Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNF alpha-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

Cryptic species in the cosmopolitan Bugulan eritina complex (Bryozoa,Cheilostomata)

Previous analyses of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and γ-proteobacterial endosymbiont diversity have suggested that the marine bryozoan Bugula neritina is a complex of three cryptic species, namely Types S, D and N. Types D and N were previously reported to have restricted distributions along California (western USA) and Delaware and Connecticut (eastern USA), respectively, whereas Type S is considered widespread in tropical, subtropical and temperate regions due to anthropogenic transport. Here, Bayesian species delimitation analysis of a data set composed of two mitochondrial (COI and large ribosomal RNA subunit [16S]) and two nuclear genes (dynein light chain roadblock type-2 protein [DYN] and voltage-dependent anion-selective channel protein [VDAC]) demonstrated that Types S, D and N correspond to three biological species. This finding was significantly supported, in spite of the combinations of priors applied for ancestral population size and root age. Furthermore, COI sequences were used to assess the introduction patterns of the cosmopolitan Type S species. Two COI haplotypes of Type S (S1a and S1d) were found occurring at a global scale. Mantel tests showed correlation between these haplotypes and local sea surface temperature tolerance. Accordingly, the distributions of Type S haplotypes may reflect intraspecific temperature tolerance variation, in addition to the role of introduction vectors. Finally, we show that the Type N may also have been introduced widely, as this species was found for the first time in Central California and north-eastern Australia.

Exome analysis of HIV patients submitted to dendritic cells therapeutic vaccine reveals an association of CNOT1 gene with response to the treatment

INTRODUCTION: With the aim of searching genetic factors associated with the response to an immune treatment based on autologous monocyte-derived dendritic cells pulsed with autologous inactivated HIV, we performed exome analysis by screening more than 240,000 putative functional exonic variants in 18 HIV-positive Brazilian patients that underwent the immune treatment. METHODS: Exome analysis has been performed using the ILLUMINA Infinium HumanExome BeadChip. zCall algorithm allowed us to recall rare variants. Quality control and SNP-centred analysis were done with GenABEL R package. An in-house implementation of the Wang method permitted gene-centred analysis. RESULTS: CCR4-NOT transcription complex, subunit 1 (CNOT1) gene (16q21), showed the strongest association with the modification of the response to the therapeutic vaccine (p=0.00075). CNOT1 SNP rs7188697 A/G was significantly associated with DC treatment response. The presence of a G allele indicated poor response to the therapeutic vaccine (p=0.0031; OR=33.00; CI=1.74-624.66), and the SNP behaved in a dominant model (A/A vs. A/G+G/G p=0.0009; OR=107.66; 95% CI=3.85-3013.31), being the A/G genotype present only in weak/transient responders, conferring susceptibility to poor response to the immune treatment. DISCUSSION: CNOT1 is known to be involved in the control of mRNA deadenylation and mRNA decay. Moreover, CNOT1 has been recently described as being involved in the regulation of inflammatory processes mediated by tristetraprolin (TTP). The TTP-CCR4-NOT complex (CNOT1 in the CCR4-NOT complex is the binding site for TTP) has been reported as interfering with HIV replication, through post-transcriptional control. Therefore, we can hypothesize that genetic variation occurring in the CNOT1 gene could impair the TTP-CCR4-NOT complex, thus interfering with HIV replication and/or host immune response. CONCLUSIONS: Being aware that our findings are exclusive to the 18 patients studied with a need for replication, and that the genetic variant of CNOT1 gene, localized at intron 3, has no known functional effect, we propose a novel potential candidate locus for the modulation of the response to the immune treatment, and open a discussion on the necessity to consider the host genome as another potential variant to be evaluated when designing an immune therapy study

Phylogenetic relationships of rock-wallabies, Petrogale (Marsupialia: Macropodidae) and their biogeographic history within Australia

