Search Results (12)

The conopeptide αD-FrXXA was previously isolated by our team from the venom of the vermivorous snail Conus fergusoni. This toxin is composed of two chains of 47 amino acids and inhibits neuronal and muscular subtypes of nAChR. In this study, we explored its effects on voltage-gated potassium channels heterologously expressed in Xenopus laevis oocytes using the two-electrode voltage-clamp technique (TEVC). At a concentration of 15 μM, αD-FrXXA was able to inhibit by 50% or more the currents of four subtypes of the Kv1 subfamily and slightly inhibit (<20%) two subtypes of the EAG subfamily. The conopeptide αD-FrXXA inhibits in a concentration-dependent manner the subtypes Kv1.3 (IC₅₀ 0.38 ± 0.06 μM) and Kv1.6 (IC₅₀ 0.52 ± 0.14 μM). The results reported here are noteworthy because this α-conopeptide behaves similarly to the α/κJ-PlXIVA conopeptide that inhibits nAChR and Kv channels. Full article

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5′-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure–function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine—a reversible inhibitor and analogue of (R)-1-phenylethylamine—a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the β-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds. Full article

RNA editing, a unique post-transcriptional modification, is observed in trypanosomatid parasites as a crucial procedure for the maturation of mitochondrial mRNAs. The editosome protein complex, involving multiple protein components, plays a key role in this process. In Trypanosoma brucei, a putative Z-DNA binding protein known as RBP7910 is associated with the editosome. However, the specific Z-DNA/Z-RNA binding activity and the interacting interface of RBP7910 have yet to be determined. In this study, we conducted a comparative analysis of the binding behavior of RBP7910 with different potential ligands using microscale thermophoresis (MST). Additionally, we generated a 3D model of the protein, revealing potential Z-α and Z-β nucleic acid-binding domains of RBP7910. RBP7910 belongs to the winged-helix–turn–helix (HTH) superfamily of proteins with an α₁α₂α₃β₁β₂ topology. Finally, using docking techniques, potential interacting surface regions of RBP7910 with notable oligonucleotide ligands were identified. Our findings indicate that RBP7910 exhibits a notable affinity for (CG)n Z-DNA, both in single-stranded and double-stranded forms. Moreover, we observed a broader interacting interface across its Z-α domain when bound to Z-DNA/Z-RNA compared to when bound to non-Z-form nucleic acid ligands. Full article

4-Coumarate: coenzyme A ligase (4CL; EC 6.2.1.12) is an important enzyme in the phenylpropanoid metabolic pathway that controls the biosynthesis of lignin and flavonoids. In this study, to identify the function of the Ag4CL3 gene of celery, the Ag4CL3 gene was cloned from celery cv. “Nanxuan Liuhe Ziqin”. Sequence analysis results showed that the Ag4CL3 gene contained an open reading frame (ORF) with a length of 1688 bp, and 555 amino acids were encoded. The Ag4CL3 protein was highly conserved among different plant species. Phylogenetic analysis demonstrated that the 4CL proteins from celery and carrot belonged to the same clade. The Ag4CL3 protein was mainly composed of 31.89% α-helixes, 18.02% extended strands, 6.67% β-turns, and 43.42% random coils, and the signal peptide was unfound. A total of 62 phosphorylation sites and a class-I superfamily of adenylate-forming domains were found. As the growth time increased, the plant height and stem thickness also increased, and the petiole lignin content increased and became lignified gradually. The relative expression levels of the Ag4CL3 gene in “Nanxuan Liuhe Ziqin” petioles were higher than those in other tissues, with the highest level occurring 70 d after sowing. The lignin contents in the transgenic Arabidopsis thaliana lines hosting the Ag4CL3 gene were higher than those in the WT. In this study, the overexpression of Ag4CL3 led to the significant upregulation of lignin biosynthesis gene expression in transgenic A. thaliana plants, except for AtPAL, AtCCR, and AtLAC. This study speculates that Ag4CL3 genes are related to lignin synthesis in A. graveolens. Full article

Chitosan is a functional ingredient that is widely used in food chemistry as an emulsifier, flocculant, antioxidant, or preservative. Chitin deacetylases (CDAs) can catalyze the hydrolysis of acetyl groups, making them useful in the clean production of chitosan. However, the high inactivity of crystalline chitin catalyzed by CDAs has been regarded as the technical bottleneck of crystalline chitin deacetylation. Here, we mined the AsCDA gene from the genome of Acinetobacter schindleri MCDA01 and identified a member of the uraD_N-term-dom superfamily, which was a novel chitin deacetylase with the highest deacetylation activity. The AsCDA gene was expressed in Escherichia coli BL21 by IPTG induction, whose activity to colloidal chitin, α-chitin, and β-chitin reached 478.96 U/mg, 397.07 U/mg, and 133.27 U/mg, respectively. In 12 h, the enzymatic hydrolysis of AsCDA removed 63.05% of the acetyl groups from α-chitin to prepare industrial chitosan with a degree of deacetylation higher than 85%. AsCDA, as a potent chitin decomposer in the production of chitosan, plays a positive role in the upgrading of the chitosan industry and the value-added utilization of chitin biological resources. Full article

The activities of 1α,25-dihydroxyvitamin D3, 1,25D₃, are mediated via its binding to the vitamin D receptor (VDR), a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily. Numerous studies have demonstrated the important role of 1,25D₃ and VDR signaling in various biological processes and associated pathologies. A wealth of information about ligand recognition and mechanism of action by structural analysis of the VDR complexes is also available. The methods used in these structural studies were mainly X-ray crystallography complemented by NMR, cryo-electron microscopy and structural mass spectrometry. This review aims to provide an overview of the current knowledge of VDR structures and also to explore the recent progress in understanding the complex mechanism of action of 1,25D₃ from a structural perspective. Full article

