Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

Structural and functional analysis of Hikeshi, a new nuclear transport receptor of Hsp70s.

Hikeshi is a nuclear transport receptor required for cell survival after stress. It mediates heat-shock-induced nuclear import of 70 kDa heat-shock proteins (Hsp70s) through interactions with FG-nucleoporins (FG-Nups), which are proteins in nuclear pore complexes (NPCs). Here, the crystal structure of human Hikeshi is presented at 1.8 Šresolution. Hikeshi forms an asymmetric homodimer that is responsible for the interaction with Hsp70s. The asymmetry of Hikeshi arises from the distinct conformation of the C-terminal domain (CTD) and the flexibility of the linker regions of each monomer. Structure-guided mutational analyses showed that both the flexible linker region and the CTD are important for nuclear import of Hsp70. Pull-down assays revealed that only full-length Hsp70s can interact with Hikeshi. The N-terminal domain (NTD) consists of a jelly-roll/ß-sandwich fold structure which contains hydrophobic pockets involved in FG-Nup recognition. A unique extended loop (E-loop) in the NTD is likely to regulate the interactions of Hikeshi with FG-Nups. The crystal structure of Hikeshi explains how Hikeshi participates in the regulation of nuclear import through the recognition of FG-Nups and which part of Hikeshi affects its binding to Hsp70. This study is the first to yield structural insight into this highly unique import receptor.

Structural basis for the selective nuclear import of the C2H2 zinc-finger protein Snail by importin ß.

Snail contributes to the epithelial-mesenchymal transition by suppressing E-cadherin in transcription processes. The Snail C2H2-type zinc-finger (ZF) domain functions both as a nuclear localization signal which binds to importin ß directly and as a DNA-binding domain. Here, a 2.5 Šresolution structure of four ZF domains of Snail1 complexed with importin ß is presented. The X-ray structure reveals that the four ZFs of Snail1 are required for tight binding to importin ß in the nuclear import of Snail1. The shape of the ZFs in the X-ray structure is reminiscent of a round snail, where ZF1 represents the head, ZF2-ZF4 the shell, showing a novel interaction mode, and the five C-terminal residues the tail. Although there are many kinds of C2H2-type ZFs which have the same fold as Snail, nuclear import by direct recognition of importin ß is observed in a limited number of C2H2-type ZF proteins such as Snail, Wt1, KLF1 and KLF8, which have the common feature of terminating in ZF domains with a short tail of amino acids.

Degradation of Polo-like Kinase 1 by the Novel Poly-Arginine N-Degron Pathway PROTAC Regulates Tumor Growth in Nonsmall Cell Lung Cancer.

Polo-like kinase 1 (PLK1), which is crucial in cell cycle regulation, is considered a promising anticancer drug target. Herein, we present the N-degron pathway-based proteolysis targeting chimera (PROTAC) for PLK1 degradation, targeting the Polo-box domain (PBD). We identified DD-2 as the most potent PROTAC that selectively induces PLK1 degradation in cancer cells, including HeLa and nonsmall cell lung cancer (NSCLC), through the N-degron pathway. DD-2 exhibited significant in vitro anticancer effects, inducing G2/M arrest and apoptosis in HeLa and NSCLC cell lines. DD-2 showed significant tumor growth inhibition in a xenograft mouse model using HeLa and NSCLC cell lines, highlighting its potential in cancer treatment. Furthermore, the combination of DD-2 with tyrosine kinase inhibitor (TKI), osimertinib, effectively suppressed tumor growth in double-mutated H1975 cell lines, emphasizing DD-2's potential in combination cancer therapies. Collectively, this study demonstrates the potential of the N-degron pathway, especially using DD-2, for targeted cancer therapies.

Crystallization and preliminary X-ray diffraction analysis of human importin ß-Snail zinc finger domain complex.

Snail is a C2H2-type zinc finger transcriptional repressor that induces epithelial-mesenchymal transition by repression of E-cadherin expression levels during embryonic development and tumour progression. Snail is imported into the nucleus by importin ß through direct binding with its four zinc finger domain. The complex between importin ß and Snail four zinc finger domain was crystallized in order to understand the nuclear transport mechanism of Snail. The constituents of the complex were separately expressed and were then co-purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 228.2, b = 77.5, c = 72.0 Å, ß = 100.9° and diffracted to 2.5 Šresolution.

Small-molecule inhibitors of ferrochelatase are antiangiogenic agents.

