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Cytochrome P450 monooxygenases (CYP, EC 1.14.14.1) belong to a large family of enzymes that catalyze the hydroxylation of various substrates. Here, we present the crystal structure of CYP105P2 isolated from Streptomyces peucetius ATCC27952 at a 2.1 Å resolution. The structure shows the presence of a pseudo-ligand molecule in the active site, which was co-purified fortuitously and is presumed to be a biphenyl derivative. Comparison with previously determined substrate-bound CYP structures showed that binding of the ligand produces large and distinctive conformational changes in α2-α3, α7-α9, and the C-terminal loop regions. This structural flexibility confirms our previous observation that CYP105P2 can accommodate a broad range of ligands. The structure complexed with a pseudo-ligand provides the first molecular view of CYP105P2-ligand interactions, and it indicates the involvement of hydrophobic residues (Pro82, Ala181, Met187, Leu189, Leu193, and Ile236) in the interactions between hydrophobic ligands and CYP105P2. These results provide useful insights into the structural changes involved in the recognition of different ligands by CYP105P2. [ABSTRACT FROM AUTHOR]

This study aimed to isolate and characterize sulfated polysaccharides (SPs) from Iridaea cordata and evaluate their anticancer activity. SPs of the Antarctic red seaweed were obtained by CaCl2 (SP1) and ethanol precipitations (SP2) following diluted acid extraction at room temperature. Yields of SP1 and SP2 were approximately 14% and 23%, respectively, of the dry weight of red seaweed. The average molecular mass of the SP1 and SP2 was estimated about 1.84 × 103 and 1.42 × 103 kDa, respectively, by size-fractionation High-Performance Liquid Chromatography (HPLC). From the High-Performance Anion- Exchange Chromatography-Pulsed Amperometric Detection (HPAEC-PAD) analysis, the main monosaccharide was galactose with glucose and fucose as minor components. The sulfate content of SP2 (40.4%) was slightly higher than that of SP1 (33.8%). The FT-IR spectra also showed characteristic band of carrageenan-like sulfated polysaccharides. Taken together the SPs are thought to be carrageenan-like sulfated galactan. The polysaccharides (SPs) from I. cordata exhibited weak antitumor activity against PC- 3 (prostate cancer), HeLa (cervical cancer), and HT-29 (human colon adenocarcinoma). To our knowledge, this is the first data on biological activity of the Antarctic red seaweed I. cordata. [ABSTRACT FROM AUTHOR]

STUDY QUESTION: Can antifreeze proteins (AFPs) from three different sources improve the efficacy of mouse oocyte vitrification? SUMMARY ANSWER: Treatment with AFPs can improve both murine oocyte quality and embryo development, and reduce reactive oxygen species (ROS) production in vitrified-warmed oocytes. WHAT IS KNOWN ALREADY: A previous study discovered that vitrification of immature oocytes and 2-cell stage embryos of mice augmented with antifreeze glycoproteins at 40 mg/ml dramatically improved the morphological integrity of the samples, suggesting that AFPs have the ability to inhibit ice formation and stabilize the plasma membrane. STUDY DESIGN, SIZE, DURATION: Metaphase II oocytes were obtained from 4-week-old BD-F1 mice. AFPs from bacteria (Flavobacterium frigoris ice-binding protein (FfIBP)), yeast (Glaciozyma sp. ice-binding protein (LeIBP)) and fish (Type III AFP) were added to the vitrification and warming solutions individually. Survival and development, meiotic spindle organization, intracellular ROS, mitochondrial activity, DNA doublestrand breaks (DSBs) and repair of damaged DNA were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Vitrification of oocytes was performed with the Cryo Top (equilibration solution: 7.5% ethylene glycol (EG) and 7.5% 1,2-propandiol (PROH) for 5 min; vitrification solution: 15% EG, 15% PROH and 0.5 M sucrose for 1 min).Warming was performed in three steps with decreasing concentrations of sucrose (1.0, 0.5 and 0.25 M sucrose). main results and the role of chance: AFP-treatment can improve murine oocyt equality and embryo development. Survival rates, cleavage rates and blastocyst rates (blastocyst per cleaved and per survived oocytes) of oocytes in AFP-treated groups were significantly higher than those in the control group[75.0, 89.0, 90.0 and 85.0% for survival rate (P = 0.012); 58.7, 89.0, 87.8 and 81.2% for cleavage rate (P = 0.003); 52.3, 87.7, 78.5 and 76.8% for blastocyst per cleaved oocytes (P<0.01); 30.7, 78.0, 68.9 and 62.4% for blastocyst per survived oocytes (P<0.01) in control, FfIBP

