Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high alpha-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K (D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K (D) of 4 mu M, and a moderate alpha-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

The sialotranscriptome of Antricola delacruzi female ticks is compatible with non-hematophagous behavior and an alternative source of food

The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari toward haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL-domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, anti-microbials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacruzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL-domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively. (C) 2012 Elsevier Ltd. All rights reserved.

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous cofactor participating in numerous redox reactions. It is also a substrate for regulatory modifications of proteins and nucleic acids via the addition of ADP-ribose moieties or removal of acyl groups by transfer to ADP-ribose. In this study, we use in-depth sequence, structure and genomic context analysis to uncover new enzymes and substrate-binding proteins in NAD-utilizing metabolic and macromolecular modification systems. We predict that Escherichia coli YbiA and related families of domains from diverse bacteria, eukaryotes, large DNA viruses and single strand RNA viruses are previously unrecognized components of NAD-utilizing pathways that probably operate on ADP-ribose derivatives. Using contextual analysis we show that some of these proteins potentially act in RNA repair, where NAD is used to remove 2'-3' cyclic phosphodiester linkages. Likewise, we predict that another family of YbiA-related enzymes is likely to comprise a novel NAD-dependent ADP-ribosylation system for proteins, in conjunction with a previously unrecognized ADP-ribosyltransferase. A similar ADP-ribosyltransferase is also coupled with MACRO or ADP-ribosylglycohydrolase domain proteins in other related systems, suggesting that all these novel systems are likely to comprise pairs of ADP-ribosylation and ribosylglycohydrolase enzymes analogous to the DraG-DraT system, and a novel group of bacterial polymorphic toxins. We present evidence that some of these coupled ADP-ribosyltransferases/ribosylglycohydrolases are likely to regulate certain restriction modification enzymes in bacteria. The ADP-ribosyltransferases found in these, the bacterial polymorphic toxin and host-directed toxin systems of bacteria such as Waddlia also throw light on the evolution of this fold and the origin of eukaryotic polyADP-ribosyltransferases and NEURL4-like ARTs, which might be involved in centrosomal assembly. We also infer a novel biosynthetic pathway that might be involved in the synthesis of a nicotinate-derived compound in conjunction with an asparagine synthetase and AMPylating peptide ligase. We use the data derived from this analysis to understand the origin and early evolutionary trajectories of key NAD-utilizing enzymes and present targets for future biochemical investigations.

Self assembly of human septin 2 into amyloid filaments

Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in beta-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures. (C) 2011 Elsevier Masson SAS. All rights reserved.

IGFBP7 participates in the reciprocal interaction between acute lymphoblastic leukemia and BM stromal cells and in leukemia resistance to asparaginase

The interaction of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells (BMSCs) has a positive impact on ALL resistance to chemotherapy. We investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL cells upon coculture with BMSCs. Coculture with stromal cells resulted in increased insulin-like growth factor (IGF)-binding protein 7 (IGFBP7) expression by ALL cells. Assays with IGFBP7 knockdown ALL and stromal cell lines, or with addition of recombinant rIGFBP7 (rIGFBP7) to the culture medium, showed that IGFBP7 acts as a positive regulator of ALL and stromal cells growth, and significantly enhances in-vitro resistance of ALL to asparaginase. In these assays, IGFBP7 function occurred mainly in an insulin-and stromal-dependent manner. ALL cells were found to contribute substantially to extracellular IGFBP7 levels in the conditioned coculture medium. Diagnostic BM plasma from children with ALL had higher levels of IGFBP7 than controls. IGFBP7, in an insulin/IGF-dependent manner, enhanced asparagine synthetase expression and asparagine secretion by BMSCs, thus providing a stromal-dependent mechanism by which IGFBP7 protects ALL cells against asparaginase in this coculture system. Importantly, higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (Cox regression model, P = 0.003) in precursor B-cell Ph(-) ALL patients (n = 147) treated with a contemporary polychemotherapy protocol.

Molecular and Electronic Structure of the Peptide Subunit of Geobacter sulfurreducens Conductive Phi from First Principles

The respiration of metal oxides by the bacterium Geobacter sulfurreducens requires the assembly of a small peptide (the GS pilin) into conductive filaments termed pili. We gained insights into the contribution of the GS pilin to the pilus conductivity by developing a homology model and performing molecular dynamics simulations of the pilin peptide in vacuo and in solution. The results were consistent with a predominantly helical peptide containing the conserved a-helix region required for pilin assembly but carrying a short carboxy-terminal random-coiled segment rather than the large globular head of other bacterial pilins. The electronic structure of the pain was also explored from first principles and revealed a biphasic charge distribution along the pilin and a low electronic HOMO-LUMO gap, even in a wet environment. The low electronic band gap was the result of strong electrostatic fields generated by the alignment of the peptide bond dipoles in the pilin's alpha-helix and by charges from ions in solution and amino acids in the protein. The electronic structure also revealed some level of orbital delocalization in regions of the pilin containing aromatic amino acids and in spatial regions of high resonance where the HOMO and LUMO states are, which could provide an optimal environment for the hopping of electrons under thermal fluctuations. Hence, the structural and electronic features of the pilin revealed in these studies support the notion of a pilin peptide environment optimized for electron conduction.

Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma

Background. Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. Material and methods. We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. Results. Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN+) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN+ with an accuracy of 80.0% (p = 0.012). Conclusions. The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our "Nodal Index" model are predictive of lymph node metastasis in OSCC.

ANTIFUNGAL ACTIVITY OF LECTINS AGAINST YEAST OF VAGINAL SECRETION

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256 mu g/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

Analysis of the mRNA expression of the S100 beta protein in adipocytes of patients with diabetes mellitus, type 2

Objective: This study aims to explore the possible relationship between the expression level of S100 beta protein mRNA with diabetes mellitus type 2 in adipocytes from patients with this disease in comparison with normoglycemic individuals. Materials and methods: Samples of adipose tissue of eight patients from the coronary section of the Institute Dante Pazzanese of Cardiology (IDPC), four in Group Diabetes and four of Normoglycemic group, were evaluated by RT-PCR real time. Results: An increase around 15 times values, between the threshold cycle (Delta Ct), of mRNA expression of S100 beta protein in adipocytes of the diabetes group was observed in comparison to the control group (p = 0.015). Conclusion: Our results indicate, for the first time, that there is coexistence of increased expression of the S100 beta and the type 2 diabetes mellitus gene. Arq Bras Endocrinol Metab. 2012;56(7):435-40

Comparison of the Neutrophil Proteome in Trauma Patients and Normal Controls

Background: Neutrophils have an impressive array of microbicidal weapons, and in the presence of a pathogen, progress from a quiescent state in the bloodstream to a completely activated state. Failure to regulate this activation, for example, when the blood is flooded with cytokines after severe trauma, causes inappropriate neutrophil activation that paradoxically, is associated with tissue and organ damage. Acidic proteomic maps of quiescent human neutrophils were analyzed and compared to those of activated neutrophils from severe trauma patients. The analysis revealed 114 spots whose measured volumes differed between activated and quiescent neutrophils, with 27 upregulated and 87 downregulated in trauma conditions. Among the identified proteins, grancalcin, S100-A9 and CACNB2 reinforce observed correlations between motility and ion flux, ANXA3, SNAP, FGD1 and Zfyve19 are involved in vesicular transport and exocytosis, and GSTP1, HSPA1 HSPA1L, MAOB, UCH-L5, and PPA1 presented evidence that activated neutrophils may have diminished protection against oxidative damage and are prone to apoptosis. These are discussed, along with proteins involved in cytoskeleton reorganization, reactive oxygen species production, and ion flux. Proteins such as Zfyve19, MAOB and albumin-like protein were described for the first time in the neutrophil. In this work we achieved the identification of several proteins potentially involved in inflammatory signaling after trauma, as well as proteins described for the first time in neutrophils.

Characterization of the mechanisms underlying the crosstalk between galectins and notch in gastric cancer

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide. Galectins form a family of β-galactosides binding proteins that recognize a variety of glycan-containing proteins at the cell surface and are overexpressed in various tumors, including gastric cancer. Galectins overexpression as well as changes in their subcellular distribution has been associated with gastric cancer progression and poor prognosis. It is not well understood, however, how the interaction between galectins and glycosylated receptors modulates tumor development and growth. Since Notch receptors and ligands contain glycan structures known to bind galectins, we aim to demonstrate that galectins expression in the tumor microenvironment may interfere with Notch signaling activation during tumor development and progression. Materials and methods Immunoprecipitation procedures with gastric cancer cell line AGS (ATCC CRL-1739) and MKN45 (ACC 409) were used to test for association between galectin-1/-3 and Notch-1 receptor. Furthermore, we transfected AGS cell line with siRNA against galectin-1/-3 or scramble using standard protocols (IDT DNA technologies), stimulate them with immobilized human recombinant delta-4 or Jagged-1 and assessed Notch-1 receptor activation. Results Galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling pathway upon activation by Delta/Jagged ligands. Galectin-1 knockdown alters Notch-1 activation induced by Delta-4 whereas galectin-3 knockdown alters jagged-1-mediated Notch-1 activation. Furthermore, we found that exogenously added galectin-3 can enhance Notch-1 activation by Jagged-1. Conclusion Our results suggest that galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling activation induced by Jagged-1 and Delta-4.

Antifungal activity of lectins against yeast of vaginal secretion

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256µg/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.