with BVDV and BRSV causes more severe respiratory and enteric disease than infection with either virus alone. Our hypothesis was that Npro modulates the innate immune responses to BVDV infection and enhances replication of BVDV or BRSV co-infection. The noncytopathic BVDV2 viruses NY93/c N- Npro 18 EGFP (a mutant with modified Npro fused with enhanced green fluorescent protein), NY93 infectious clone (NY93/c), wild-type NY93-BVDV2 (NY93-wt), and BRSV were evaluated in this study. The objectives of this study were: (1) to characterize the replication kinetics and IFN-I induction in Madin-Darby bovine kidney (MDBK) cells following infection with each of the BVDV isolates, and (2) to characterize the influence of BVDV-mediated IFN-I antagonism on enhancement of BRSV replication in bovine turbinate (BT) cells. NY93/c N- Npro 18 EGFP replicated 0.4 – 1.6 TCID50 logs lower than NY93-wt in MDBK cells. NY93/c N- Npro 18 EGFP-infected MDBK cells synthesized IFN-I significantly higher than NY93/c- and NY93-wt-infected MDBK cells. BT cells co-infected with NY93/c N- Npro 18 EGFP/BRSV or NY93-wt/BRSV were evaluated to determine the effects of co-infection on BRSV replication and IFN-I induction in BT cells. BRSV RNA levels in NY93-wt/BRSV co-infected BT cells were 2.49, 2.79, and 2.89 copy number logs significantly greater than in NY93/c N- Npro 18 EGFP/BRSV co-infected BT cells on days 5, 7, and 9 post-infection, respectively. BVDV RNA levels in NY93/c N- Npro 18 EGFP-infected BT cells were 1.64 – 4.38 copy number logs lower than in NY93-wt-infected BT cells. NY93/c N- Npro 18 EGFP single and co-infected BT cells synthesized IFN-I significantly higher than NY93-wt single and co-infected BT cells. In summary, these findings suggest: (1) NY93/c N- Npro 18 EGFP BVDV2 induced higher levels of IFN-I than BVDV2-wt and may be useful as a safer, replicating BVDV vaccine, and (2) Enhancement of BRSV infection by BVDV co-infection is mediated by antagonism of IFN-I.

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo-and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

Maturation of humoral immune responses : Studies on the effects of antigen type, apoptosis and age

The humoral immune response is dependent on the formation of antibodies. Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the germinal center (GC) reaction. The GC is the main site for class switch, somatic hypermutation and generation of memory cells. Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent antigens (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the T cell compartment is more affected by age than the B cell compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a plasma cell biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs. B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, lpr mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.

Characterization of L-type Calcium Channel Binding-Site of a new class of Calcium modulators by a Multidisciplinary approach

Many potential diltiazem related L-VDCC blockers were developed using a multidisciplinary approach. This current study was to investigate and compare diltiazem with to the newly developed compounds by mouse Langendorff-perfused heart, Ca2+-transient and on recombinant L-VDCC. Twenty particular compounds were selected by the ligand-based virtual screening procedure (LBVS). From these compounds, five of them (5b, M2, M7, M8 and P1) showed a potent and selective inotropic activity on guinea-pig left atria driven 1 Hz. Further assays displayed an interesting negative inotropic effect of M2, M8, P1 and M7 on guinea pig isolated left papillary muscle driven at 1 Hz, a relevant vasorelaxant activity of 5b, M2, M7, M8 and P1 on K+-depolarized guinea-pig ileum longitudinal smooth muscle and a significant inhibition of contraction of 5b, M2, M8 and P1 on carbachol stimulated ileum longitudinal smooth muscle. Wild-type human heart and rabbit lung α1 subunits were expressed (combined with the regulatory α2δ and β3 subunits) in Xenopus Leavis oocytes using a two-electrode voltage clamp technique. Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. Previous radioligand binding assays revealed a complex interaction with the benzothiazepine binding site for M2, M7 and M8. (Carosati E. et al. J. Med Chem. 2006, 49; 5206). In agreement with this findings, the relative order of increased rates of contraction and relaxation at lower concentrations s(≤10-6M) in unpaced hearts was M7>M2>M8>P1. Similar increases in Ca2+ transient were observed in cardiomyocytes. Diltiazem showed negative inotropic effects whereas 5b had no significant effect. Diltiazem blocks Ca2+current in a use-dependent manner and facilitates the channel by accelerating the inactivation and decelerating the recovery from inactivation. In contrast to diltiazem, the new analogs had no pronounced use-dependence. Application of 100 μM M8, M2 showed ~ 10% tonic block; in addition, M8, M2 and P1 shifted the steady state inactivation in hyperpolarized direction and the current inactivation time was significantly decreased compared with control (219.6 ± 11.5 ms, 226 ± 14.5 vs. 269 ± 12.9 vs. 199.28 ± 8.19 ms). Contrary to diltiazem, the recovery from the block by M8 and M2 was comparable to control. Only P1 showed a significantly decrease of the time for the recovery from inactivation. All of the compounds displayed the same sensitivity on the Ca2+ channel rabbit lung α1 except P1. Taken together, these findings suggest that M8, M2 and P1 might directly decrease the binding affinity or allow rapid dissociation from the benzothiazepine binding site.

The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

Isolated GH deficiency type II: knockdown of the harmful Delta3GH recovers wt-GH secretion in rat tumor pituitary cells

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

Isolated growth hormone deficiency type 2: from gene to therapy

Isolated growth hormone deficiency type-2 (IGHD-2), the autosomal-dominant form of GH deficiency, is mainly caused by specific splicing mutations in the human growth hormone (hGH) gene (GH-1). These mutations, occurring in and around exon 3, cause complete exon 3 skipping and produce a dominant-negative 17.5 kD GH isoform that reduces the accumulation and secretion of wild type-GH (wt-GH). At present, patients suffering from IGHD-2 are treated with daily injections of recombinant human GH (rhGH) in order to reach normal height. However, this type of replacement therapy, although effective in terms of growth, does not prevent toxic effects of the 17.5-kD mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. Considering a well-known correlation between the clinical severity observed in IGHD-2 patients and the increased expression of the 17.5-kD isoform, therapies that specifically target this isoform may be useful in patients with GH-1 splicing defects. This chapter focuses on molecular strategies that could represent future directions for IGHD-2 treatment.

Type I IFN induction in response to Listeria monocytogenes in human macrophages: evidence for a differential activation of IFN regulatory factor 3 (IRF3)

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.

Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.