Pathogen-induced reorganization of the host cell cytoskeleton is a common strategy utilized in host cell invasion by many facultative intracellular bacteria, such as Shigella, Listeria, enteroinvasive E. coli and Salmonella. Shigella is an enteroinvasive intracellular pathogen that preferentially infects human epithelial cells and causes bacillary dysentery. Invasion of Shigella into intestinal epithelial cells requires extensive remodeling of the actin cytoskeleton with the aid of pathogenic effector proteins injected into the host cell by the activity of the type III secretion system. These so-called Shigella invasins, including IpaA, IpaC, IpgB1, IpgB2 and IpgD, modulate the actin-regulatory system in a concerted manner to guarantee efficient entry of the bacteria into host cells. [ABSTRACT FROM AUTHOR]

Ice-binding proteins (IBPs) inhibit ice growth through direct interaction with ice crystals to permit the survival of polar organisms in extremely cold environments. FfIBP is an ice-binding protein encoded by the Antarctic bacterium Flavobacterium frigoris PS1. The X-ray crystal structure of FfIBP was determined to 2.1 Å resolution to gain insight into its ice- binding mechanism. The refined structure of FfIBP shows an intramolecular disulfide bond, and analytical ultracentrifugation and analytical size-exclusion chromatography show that it behaves as a monomer in solution. Sequence alignments and structural comparisons of IBPs allowed two groups of IBPs to be defined, depending on sequence differences between the α2 and 4 loop regions and the presence of the disulfide bond. Although FfIBP closely resembles Leucosporidium (recently re-classified as Glaciozyma) IBP (LeIBP) in its amino-acid sequence, the thermal hysteresis (TH) activity of FfIBP appears to be tenfold higher than that of LeIBP. A comparison of the FfIBP and LeIBP structures reveals that FfIBP has different ice-binding residues as well as a greater surface area in the ice-binding site. Notably, the ice-binding site of FfIBP is composed of a T-A/G-X-T/N motif, which is similar to the ice-binding residues of hyperactive antifreeze proteins. Thus, it is proposed that the difference in TH activity between FfIBP and LeIBP may arise from the amino-acid composition of the ice-binding site, which correlates with differences in affinity and surface complementarity to the ice crystal. In conclusion, this study provides a molecular basis for understanding the antifreeze mechanism of FfIBP and provides new insights into the reasons for the higher TH activity of FfIBP compared with LeIBP. [ABSTRACT FROM AUTHOR]

Pathogenic Rickettsia species cause high morbidity and mortality, especially R. prowazekii, the causative agent of typhus. Like many intracellular pathogens, Rickettsia exploit the cytoskeleton to enter and spread within the host cell. Here we report that the cell surface antigen sca4 of Rickettsia co-localizes with vinculin in cells at sites of focal adhesions in sca4-transfected cells and that sca4 binds to and activates vinculin through two vinculin binding sites (VBSs) that are conserved across all Rickettsia. Remarkably, this occurs through molecular mimicry of the vinculin-talin interaction that is also seen with the IpaA invasin of the intracellular pathogen Shigella, where binding of these VBSs to the vinculin seven-helix bundle head domain (Vh1) displaces intramolecular interactions with the vinculin tail domain that normally clamp vinculin in an inactive state. Finally, the vinculin sca4-VBS crystal structures reveal that vinculin adopts a new conformation when bound to the C-terminal VBS of sca4. Collectively, our data define the mechanism by which sca4 activates vinculin and interacts with the actin cytoskeleton, and they suggest important roles for vinculin in Rickettsia pathogenesis. [ABSTRACT FROM AUTHOR]

Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated DLeu954 metavinculin deletion mutant. These structures reveal that an a-helix (H19) and extended coil of the metavinculin insert replace a-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated DLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H19 a-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions. [ABSTRACT FROM AUTHOR]

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-Å resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at -1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at -3 and -5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved βB/βC loop and N-terminal βA strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal βA strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies. [ABSTRACT FROM AUTHOR]

Plasmid Achromobacter secretion (PAS) factor is a putative secretion factor that induces the secretion of periplasmic proteins. PAS factor from Vibrio vulnificus was crystallized at 294 K by the hanging drop vapor- diffusion method. It was isolated as a monomer during the purification procedures. The native crystal belongs to the F222 space group with unit cell parameters a = 56.1, b = 74.4, c = 80.0 Å, α = β = γ = 90°. The crystal was soaked in cryoprotectant containing 1 M NaBr for 1 h for MAD phasing. The diffraction limit of the Br-MAD data set was 1.9 Å using synchrotron Xray irradiation at beam line BL-18B at the Photon Factory, Japan. [ABSTRACT FROM AUTHOR]

GluR0 from Nostoc punctiforme ( NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor. The ligand-binding core of NpGluR0 was crystallized at 294 K using the hanging-drop vapour-diffusion method. The l-­glutamate-complexed crystal belongs to space group C2221, with unit-cell parameters a = 78.0, b = 145.1, c = 132.1 Å. The crystals contain three subunits in the asymmetric unit, with a VM value of 2.49 Å3 Da−1. The diffraction limit of the l-glutamate complex data set was 2.1 Å using synchrotron X-ray radiation at beamline BL-4A of the Pohang Accelerator Laboratory (Pohang, Korea). [ABSTRACT FROM AUTHOR]

Abstract Low-molecular-weight heparin (LMWH), derived from unfractionated heparin (UFH), has enhanced anticoagulant efficacy, long duration of action, and extended half-life. Patients receiving LMWH for preventive therapies would strongly benefit from its long-term effects, however, achieving this is challenging. Here, we design and evaluate a nanoengineered LMWH and octadecylamine conjugate (LMHO) that can act for a long time while maintaining close to 97 ± 3% of LMWH activity via end-specific conjugation of the reducing end of LMWH. LMHO can self-assemble into nanoparticles with an average size of 105 ± 1.7 nm in water without any nanocarrier and can be combined with serum albumin, resulting in a lipid-based albumin shuttling effect. Such molecules can circulate in the bloodstream for 4–5 days. We corroborate the self-assembly capability of LMHO and its interaction with albumin through molecular dynamics (MD) simulations and transmission electron microscopy (TEM) analysis. This innovative approach to carrier-free polysaccharide delivery, enhanced by nanoengineered albumin shuttling, represents a promising platform to address limitations in conventional therapies.

Shank proteins are a new family of scaffold proteins interacting with various membrane and cytoplasmic proteins. Shank contains multiple protein-protein interaction sites, including ankyrin repeats, an SH3 domain, a PDZ domain, a long proline-rich region and an SAM domain. The PDZ domain of Shank binds to the C-terminus of guanylate kinase-associated protein (GKAP). The PDZ domain of Shankl from Rattus norvegicus and its complex with the C-terminal octapeptide of GKAP were crystallized at 294 K using polyethylene glycol 20 000 and 6000 as precipitants. Diffraction data sets from a peptide-free crystal and a complex crystal were collected to 1.8 and 3.2 Å resolution, respectively, using synchrotron radiation. The peptide-free crystal belongs to space group P2[sub 1], with unit-cell parameters a = 42.0, b - 50.3, c = 51.8 Å, β = 106.3°. The complex crystal belongs to space group P2[sub 1]2[sub 1]2[sub 1], with unit-cell parameters a = 89.4, b = 97.5, c = 108.3 Å. [ABSTRACT FROM AUTHOR]

The sixth PDZ domain from GRIPI and its complex with the octapeptide of the liprin-α1 C-terminus were crystallized at 294 K by the hanging-drop vapour-diffusion method. The native crystal belongs to space group P6122 (or P6522), with unit-cell parameters a = b = 40.3, c = 222.9 Å. The complex crystal belongs to space group R32, with unit-cell parameters a = b = 117.8, c = 102.0 Å. Native and peptide-complex diffraction data were collected to resolutions of 1.5 and 1.8 Å, respectively, using synchrotron X-rays. [ABSTRACT FROM AUTHOR]