Activity of the heme synthesis enzyme ferrochelatase (FECH) is implicated in multiple diseases. In particular, it is a mediator of neovascularization in the eye and thus an appealing therapeutic target for preventing blindness. However, no drug-like direct FECH inhibitors are known. Here, we set out to identify small-molecule inhibitors of FECH as potential therapeutic leads using a high-throughput screening approach to identify potent inhibitors of FECH activity. A structure-activity relationship study of a class of triazolopyrimidinone hits yielded drug-like FECH inhibitors. These compounds inhibit FECH in cells, bind the active site in cocrystal structures, and are antiangiogenic in multiple in vitro assays. One of these promising compounds was antiangiogenic in vivo in a mouse model of choroidal neovascularization. This foundational work may be the basis for new therapeutic agents to combat not only ocular neovascularization but also other diseases characterized by FECH activity.

Selective enrichment and mass spectrometric identification of nitrated peptides using fluorinated carbon tags.

Protein tyrosine nitration (PTN) is a post-translational modification that is related to several acute or chronic diseases. PTN introduces a nitro group in the ortho position of the phenolic hydroxyl group of tyrosine residues. PTN has been shown to be involved in the pathogenesis of inflammatory responses, cancers, and neurodegenerative and age-related disorders. Furthermore, it has been proposed that PTN regulates signal cascades related to nitric oxide (NO·) production and NO-mediated processes. Although nitrated proteins as markers of oxidative stress are confirmed by immunological assays in various affected cells or tissues, it is not known how many different types of proteins in living cells are nitrated. Since protein nitration is a low-abundance post-translational modification, development of an effective enrichment method for nitrated proteins is needed to detect nitrated peptides or proteins from the limited amount of pathophysiological samples. In the present study, we developed an enrichment method using specific chemical tagging. Nitroproteome profiling using chemical tagging and mass spectrometry was validated by model proteins. Furthermore, we successfully identified numerous nitrated proteins from the Huh7 human hepatoma cell line.

Crystallization and preliminary X-ray crystallographic study of the human MST2 SARAH domain.

The SARAH domain at the C-terminus of human MST2 (residues 436-484) was overproduced and purified using an Escherichia coli expression system. The purified domain was crystallized using the hanging-drop vapour-diffusion technique. Two crystal forms were obtained. The crystals belonged to space group P2, with unit-cell parameters a = 62.0, b = 119.2, c = 62.0 Å, α = 90.0, ß = 90.5, γ = 90.0°, or to space group P6(1)22, with unit-cell parameters a = 54.5, b = 54.5, c = 303.1 Å. These crystals diffracted to 2.7 and 3.0 Å resolution, respectively.

Structural basis for nuclear import complex dissociation by RanGTP.

Nuclear protein import is mediated mainly by the transport factor importin-beta that binds cytoplasmic cargo, most often via the importin-alpha adaptor, and then transports it through nuclear pore complexes. This active transport is driven by disassembly of the import complex by nuclear RanGTP. The switch I and II loops of Ran change conformation with nucleotide state, and regulate its interactions with nuclear trafficking components. Importin-beta consists of 19 HEAT repeats that are based on a pair of antiparallel alpha-helices (referred to as the A- and B-helices). The HEAT repeats stack to yield two C-shaped arches, linked together to form a helicoidal molecule that has considerable conformational flexibility. Here we present the structure of full-length yeast importin-beta (Kap95p or karyopherin-beta) complexed with RanGTP, which provides a basis for understanding the crucial cargo-release step of nuclear import. We identify a key interaction site where the RanGTP switch I loop binds to the carboxy-terminal arch of Kap95p. This interaction produces a change in helicoidal pitch that locks Kap95p in a conformation that cannot bind importin-alpha or cargo. We suggest an allosteric mechanism for nuclear import complex disassembly by RanGTP.

Characterization of acetohydroxyacid synthase I from Escherichia coli K-12 and identification of its inhibitors.

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS-PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg(+2), ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 µM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.

Chemical approach for specific enrichment and mass analysis of nitrated peptides.

The analysis and detection of 3-nitrotyrosine are biologically and clinically important because protein tyrosine nitration is known to be involved in a number of biological phenomena such as cellular signal transduction, pathogenesis of inflammatory responses, and age-related disorders. However, the main obstacles in the study are low abundance of nitrated species and lack of efficient enrichment methods. Here in, we suggest a new chemical approach to analyze nitrated peptides using mass spectrometry by incorporating specific tagging groups in the peptides through simple chemical transformations. Nitro groups on tyrosine side chains of nitrated peptides were subjected to reduction to give rise to amine which was further converted to metal-chelating motif. Mass analyses verified that Ni(2+)-NTA magnetic agarose beads selectively captured and isolated the modified peptides, i.e., nitrated peptides, by strong and specific metal chelating interactions. We further demonstrated the utility of our approach by detection of nitrated peptides in complex samples such as tryptic peptide mixtures of bovine serum albumin (BSA) and a HeLa cell lysate.

