Mechano-Redox Control of Mac-1 De-Adhesion by PDI Promotes Directional Movement Under Flow.

BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

Introducing ADNP and SIRT1 as new partners regulating microtubules and histone methylation.

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation and function. As such, de novo mutations in ADNP lead to the autistic ADNP syndrome and somatic ADNP mutations may drive Alzheimer's disease (AD) tauopathy. Sirtuin 1 (SIRT1) is positively associated with aging, the major risk for AD. Here, we revealed two key interaction sites for ADNP and SIRT1. One, at the microtubule end-binding protein (EB1 and EB3) Tau level, with EB1/EB3 serving as amplifiers for microtubule dynamics, synapse formation, axonal transport, and protection against tauopathy. Two, on the DNA/chromatin site, with yin yang 1, histone deacetylase 2, and ADNP, sharing a DNA binding motif and regulating SIRT1, ADNP, and EB1 (MAPRE1). This interaction was linked to sex- and age-dependent altered histone modification, associated with ADNP/SIRT1/WD repeat-containing protein 5, which mediates the assembly of histone modification complexes. Single-cell RNA and protein expression analyses as well as gene expression correlations placed SIRT1-ADNP and either MAPRE1 (EB1), MAPRE3 (EB3), or both in the same mouse and human cell; however, while MAPRE1 seemed to be similarly regulated to ADNP and SIRT1, MAPRE3 seemed to deviate. Finally, we demonstrated an extremely tight correlation for the gene transcripts described above, including related gene products. This correlation was specifically abolished in affected postmortem AD and Parkinson's disease brain select areas compared to matched controls, while being maintained in blood samples. Thus, we identified an ADNP-SIRT1 complex that may serve as a new target for the understanding of brain degeneration.

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent.

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO2 as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin-mediated drug-delivery of RGD-modified DLDH (DLDHRGD ), leading to apoptotic cell death. In these experiments, DLDHRGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA-binding capability of this enzyme. To explore this interaction under cell-free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel-shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA-binding. A Docking algorithm for prediction of protein-DNA complexes, Paradoc, identified a putative DNA binding site at the C-terminus of DLDH. Our finding that TiO2 -bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.

R269C variant of ESR1: high prevalence and differential function in a subset of pancreatic cancers.

BACKGROUND: Estrogen receptor α (ESR1) plays a critical role in promoting growth of various cancers. Yet, its role in the development of pancreatic cancer is not well-defined. A less studied region of ESR1 is the hinge region, connecting the ligand binding and DNA domains. rs142712646 is a rare SNP in ESR1, which leads to a substitution of arginine to cysteine at amino acid 269 (R269C). The mutation is positioned in the hinge region of ESR1, hence may affect the receptor structure and function. We aimed to characterize the activity of R269C-ESR1 and study its role in the development of pancreatic cancer. METHODS: Transcriptional activity was evaluated by E2-response element (ERE) and AP1 -luciferase reporter assays and qRT-PCR. Proliferation and migration were assessed using MTT and wound healing assays. Gene-expression analysis was performed using RNAseq. RESULTS: We examined the presence of this SNP in various malignancies, using the entire database of FoundationOne and noted enrichment of it in a subset of pancreatic non-ductal adenocarcinoma (n = 2800) compared to pancreatic ductal adenocarcinoma (PDAC) as well as other tumor types (0.53% vs 0.29%, p = 0.02). Studies in breast and pancreatic cancer cells indicated cell type-dependent activity of ESR1 harboring R269C. Thus, expression of R269C-ESR1 enhanced proliferation and migration of PANC-1 and COLO-357 pancreatic cancer cells but not of MCF-7 breast cancer cells. Moreover, R269C-ESR1 enhanced E2-response elements (ERE) and AP1-dependent transcriptional activity and increased mRNA levels of ERE and AP1-regulated genes in pancreatic cancer cell lines, but had a modest effect on MCF-7 breast cancer cells. Accordingly, whole transcriptome analysis indicated alterations of genes associated with tumorigenicity in pancreatic cancer cells and upregulation of genes associated with cell metabolism and hormone biosynthesis in breast cancer cells. CONCLUSIONS: Our study shed new light on the role of the hinge region in regulating transcriptional activity of the ER and indicates cell-type specific activity, namely increased activity in pancreatic cancer cells but reduced activity in breast cancer cells. While rare, the presence of rs142712646 may serve as a novel genetic risk factor, and a possible target for therapy in a subset of non-ductal pancreatic cancers.

