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.

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

Molecular characterization of a mariner-like element in the Atta sexdens rubropilosa genome

Mobile elements are widely present in eukaryotic genomes. They are repeated DNA segments that are able to move from one locus to another within the genome. They are divided into two main categories, depending on their mechanism of transposition, involving RNA (class I) or DNA (class II) molecules. The mariner-like elements are class II transposons. They encode their own transposase, which is necessary and sufficient for transposition in the absence of host factors. They are flanked by a short inverted terminal repeat and a TA dinucleotide target site, which is duplicated upon insertion. The transposase consists of two domains, an N-terminal inverted terminal repeat binding domain and a C-terminal catalytic domain. We identified a transposable element with molecular characteristics of a mariner-like element in Atta sexdens rubropilosa genome. Identification started from a PCR with degenerate primers and queen genomic DNA templates, with which it was possible to amplify a fragment with mariner transposable-element homology. Phylogenetic analysis demonstrated that this element belongs to the mauritiana subfamily of mariner-like elements and it was named Asmar1. We found that Asmar1 is homologous to a transposon described from another ant, Messor bouvieri. The predicted transposase sequence demonstrated that Asmar1 has a truncated transposase ORF. This study is part of a molecular characterization of mobile elements in the Atta spp genome. Our finding of mariner-like elements in all castes of this ant could be useful to help understand the dynamics of mariner-like element distribution in the Hymenoptera.

Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber

Background: Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra-and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. Results: Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U. mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2: 1 or 4: 1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber. Conclusion: Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) gamma Activators and Pan-PPAR Partial Agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) gamma to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPAR gamma ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPAR gamma LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPAR gamma LBD, stronger partial agonists with full length PPAR gamma and exhibit full blockade of PPAR gamma phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPAR gamma also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/beta-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPAR gamma modulators with useful clinical profiles among natural products.

Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a target for treatment of type II diabetes and other conditions. PPAR gamma full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPAR gamma but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPAR gamma agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPAR gamma target genes in 3T3-L1 cells (LPL, ORL1, and CEBP alpha) and PPAR gamma-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPAR gamma ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPAR gamma LBD conformer and occupies a space near the beta-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPAR gamma, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Omega-loop among H2', H3, and the beta-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPAR gamma-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPAR gamma modulators.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

A novel DAX1/NR0B1 mutation in a patient with adrenal hypoplasia congenita and hypogonadotropic hypogonadism

We report a case of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) due to a novel DAX1 mutation. A 19-month-old boy with hyperpigmentation and failure to thrive came to our service for investigation. Three brothers of the patient had died due to adrenal failure, and a maternal cousin had adrenal insufficiency. Adrenoleukodystrophy was excluded. MRI showed normal pituitary and hypothalamus. Plasma hormone evaluation revealed high ACTH (up to 2,790 pg/mL), and low levels of androstenedione, DHEA-S, 11-deoxycortisol, and cortisol. At 14 years of age the patient was still prepubescent, his weight was 43.6 kg (SDS: -0.87) and his height was 161 cm (SDS: -0.36), with normal body proportions. In the GnRH test, basal and maximum values of LH and FSH were respectively 0.6/2.1 and < 1.0/< 1.0 U/L. Molecular investigation identified a novel mutation that consists of a deletion of codon 372 (AAC; asparagine) in exon 1 of DAX1. This mutation was not found in a study of 200 alleles from normal individuals. Prediction site analysis indicated that this alteration, located in the DAX1 ligand-binding domain, may damage DAX1 protein. We hypothesize that the novel (p.Asp372del) DAX1 mutation might be able to cause a disruption of DAX1 function, and is probably involved in the development of AHC and HH in this patient. Arq Bras Endocrinol Metab. 2012;56(8):496-500

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.

Analysis of Schistosoma mansoni genes shared with Deuterostomia and with possible roles in host interactions

Abstract Background: Schistosoma mansoni is a blood helminth parasite that causes schistosomiasis, a disease that affects 200 million people in the world. Many orthologs of known mammalian genes have been discovered in this parasite and evidence is accumulating that some of these genes encode proteins linked to signaling pathways in the parasite that appear to be involved with growth or development, suggesting a complex co-evolutionary process. Results: In this work we found 427 genes conserved in the Deuterostomia group that have orthologs in S. mansoni and no members in any nematodes and insects so far sequenced. Among these genes we have identified Insulin Induced Gene (INSIG), Interferon Regulatory Factor (IRF) and vasohibin orthologs, known to be involved in mammals in mevalonate metabolism, immune response and angiogenesis control, respectively. We have chosen these three genes for a more detailed characterization, which included extension of their cloned messages to obtain full-length sequences. Interestingly, SmINSIG showed a 10-fold higher expression in adult females as opposed to males, in accordance with its possible role in regulating egg production. SmIRF has a DNA binding domain, a tryptophan-rich N-terminal region and several predicted phosphorylation sites, usually important for IRF activity. Fourteen different alternatively spliced forms of the S. mansoni vasohibin (SmVASL) gene were detected that encode seven different protein isoforms including one with a complete C-terminal end, and other isoforms with shorter C-terminal portions. Using S. mansoni homologs, we have employed a parsimonious rationale to compute the total gene losses/gains in nematodes, arthropods and deuterostomes under either the Coelomata or the Ecdysozoa evolutionary hypotheses; our results show a lower losses/gains number under the latter hypothesis. Conclusion: The genes discussed which are conserved between S. mansoni and deuterostomes, probably have an ancient origin and were lost in Ecdysozoa, being still present in Lophotrochozoa. Given their known functions in Deuterostomia, it is possible that some of them have been co-opted to perform functions related (directly or indirectly) to host adaptation or interaction with host signaling processes.

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

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

Plasmodium vivax Duffy binding protein: baseline antibody responses and parasite polymorphisms in a well-consolidated settlement of the Amazon Region

Objective To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) a leading malaria vaccine candidate in a well-consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBPII) within the local malaria parasite population. Methods Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBPII. Results The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti-PvDBP IgG antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) with subjects age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBPII diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement. Conclusion The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBPII diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.

Identification of Regions Involved in Substrate Binding and Dimer Stabilization within the Central Domains of Yeast Hsp40 Sis1

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Tip indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.

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

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