Abstract Background Despite the effectiveness of glucagon-like peptide-1 agonist (GLP-1A) in the treatment of diabetes, its large molecular weight and high hydrophilicity result in poor cellular permeability, thus limiting its oral bioavailability. To address this, we developed a chimeric GLP-1A that targets transporter-mediated endocytosis to enhance cellular permeability to GLP-1A by utilizing the transporters available in the intestine, particularly the apical sodium-dependent bile acid transporter (ASBT). Methods In silico molecular docking and molecular dynamics simulations were used to investigate the binding interactions of mono-, bis-, and tetra-deoxycholic acid (DOCA) (monoDOCA, bisDOCA, and tetraDOCA) with ASBT. After synthesizing the chimeric GLP-1A-conjugated oligomeric DOCAs (mD-G1A, bD-G1A, and tD-G1A) using a maleimide reaction, in vitro cellular permeability and insulinotropic effects were assessed. Furthermore, in vivo oral absorption in rats and hypoglycemic effect on diabetic db/db mice model were evaluated. Results In silico results showed that tetraDOCA had the lowest interaction energy, indicating high binding affinity to ASBT. Insulinotropic effects of GLP-1A-conjugated oligomeric DOCAs were not different from those of GLP-1A-Cys or exenatide. Moreover, bD-G1A and tD-G1A exhibited improved in vitro Caco-2 cellular permeability and showed higher in vivo bioavailability (7.58% and 8.63%) after oral administration. Regarding hypoglycemic effects on db/db mice, tD-G1A (50 μg/kg) lowered the glucose level more than bD-G1A (50 μg/kg) compared with the control (35.5% vs. 26.4%). Conclusion GLP-1A was conjugated with oligomeric DOCAs, and the resulting chimeric compound showed the potential not only for glucagon-like peptide-1 receptor agonist activity but also for oral delivery. These findings suggest that oligomeric DOCAs can be used as effective carriers for oral delivery of GLP-1A, offering a promising solution for enhancing its oral bioavailability and improving diabetes treatment. Graphical Abstract

In cancer therapy, photodynamic therapy (PDT) has attracted significant attention due to its high potential for tumor-selective treatment. However, PDT agents often exhibit poor physicochemical properties, including solubility, necessitating the development of nanoformulations. In this study, we developed two cationic peptide-based self-assembled nanomaterials by using a PDT agent, chlorin e6 (Ce6). To manufacture biocompatible nanoparticles based on peptides, we used the cationic poly-L-lysine peptide, which is rich in primary amines. We prepared low- and high-molecular-weight poly-L-lysine, and then evaluated the formation and performance of nanoparticles after chemical conjugation with Ce6. The results showed that both molecules formed self-assembled nanoparticles by themselves in saline. Interestingly, the high-molecular-weight poly-L-lysine and Ce6 conjugates (HPLCe6) exhibited better self-assembly and PDT performance than low-molecular-weight poly-L-lysine and Ce6 conjugates (LPLCe6). Moreover, the HPLCe6 conjugates showed superior cellular uptake and exhibited stronger cytotoxicity in cell toxicity experiments. Therefore, it is functionally beneficial to use high-molecular-weight poly-L-lysine in the manufacturing of poly-L-lysine-based self-assembling biocompatible PDT nanoconjugates.

