Enzymatic Antioxidant Signatures in Hyperthermophilic Archaea

To fight reactive oxygen species (ROS) produced by both the metabolism and strongly oxidative habitats, hyperthermophilic archaea are equipped with an array of antioxidant enzymes whose role is to protect the biological macromolecules from oxidative damage. The most common ROS, such as superoxide radical (O2-.) and hydrogen peroxide (H2O2), are scavenged by superoxide dismutase, peroxiredoxins, and catalase. These enzymes, together with thioredoxin, protein disulfide oxidoreductase, and thioredoxin reductase, which are involved in redox homeostasis, represent the core of the antioxidant system. In this review, we offer a panorama of progression of knowledge on the antioxidative system in aerobic or microaerobic (hyper)thermophilic archaea and possible industrial applications of these enzymes

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

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

Investigating the properties of TBA variants with twin thrombin binding domains

In this paper, we report studies concerning thrombin binding aptamer (TBA) dimeric derivatives in which the 3'-ends of two TBA sequences have been joined by means of linkers containing adenosine or thymidine residues and/or a glycerol moiety. CD and electrophoretic investigations indicate that all modified aptamers are able to form G-quadruplex domains resembling that of the parent TBA structure. However, isothermal titration calorimetry measurements of the aptamer/thrombin interaction point to different affinities to the target protein, depending on the type of linker. Consistently, the best ligands for thrombin show anticoagulant activities higher than TBA. Interestingly, two dimeric aptamers with the most promising properties also show far higher resistances in biological environment than TBA

Mapping key interactions in the dimerization process of HBHA from Mycobacterium tuberculosis, insights into bacterial agglutination

AbstractHBHA is a cell-surface protein implicated in the dissemination of Mycobacterium tuberculosis (Mtb) from the site of primary infection. Its N-terminal coiled-coil region is also involved in bacterial agglutination. However, despite the importance of HBHA dimerization in agglutination, protein regions involved in dimerization are hitherto not known. Here, we mapped these regions by coupling peptide synthesis, biochemical and computational analyses, and identified structural determinants for HBHA monomer–monomer recognition. Importantly, we obtained the first molecule able to induce HBHA dimer disaggregation at 37°C, the typical growth temperature of Mtb. This result provides new opportunities towards the development of Mtb anti-aggregation molecules with therapeutic interest.Structured summary of protein interactionsHBHA and HBHA bind by molecular sieving (View interaction)HBHA and H1 peptide bind by competition binding (View Interaction)HBHA and H1ext peptide bind by competition binding (View Interaction)HBHA and H2ext peptide bind by competition binding (View Interaction)HBHA and H2 peptide bind by competition binding (View Interaction)HBHA and H2ext peptide bind by competition binding (View Interaction)HBHA and HBHA bind by blue native page (View interaction

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

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

Cystatin B Involvement in Synapse Physiology of Rodent Brains and Human Cerebral Organoids

Cystatin B (CSTB) is a ubiquitous protein belonging to a superfamily of protease inhibitors. CSTB may play a critical role in brain physiology because its mutations cause progressive myoclonic epilepsy-1A (EPM1A), the most common form of progressive myoclonic epilepsy. However, the molecular mechanisms underlying the role of CSTB in the central nervous system (CNS) are largely unknown. To investigate the possible involvement of CSTB in the synaptic plasticity, we analyzed its expression in synaptosomes as a model system in studying the physiology of the synaptic regions of the CNS. We found that CSTB is not only present in the synaptosomes isolated from rat and mouse brain cortex, but also secreted into the medium in a depolarization-controlled manner. In addition, using biorthogonal noncanonical amino acid tagging (BONCAT) procedure, we demonstrated, for the first time, that CSTB is locally synthesized in the synaptosomes. The synaptic localization of CSTB was confirmed in a human 3D model of cortical development, namely cerebral organoids. Altogether, these results suggest that CSTB may play a role in the brain plasticity and open a new perspective in studying the involvement of CSTB deregulation in neurodegenerative and neuropsychiatric diseases