Genomic Analysis of Natural Rough Brucella melitensis Rev.1 Vaccine Strains: Identification and Characterization of Mutations in Key Genes Associated with Bacterial LPS Biosynthesis and Virulence.

Brucella species are facultative intracellular bacteria that cause brucellosis, a zoonotic world-wide disease. The live attenuated B. melitensis Rev.1 vaccine strain is widely used for the control of brucellosis in the small ruminant population. However, Rev.1 induces antibodies against the O-polysaccharide (O-PS) of the smooth lipopolysaccharide thus, it is difficult to differentiate between infected and vaccinated animals. Hence, rough Brucella strains lacking the O-PS have been introduced. In the current study, we conducted a comprehensive comparative analysis of the genome sequence of two natural Rev.1 rough strains, isolated from sheep, against that of 24 Rev.1 smooth strains and the virulent reference strain B. melitensis 16M. We identified and characterized eight vital mutations within highly important genes associated with Brucella lipopolysaccharide (LPS) biosynthesis and virulence, which may explain the mechanisms underlying the formation of the Rev.1 rough phenotype and may be used to determine the mechanism underlying virulence attenuation. Further complementation studies aimed to estimate the specific role of these mutations in affecting Brucella morphology and virulence will serve as a basis for the design of new attenuated vaccines for animal immunization against brucellosis.

The myelin proteolipid plasmolipin forms oligomers and induces liquid-ordered membranes in the Golgi complex.

Myelin comprises a compactly stacked massive surface area of protein-poor thick membrane that insulates axons to allow fast signal propagation. Increasing levels of the myelin protein plasmolipin (PLLP) were correlated with post-natal myelination; however, its function is unknown. Here, the intracellular localization and dynamics of PLLP were characterized in primary glial and cultured cells using fluorescently labeled PLLP and antibodies against PLLP. PLLP localized to and recycled between the plasma membrane and the Golgi complex. In the Golgi complex, PLLP forms oligomers based on fluorescence resonance energy transfer (FRET) analyses. PLLP oligomers blocked Golgi to plasma membrane transport of the secretory protein vesicular stomatitis virus G protein (VSVG), but not of a VSVG mutant with an elongated transmembrane domain. Laurdan staining analysis showed that this block is associated with PLLP-induced proliferation of liquid-ordered membranes. These findings show the capacity of PLLP to assemble potential myelin membrane precursor domains at the Golgi complex through its oligomerization and ability to attract liquid-ordered lipids. These data support a model in which PLLP functions in myelin biogenesis through organization of myelin liquid-ordered membranes in the Golgi complex.

Homozygous MED25 mutation implicated in eye-intellectual disability syndrome.

Genetic syndromes involving both brain and eye abnormalities are numerous and include syndromes such as Warburg micro syndrome, Kaufman oculocerebrofacial syndrome, Cerebro-oculo-facio-skeletal syndrome, Kahrizi syndrome and others. Using exome sequencing, we have been able to identify homozygous mutation p.(Tyr39Cys) in MED25 as the cause of a syndrome characterized by eye, brain, cardiac and palatal abnormalities as well as growth retardation, microcephaly and severe intellectual disability in seven patients from four unrelated families, all originating from the same village. The protein encoded by MED25 belongs to Mediator complex or MED complex, which is an evolutionary conserved multi-subunit RNA polymerase II transcriptional regulator complex. The MED25 point mutation is located in the von Willebrand factor type A (MED25 VWA) domain which is responsible for MED25 recruitment into the Mediator complex; co-immunoprecipitation experiment demonstrated that this mutation dramatically impairs MED25 interaction with the Mediator complex in mammalian cells.

Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB.

Pseudomonas putida GPo1 alkane hydroxylase (AlkB) is an integral membrane protein that catalyses the hydroxylation of medium-chain alkanes (C3-C12). 1-Octyne irreversibly inhibits this non-haem di-iron mono-oxygenase under turnover conditions, suggesting that it acts as a mechanism-based inactivator. Upon binding to the active site, 1-octyne is postulated to be oxidized to an oxirene that rapidly rearranges to a reactive ketene which covalently acylates nearby residues, resulting in enzyme inactivation. In analysis of inactivated AlkB by LC-MS/MS, several residues exhibited a mass increase of 126.1 Da, corresponding to the octanoyl moiety derived from oxidative activation of 1-octyne. Mutagenesis studies of conserved acylated residues showed that Lys18 plays a critical role in enzyme function, as a single-point mutation of Lys18 to alanine (K18A) completely abolished enzymatic activity. Finally, we present a computational 3D model structure of the transmembrane domain of AlkB, which revealed the overall packing arrangement of the transmembrane helices within the lipid bilayer and the location of the active site mapped by the 1-octyne modifications.

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

Association between variants of 5-hydroxytryptamine receptor 3C (HTR3C) and chemotherapy-induced symptoms in women receiving adjuvant treatment for breast cancer.

Administration of chemotherapy is associated with a wide array of symptoms affecting quality of life. Genetic risk factors for severity of chemotherapy-induced symptoms have not been determined. The present study aimed to explore the associations between polymorphisms in candidate genes and chemotherapy-induced symptoms. Women treated with at least two cycles of adjuvant doxorubicin and cyclophosphamide, with or without paclitaxel for early breast cancer (n = 105) completed the memorial symptom assessment scale and provided blood for genotyping. DNA was extracted from peripheral blood leukocytes and assayed for single nucleotide polymorphisms (SNPs) in GTP cyclohydrolase 1 (GCH1, rs10483639, rs3783641, and rs8007267), catecholamine-o-methyltransferase (COMT, rs4818), and 5-hydroxytryptamine (serotonin) receptor 3C (HTR3C, rs6766410, and rs6807362). Genotyping of HTR3C revealed a significant association between the presence of rs6766410 and rs6807362 SNPs (K163 and G405 variants) and increased severity of symptoms (p = 0.0001 and p = 0.007, respectively). Multiple regressions revealed that rs6766410 and rs6807362, but not age or stage at diagnosis, predicted severity of symptoms (p = 0.001 and p = 0.006, respectively) and explained 12 % of the variance in each regression model. No association was found between the genetic variants of CGH1 or COMT and symptom score. Our study indicates, for the first time, an association between variants of HTR3C and severity of chemotherapy-induced symptoms. Analyzing these genetic variants may identify patients at increased risk for the development of chemotherapy-induced symptoms and targeting the serotonin pathway may serve as a novel treatment strategy for these patients.

Insulin-like growth factor-I receptor (IGF-IR) translocates to nucleus and autoregulates IGF-IR gene expression in breast cancer cells.