We report the draft genome sequence of Paenisporosarcina sp. strain TG-20, which is 4.12 Mb in size and consists of 4,071 protein-coding genes and 76 RNA genes. The genome sequence of Paenisporosarcina sp. TG-20 may provide useful information about molecular adaptations that enhance survival in icy subsurface environments. [ABSTRACT FROM AUTHOR]

CYP105D18 supports H2O² as an oxygen surrogate for catalysis well and shows high H2O² resistance capacity. We report the hydroxylation of different steroids using H2O² as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2b-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. [ABSTRACT FROM AUTHOR]

The members of Microbacterium isolated from different environments are known to form peptidoglycan. In this study, we compared the biofilm-forming abilities of Microbacterium sp. PAMC22086 (PAMC22086), which was isolated from the soil in the South Shetland Islands and Microbacterium sp. PAMC21962 (PAMC21962), which was isolated from algae in the South Shetland Islands. The analysis of average nucleotide identity and phylogeny of PAMC22086 revealed a 97% similarity to Microbacterium oxydans VIU2A, while PAMC21962 showed a 99.1% similarity to Microbacterium hominis SGAir0570. For the comparative genomic analysis of PAMC22086 and PAMC21962, the genes related to biofilm formation were identified using EggNOG and KEGG pathway databases. The genes possessed by both PAMC22086 and PAMC21962 are cpdA, phnB, rhlC, and glgC, which regulate virulence, biofilm formation, and multicellular structure. Among the genes indirectly involved in biofilm formation, unlike PAMC21962, PAMC22086 possessed csrA, glgC, and glgB, which are responsible for attachment and glycogen biosynthesis. Additionally, in PAMC22086, additional functional genes rsmA, which is involved in mobility and polysaccharide production, and dksA, GTPase, and oxyR, which play roles in cell cycle and stress response, were identified. In addition, the biofilm-forming ability of the two isolates was examined in vivo using the standard crystal violet staining technique, and morphological differences in the biofilm were investigated. It is evident from the different distribution of biofilm-associated genes between the two strains that the bacteria can survive in different niches by employing distinct strategies. Both strains exhibit distinct morphologies. PAMC22086 forms a biofilm that attaches to the side, while PAMC21962 indicates growth starting from the center. The biofilm formation-related genes in Microbacterium are not well understood. However, it has been observed that Microbacterium species form biofilm regardless of the number of genes they possess. Through comparison between different Microbacterium species, it was revealed that specific core genes are involved in cell adhesion, which plays a crucial role in biofilm formation. This study provides a comprehensive profile of the Microbacterium genus’s genomic features and a preliminary understanding of biofilm in this genus, laying the foundation for further research.

Numerous cathepsin B inhibitors have been developed and are under investigation as potential cancer treatments. They have been evaluated for their ability to inhibit cathepsin B activity and reduce tumor growth. However, they have shown critical limitations, including low anticancer efficacy and high toxicity, due to their low selectivity and delivery problems. In this study, we developed a novel peptide and drug conjugate (PDC)-based cathepsin B inhibitor using cathepsin-B-specific peptide (RR) and bile acid (BA). Interestingly, this RR and BA conjugate (RR–BA) was able to self-assemble in an aqueous solution, and as a result, it formed stable nanoparticles. The nano-sized RR–BA conjugate showed significant cathepsin B inhibitory effects and anticancer effects against mouse colorectal cancer (CT26) cells. Its therapeutic effect and low toxicity were also confirmed in CT26 tumor-bearing mice after intravenous injection. Therefore, based on these results, the RR–BA conjugate could be developed as an effective anticancer drug candidate for inhibiting cathepsin B in anticancer therapy.

[This corrects the article DOI: 10.1371/journal.pone.0158841.].

The psychrophilic bacterium Paenisporosarcina sp. TG-14 was isolated from sediment-laden stratified basal ice from Taylor Glacier, McMurdo Dry Valleys, Antarctica. Here we report the draft genome sequence of this strain, which may provide useful information on the cold adaptation mechanism in extremely variable environments. [ABSTRACT FROM AUTHOR]

The psychrophilic bacterium Moritella dasanensis strain ArB 0140 was isolated near a glacier in Kongsfjorden, Svalbard Archipelago, Norway. Here we report a 4.89-Mb draft genome sequence of Moritella dasanensis ArB 0140, which could provide comprehensive information on a psychrophilic mechanism in extreme environments. [ABSTRACT FROM AUTHOR]

Describes the crystal structure of glutamate receptor-interacting protein (GRIP) 1 PDZ6-peptide complex. Emergence of the structural basis for class II PDZ target recognition and PDZ domain-mediated multimerization; Reorientation of helix alpha B; Formation of GRIP multimers and efficient clustering of GRIP-binding proteins.