The rock-wallaby genus Petrogale comprises a group of habitat-specialist macropodids endemic to Australia. Their restriction to rocky outcrops, with infrequent interpopulation dispersal, has been suggested as the cause of their recent and rapid diversification. Molecular phylogenetic relationships within and among species of Petrogale were analysed using mitochondrial (cytochrome oxidase c subunit 1, cytochrome b. NADH dehydrogenase subunit 2) and nuclear (omega-globin intron, breast and ovarian cancer susceptibility gene) sequence data with representatives that encompassed the morphological and chromosomal variation within the genus, including for the first time both Petrogale concinna and Petrogale purpureicollis. Four distinct lineages were identified, (1) the brachyotis group, (2) Petrogale persephone, (3) Petrogale xanthopus and (4) the lateralis-penicillata group. Three of these lineages include taxa with the ancestral karyotype (2n = 22). Paraphyletic relationships within the brachyotis group indicate the need for a focused phylogeographic study. There was support for P. purpureicollis being reinstated as a full species and P. concinna being placed within Petrogale rather than in the monotypic genus Peradorcas. Bayesian analyses of divergence times suggest that episodes of diversification commenced in the late Miocene-Pliocene and continued throughout the Pleistocene. Ancestral state reconstructions suggest that Petrogale originated in a mesic environment and dispersed into more arid environments, events that correlate with the timing of radiations in other arid zone vertebrate taxa across Australia. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats

Abstract Background: Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. Findings: By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. Conclusions: SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats

Background Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. Findings By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. Conclusions SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects

Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous gamma H2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the alpha-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-alpha primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.

The dengue virus non-structural 1 protein: risks and benefits

The dengue virus (DENV) non-structural 1 (NS1) protein plays a critical role in viral RNA replication and has a central position in DENV pathogenesis. DENV NS1 is a glycoprotein expressed in infected mammalian cells as soluble monomers that dimerize in the lumen of the endoplasmic reticulum; NS1 is subsequently transported to the cell surface, where it remains membrane associated or is secreted into the extracellular milieu as a hexameric complex. During the last three decades, the DENV NS1 protein has also been intensively investigated as a potential target for vaccines and antiviral drugs. In addition, NS1 is the major diagnostic marker for dengue infection. This review highlights some important issues regarding the role of NS1 in DENV pathogenesis and its biotechnological applications, both as a target for the development of safe and effective vaccines and antiviral drugs and as a tool for the generation of accurate diagnostic methods

Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-alpha production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1

Background: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. Objective: To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of beta-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Methods: Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. Results: PBMCs from patients with APECED syndrome had reduced TNF-alpha responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-a release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. Conclusion: AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome. (J Allergy Clin Immunol 2012;129:464-72.)

Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

Background: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3 >rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

Upregulation of hsa-miR-125b in HTLV-1 asymptomatic carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis patients

The retrovirus human T lymphotropic virus type 1 (HTLV-1) promotes spastic paraparesis, adult T cell leukaemia and other diseases. Recently, some human microRNAs (miRNAs) have been described as important factors in host-virus interactions. This study compared miRNA expression in control individuals, asymptomatic HTLV-1 carriers and HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis patients. The proviral load and Tax protein expression were measured in order to characterize the patients. hsa-miR-125b expression was significantly higher in patients than in controls (p = 0.0285) or in the HAM group (p = 0.0312). Therefore, our findings suggest that miR-125b expression can be used to elucidate the mechanisms of viral replication and pathogenic processes.

Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in the unprotonated form, the protein structure is very stable for ionic strengths ranging from 0 to 225 mM. Therefore, our findings support the hypothesis that the histidines constitute the hot points that induce configurational changes of E protein in acid pH, and give extra motivation to the development of new ideas for antivirus compound design.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Assessing the benefits of rosiglitazone in women with polycystic ovary syndrome through its effects on insulin-like growth factor 1, insulin-like growth factor-binding protein-3 and insulin resistance: a pilot study

Federal University of Sao Paulo