The insulin-like growth factor (IGF) system plays an important role in mammary gland biology as well as in the etiology of breast cancer. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I and IGF-II, has emerged in recent years as a promising therapeutic target. The IGF and estrogen signaling pathways act in a synergistic manner in breast epithelial cells. The present study was aimed at investigating 1) the putative translocation of IGF-IR and the related insulin receptor (IR) to the nucleus in breast cancer cells, 2) the impact of IGF-IR and IR levels on IGF-IR biosynthesis in estrogen receptor (ER)-positive and ER-depleted breast cancer cells, and 3) the potential transcription factor role of IGF-IR in the specific context of IGF-IR gene regulation. We describe here a novel mechanism of autoregulation of IGF-IR gene expression by cellular IGF-IR, which is seemingly dependent on ER status. Regulation of the IGF-IR gene by IGF-IR protein is mediated at the level of transcription, as demonstrated by 1) binding assays (DNA affinity chromatography and ChIP) showing specific IGF-IR binding to IGF-IR promoter DNA and 2) transient transfection assays showing transactivation of the IGF-IR promoter by exogenous IGF-IR. The IR is also capable of translocating to the nucleus and binding the IGF-IR promoter in ER-depleted, but not in ER-positive, cells. However, transcription factors IGF-IR and IR display diametrically opposite activities in the context of IGF-IR gene regulation. Thus, whereas IGF-IR stimulated IGF-IR gene expression, IR inhibited IGF-IR promoter activity. In summary, we have identified a novel mechanism of IGF-IR gene autoregulation in breast cancer cells. The clinical implications of these findings and, in particular, the impact of IGF-IR/IR nuclear localization on targeted therapy require further investigation.

The type III effector HsvG of the gall-forming Pantoea agglomerans mediates expression of the host gene HSVGT.

The type III effector HsvG of the gall-forming Pantoea agglomerans pv. gypsophilae is a DNA-binding protein that is imported to the host nucleus and involved in host specificity. The DNA-binding region of HsvG was delineated to 266 amino acids located within a secondary structure region near the N-terminus of the protein but did not display any homology to canonical DNA-binding motifs. A binding site selection procedure was used to isolate a target gene of HsvG, named HSVGT, in Gypsophila paniculata. HSVGT is a predicted acidic protein of the DnaJ family with 244 amino acids. It harbors characteristic conserved motifs of a eukaryotic transcription factor, including a bipartite nuclear localization signal, zinc finger, and leucine zipper DNA-binding motifs. Quantitative real-time polymerase chain reaction analysis demonstrated that HSVGT transcription is specifically induced in planta within 2 h after inoculation with the wild-type P. agglomerans pv. gypsophilae compared with the hsvG mutant. Induction of HSVGT reached a peak of sixfold at 4 h after inoculation and progressively declined thereafter. Gel-shift assay demonstrated that HsvG binds to the HSVGT promoter, indicating that HSVGT is a direct target of HsvG. Our results support the hypothesis that HsvG functions as a transcription factor in gypsophila.

Multiple congenital anomalies-hypotonia-seizures syndrome is caused by a mutation in PIGN.

BACKGROUND: This study reports on a hitherto undescribed autosomal recessive syndrome characterised by dysmorphic features and multiple congenital anomalies together with severe neurological impairment, chorea and seizures leading to early death, and the identification of a gene involved in the pathogenesis of the disease. METHODS: Homozygosity mapping was performed using Affymetrix Human Mapping 250k NspI arrays. Sequencing of all coding exons of the candidate genes was performed with primer sets designed using the Primer3 program. Fluorescence activated cell sorting was performed using conjugated antibody to CD59. Staining, acquisition and analysis were performed on a FACSCalibur flow cytometer. RESULTS: Using homozygosity mapping, the study mapped the disease locus to 18q21.32-18q22.1 and identified the disease-causing mutation, c.2126G→A (p.Arg709Gln), in PIGN, which encodes glycosylphosphatidylinositol (GPI) ethanolamine phosphate transferase 1, a protein involved in GPI-anchor biosynthesis. Arginine at the position 709 is a highly evolutionarily conserved residue located in the PigN domain. The expression of GPI linked protein CD59 on fibroblasts from patients as compared to that in a control individual showed a 10-fold reduction in expression, confirming the pathogenic consequences of the mutation on GPI dependent protein expression. CONCLUSIONS: The abundant expression of PIGN in various tissues is compatible with the diverse phenotypic features observed in the patients and with the involvement of multiple body systems. The presence of developmental delay, hypotonia, and epilepsy combined with multiple congenital anomalies, especially anorectal anomalies, should lead a clinician to suspect a GPI deficiency related disorder.

Biochemical and structural insights into Rab12 interactions with RILP and its family members.

