Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/ target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protei

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx: a novel peculiar member of the winged helix–turn–helix transcription factor family

The hybrid plasmid virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 aminoacid protein, namely Stf76, that does not show significant sequence homology with any protein with known three-dimensional structure. The recombinant protein recognises specifically two DNA binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. CD, spectrofluorimetric, light scattering and ITC experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix–turn–helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog

The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules

Background: Pathogenic bacteria easily develop resistance to conventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa.Results: A novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid-virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets.Conclusions: VLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs

MucR binds multiple target sites in the promoter of its own gene and is a heat-stable protein: Is MucR a H-NS-like protein?

The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc-finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti. MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well-known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat-stable protein. Overall, the results of this study suggest that MucR might resemble a H-NS protein

Molecular Basis for Non-Covalent, Non-Competitive FAAH Inhibition

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation

Medulloblastoma tumor suppressor factor RENKCTD11: structure and function based design and development of new compounds of theraupetical interest.

Hedgehog (Hh) signaling is suggested to be a major oncogenic pathway in medulloblastoma, which arises from aberrant development of cerebellar granule progenitors. Hh signaling is regulated by ubiquitin ligases E3, that process the downstream transcription factors Gli via dual Cullin-based ubiquitin-dependent pathways. Interestingly, Cullin3 has been shown to require proteins containing BTB (Broad Complex, Tramtrack and Bric a Brac) domain to target substrates. Recently a human BTB protein has been identified, RENKCTD11, which displays allelic deletion as well as significantly, reduced expression in medulloblastoma. Data in vitro have suggested that RENKCTD11 binds Cullin3 and indirectly promotes the degradation of Gli1 thus inhibiting the transactivation of the Hedgehog target genes. Although the available literature data clearly show the role of RENKCTD11 as tumour suppressor, its biochemical properties remain to be defined. In this context we have undertaken a structural and functional study on RENKCTD11 protein. Human genomic region coding for RENKCTD11 has been amplified by PCR, cloned and over-expressed in Escherichia coli in soluble form, as a fusion product with Maltose Binding Protein (MBP). The protein has been purified to homogeneity by only one purification step utilizing the presence of His-tag. Size exclusion chromatography allowed to structurally characterize RENKCTD11 as an homotetramer in agreement with in silico analysis. The TEV-digested protein sample was extremely unstable. The alignment of RENKCTD11 sequence with sequence databases shows that a region of almost one hundred residues presents a significant degree of similarity with the POZ/BTB domain of potassium (kv4) channel. Bioinformatic analyses have allowed to identify the exact extension of POZ/BTB domain of RENKCTD11. It has been cloned, expressed as a fusion product with Thioredoxin-A (TrxA), purified in soluble form as a tetramer and characterized by mass spectroscopy, circular dichroism and light scattering. The homogeneous protein has been used for crystallization tests. Site-directed mutagenesis has been used to validate the ionic interactions responsible of the POZ/BTB domain tetrameric form. The mutant D69A/R74A/N78A/R81A resulted strongly unstable. These data confirmed the key role of these residues in the stabilization of the tetrameric form of the protein. The experimental evidence that POZ/BTB of RENKCTD11 forms a stable tetramer in solution prompted us to check the possibility to generate by homology modelling a RENKCTD11 POZ/BTB-Cul3 complex with a 4:4 stoichiometry. A peptide corresponding to residues 49-68 (NH2-NSGLSFEELYRNAYTMVLHK-COOH) of Cul3 has been individuated as responsible of interaction with the domain POZ/BTB of RENKCTD11. The peptide binds POZ/BTB of RENKCTD11 with an apparent affinity constant of 0.8 μM. Then, we tried to investigate whether the same region of Cul3 was responsible for interaction with POZ/BTB domains of other KCTD proteins. It was chosen as the starting point KCTD5, a cytosolic protein whose crystal structure has been recently deposited. The POZ/BTB domain of KCTD5 with five aminoacids added at N-terminus (region 40-151) was expressed in E. coli and obtained in large amount. The results obtained by ELISA show that this POZ/BTB domain binds the helix 49-68 of Cul3 with an affinity similar to that of the BTB/POZ of RENKCTD11. Finally we present a mechanism of function of RENKCTD11 applicable to other members of KCTD family that, interacting with the ubiquitin-ligase-E3, regulate some of the most important cellular metabolic pathways by their POZ/BTB domains

Exploring the Molecular Interactions of Symmetrical and Unsymmetrical Selenoglycosides with Human Galectin-1 and Galectin-3

Galectins (Gals) are small cytosolic proteins that bind β-galactoside residues via their evolutionarily conserved carbohydrate recognition domain. Their dysregulation has been shown to be associated with many diseases. Consequently, targeting galectins for clinical applications has become increasingly relevant to develop tailored inhibitors selectively for one galectin. Accordingly, binding studies providing the molecular details of the interaction between galectin and inhibitor may be useful for the rational design of potent and selective antagonists. Gal-1 and Gal-3 are among the best-studied galectins, mainly for their roles in cancer progression; therefore, the molecular details of their interaction with inhibitors are demanded. This work gains more value by focusing on the interaction between Gal-1 and Gal-3 with the selenylated analogue of the Gal inhibitor thiodigalactose, characterized by a selenoglycoside bond (SeDG), and with unsymmetrical diglycosyl selenides (unsym(Se). Gal-1 and Gal-3 were produced heterologously and biophysically characterized. Interaction studies were performed by ITC, NMR spectroscopy, and MD simulation, and thermodynamic values were discussed and integrated with spectroscopic and computational results. The 3D complexes involving SeDG when interacting with Gal-1 and Gal-3 were depicted. Overall, the collected results will help identify hot spots for the design of new, better performing, and more specific Gal inhibitors

Considerations on the mechanics of failure of the infinite slope

This paper focuses on the mechanics of failure of the infinite slope due to groundwater table rising. To this end, the soil is modelled either as an elastic perfectly plastic or strain-softening non-associative material, obeying the Mohr-Coulomb failure criterion. Closed-form expressions of stress and strain are reported, proving that the operative shear stress along the failure surface at the base of the landslide could be less than the available shear strength and that this may be theoretically justified, not being necessarily due to softening, progressive failure or whatever other deterioration mechanism. The analysis shows that the mechanical slope response depends on the formation of a shear band at the base of the slope. This process is analysed from first yielding up to slope failure

When soil heterogeneity helps the geotechnical design: the case of drainage trenches

Experience shows that often even advanced geotechnical models cannot fully match the hydraulic or mechanical soil response due to unexpected variability of both soil structure and properties even within short distances. Engineers are then called to assess the influence of soil variability in the solution of geotechnical problems. This paper shows that in some cases, as in the design of drainage trenches, unforeseen layering favours a better soil response than in homogeneous conditions making the drainage system more efficient than foreseen

The BTB domains of the potassium channel tetramerization domain proteins prevalently assume pentameric states

Potassium channel tetramerization domain-containing (KCTD) proteins are involved in fundamental physio-pathological processes. Here, we report an analysis of the oligomeric state of the Bric-à-brack, Tram-track, Broad complex (BTB) domains of seven distinct KCTDs belonging to five major clades of the family evolution tree. Despite their functional and sequence variability, present electron microscopy data highlight the occurrence of well-defined pentameric states for all domains. Our data also show that these states coexist with alternative forms which include open pentamers. Thermal denaturation analyses conducted using KCTD1 as a model suggest that, in these proteins, different domains cooperate to their overall stability. Finally, negative-stain electron micrographs of KCTD6$^{BTB}$ in complex with Cullin3 show the presence of assemblies with a five-pointed pinwheel shape