The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are known to bind ssDNA. Here, we report the identification and characterization of Sul7s, a novel winged-helix single-stranded DNA binding protein family highly conserved in Sulfolobaceae. Sul7s from Sulfolobus islandicus binds ssDNA with an affinity approximately 15-fold higher than that for dsDNA in vitro. It prefers binding oligo(dT)₃₀ over oligo(dC)₃₀ or a dG-rich 30-nt oligonucleotide, and barely binds oligo(dA)₃₀. Further, binding by Sul7s inhibits DNA strand annealing, but shows little effect on the melting temperature of DNA duplexes. The solution structure of Sul7s determined by NMR shows a winged helix-turn-helix fold, consisting of three α-helices, three β-strands, and two short wings. It interacts with ssDNA via a large positively charged binding surface, presumably resulting in ssDNA deformation. Our results shed significant light on not only non-OB fold single-stranded DNA binding proteins in Archaea, but also the divergence of the winged-helix proteins in both function and structure during evolution. Full article

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na⁺, K⁺-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly⁹−Asn²⁸; α3, His³⁴−Gly³⁸; and α5, Glu⁶²−Arg⁷²), and a π-helix (π4, Cys⁴³−Tyr⁵⁴), and is structurally constrained by a pattern of disulfide bonds (Cys⁷–Cys⁴³, Cys²³–Cys³⁹, and Cys²⁶–Cys⁵²), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na⁺, K⁺-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity. Full article

The nuclear receptor (NR) superfamily consists of 48 members that are divided into seven subfamilies. NRs are transcription factors that play an important role in a number of biological processes. The NR superfamily includes androgen receptor, which is a key player in prostate cancer pathogenesis, suggesting the functional roles of other NRs in prostate cancer. The findings on the roles of NRs in prostate cancer thus far have shown that several NRs such as vitamin D receptor, estrogen receptor β, and mineralocorticoid receptor play antioncogenic roles, while other NRs such as peroxisome proliferator-activated receptor γ and estrogen receptor α as well as androgen receptor play oncogenic roles. However, the roles of other NRs in prostate cancer remain controversial or uninvestigated. Further research on the role of NRs in prostate cancer is required and may lead to the development of novel preventions and therapeutics for prostate cancer. Full article

Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2-b]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2-b]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2-b]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2-b]indole-based CK2 inhibitor described yet (IC₅₀ = 25 nM) is 5-isopropyl-4-(3-methylbut-2-enyl-oxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p). Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α′, the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α′, but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydrogen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium. Full article

The nucleotide-binding domain, leucine-rich repeat/pyrin domain-containing-3 (NALP3) inflammasome, which is required for synthesis of interleukin-1β, has been implicated in the pathogenesis of several autoinflammatory syndromes. This review of the literature summarizes the interconnectedness of NALP3 inflammasome with some of these disorders. Familial Mediterranean fever results from a mutation in the Mediterranean fever (MEFV) gene, which encodes the pyrin protein. Previous study results suggest that pyrin suppresses caspase-1 activation, perhaps by competing for the adaptor protein, termed, pyrin domain of apoptosis/speck-like protein containing a caspase-recruitment domain (ACS) which therefore interferes with NALP3 inflammasome activation. The nucleotide-binding domain, leucine-rich repeat/pyrin domain-containing-3 (NALP3) inflammasome is constitutively activated in cryopyrin-associated periodic syndromes due to gain-of-function mutations resulting from point mutations within the neuronal apoptosis inhibitor protein/class 2 transcription factor/heterokaryon incompatibility/telomerase-associated protein-1 (NACHT) domain of the NALP3 protein. Pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is caused by mutations in the genes encoding proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1). These PSTPIP1 mutants are thought to bind to pyrin causing an increase in the pyrin domain of apoptosis/speck-like protein containing a caspase-recruitment domain (ASC) pyroptosome assembly leading to procaspase-1 recruitment and therefore its activation. Hyperimmunoglublinemia D syndrome is caused by mevalonate kinase (MVK) deficiency, which may be affected by protein accumulation that leads to NALP3 inflammasome activation. Tumor necrosis factor receptor–associated periodic syndrome is associated with mutations in the tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) gene which decreases the level of soluble tumor necrosis factor receptor-1 (TNFR1) leading to neutralization of tumor necrosis factor (TNF)-α. In general, these autoinflammatory disorders have shown a clinical response to interleukin-1 (IL-1) antagonists, suggesting that the NALP3 inflammasome serves a critical role in their pathogenesis. Full article

Vitamin D Receptor (VDR) belongs to the nuclear receptor (NR) superfamily. Whereas the structure of the ligand binding domain (LBD) of VDR has been determined in great detail, the role of its amino acid residues in stabilizing the structure and ligand triggering conformational change is still under debate. There are 13 α-helices and one β-sheet in the VDR LBD and they form a three-layer sandwich structure stabilized by 10 residues. Thirty-six amino acid residues line the ligand binding pocket (LBP) and six of these residues have hydrogen-bonds linking with the ligand. In 1α,25-dihydroxyvitamin D₃ signaling, H3 and H12 play an important role in the course of conformational change resulting in the provision of interfaces for dimerization, coactivator (CoA), corepressor (CoR), and hTAF_II 28. In this paper we provide a detailed description of the amino acid residues stabilizing the structure and taking part in conformational change of VDR LBD according to functional domains. Full article