Alongside its biosynthetic functions, the small GTPase Rab12 negatively regulates mast cell (MC) exocytosis by its interaction with RILP to promote retrograde transport of the MC secretory granules. Given the role of Rab effectors in mediating Rab functions, in this study we used biochemical and in silico tools to decipher Rab12 interactions with its RILP family effectors. We show that Rab12 interacts with RILP, RILP-L1 and RILP-L2 independently of each other, whereby lysine-71, in mouse Rab12, is critical for Rab12 interactions with RILP-L1 or RILP-L2, but is dispensable for the binding of RILP. Focusing on RILP, and relying on molecular dynamics simulations, functional mutational analyses and peptide inhibition assays, we propose a model for the Rab12-RILP complex, consisting of a RILP homodimer and a single molecule of active Rab12, that interacts with the RILP homology domain (RHD) of one RILP monomer and a C-terminal threonine in the other monomer via its switch I and switch II regions. Mutational analyses of RILP RHD also demonstrate its involvement in the regulation of MC secretory granule transport. Jointly, our results provide structural and functional insights into the Rab12-RILP complex on the basis of which new tools could be generated for decoding Rab12 functions.

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers.

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER-ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins' role in ER-to-Golgi transport.

Independent and cooperative motions of the Kv1.2 channel: voltage sensing and gating.

Voltage-gated potassium (Kv) channels, such as Kv1.2, are involved in the generation and propagation of action potentials. The Kv channel is a homotetramer, and each monomer is composed of a voltage-sensing domain (VSD) and a pore domain (PD). We analyzed the fluctuations of a model structure of Kv1.2 using elastic network models. The analysis suggested a network of coupled fluctuations of eight rigid structural units and seven hinges that may control the transition between the active and inactive states of the channel. For the most part, the network is composed of amino acids that are known to affect channel activity. The results suggested allosteric interactions and cooperativity between the subunits in the coupling between the motion of the VSD and the selectivity filter of the PD, in accordance with recent empirical data. There are no direct contacts between the VSDs of the four subunits, and the contacts between these and the PDs are loose, suggesting that the VSDs are capable of functioning independently. Indeed, they manifest many inherent fluctuations that are decoupled from the rest of the structure. In general, the analysis suggests that the two domains contribute to the channel function both individually and cooperatively.

Staphylococcus aureus NrdH redoxin is a reductant of the class Ib ribonucleotide reductase.

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.

Genetic and antigenic characterization of sigma C protein from avian reovirus.

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine.

Systems Analysis of Insulin and IGF1 Receptors Networks in Breast Cancer Cells Identifies Commonalities and Divergences in Expression Patterns.

Insulin and insulin-like growth factor-1 (IGF1), acting respectively via the insulin (INSR) and IGF1 (IGF1R) receptors, play key developmental and metabolic roles throughout life. In addition, both signaling pathways fulfill important roles in cancer initiation and progression. The present study was aimed at identifying mechanistic differences between INSR and IGF1R using a recently developed bioinformatics tool, the Biological Network Simulator (BioNSi). This application allows to import and merge multiple pathways and interaction information from the KEGG database into a single network representation. The BioNsi network simulation tool allowed us to exploit the availability of gene expression data derived from breast cancer cell lines with specific disruptions of the INSR or IGF1R genes in order to investigate potential differences in protein expression that might be linked to biological attributes of the specific receptor networks. Modeling-generated information was corroborated by experimental and biological assays. BioNSi analyses revealed that the expression of 75 and 71 genes changed during simulation of IGF1R-KD and INSR-KD, compared to control cells, respectively. Out of 16 proteins that BioNSi analysis was based on, validated by Western blotting, nine were shown to be involved in DNA repair, eight in cell cycle checkpoints, six in proliferation, eight in apoptosis, seven in oxidative stress, six in cell migration, two in energy homeostasis, and three in senescence. Taken together, analyses identified a number of commonalities and, most importantly, dissimilarities between the IGF1R and INSR pathways that might help explain the basis for the biological differences between these networks.