Structural basis of sugar-recognizing ubiquitin ligase.

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.

Role of protein tyrosine nitration in neurodegenerative diseases and atherosclerosis.

Nitric oxide generates reactive nitrosative species, such as peroxynitrite (ONOO(-)) that may be involved in a number of diseases. ONOO(-) can mediate protein tyrosine nitration which causes structural changes of affected proteins and leads to their inactivation. Various proteomics and immunological methods including mass spectrometry combined with both liquid and 2-D PAGE, and immunodetection have been employed to identify and characterize nitrated proteins from pathological samples. This review presents the pahtobiological roles of the pathogenic posttranslational modification in neurodegenerative diseases and atherosclerosis.

Antitumor activity of cytokine-induced killer cells in nude mouse xenograft model.

Malignant glioma is the most common primary brain tumor in adults and the median survival for patients is less than a year. Despite aggressive treatments including surgical resection, radiotherapy, and chemotherapy, only modest improvement has been achieved in the survival of patients with glioma. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against human glioma cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The number of cells increased more than 200-fold and the viability was >90%. The resulting populations were consisted of 96% CD3(+), 2% CD3(-)CD56(+), 68% CD3(+)CD56(+), 2% CD4(+), <1% CD4(+)CD56(+), 80% CD8(+), and 49% CD8(+)CD56(+). This heterogeneous cell population was called as CIK cells. At an effector-target cell ratio of 30:1, CIK cells destroyed 43% of U-87 MG human glioma cells, as measured by the (51)Cr-release assay. In addition, CIK cells at doses of 0.3, 1, and 3 million cells per mouse inhibited 23%, 40%, and 50% of U-87 MG tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for glioma cancer patients.

Temperature-dependency urease activity in Vibrio parahaemolyticus is related to transcriptional activator UreR.

Vibrio parahaemolyticus possessing urease-positive property is relatively rare, but such strains consistently exhibit the TDH-related hemolysin (TRH) gene. In this study, we examined the effects of incubation temperature on urease activity expression, using the TH3996 and AQ4673 strains where the enzyme activity is known to be temperature-dependent and -independent, respectively. In the TH3996 strain, beta-galactosidase activity was 4.4-fold lower after 30 degrees C cultivation than after 37 degrees in a ureR-lacZ fusion strain, but temperature dependency was not found in ureD- or nikA-lacZ fusion strains. However, ureR-, ureD-, and nikA-lacZ fusions of the AQ4673 strain was not influenced by incubation temperature. We compared the promoter sequences of ureR between the above two strains. Intriguingly, we detected mismatches of two nucleotides between the two strains located at positions -66 and -108 upstream of the methionine initiation codon for UreR. Additionally, urease activity was not affected by culture temperature at either 30 degrees or 37 degrees by allelic introduction of the AQ4673 ureR gene into the TH3996 ureR deletion mutant. Taken together, our study demonstrates that the transcriptional factor UreR is involved in the temperature dependency of urease activity, and two nucleotides within the ureR promoter region are of particular importance for the urease activity dependency of V. parahaemolyticus.

Effects of the aryl linker and the aromatic substituent on the anti-HCV activities of aryl diketoacid (ADK) analogues.

Based on our pharmacophore model of the aryl diketoacids (ADKs), we designed and prepared a series of novel ADK analogues, which showed potent inhibitory activities against the NS5B polymerase in the submicromolar range. Pharmacophore-guided docking study revealed that the antiviral activities of the ADKs are highly dependent upon the aryl linker as well as the size and position of the aromatic substituent. It is of another importance that, unlike previously reported ADKs, three ADK analogues synthesized in this study effectively blocked Hepatitis C Virus (HCV) replication in the replicon systems.

Crystal structure of tuguaconitine, a diterpene alkaloid from Aconitum sibiricum.

The three-dimensional structure of a natural product, tuguaconitine (C23H35NO7) from Aconitumrn sibiricum was determined by single crystal X-ray diffraction analysis. The compound was recrystallized from a methanol solution into orthorhombic crystal system, with a = 18.093(4), b = 12.075(11), c = 9.824(4) A, Dm = 1.35, Dx= 1.354 g/cm3, space group P2(1)2(1)2(1), and Z=4. The structure was solved by direct method and refined by least-squares procedure to the final R value of 0.046 for 1495 observed reflections with I > or = 2sigma(I). Aconitines are diterpene alkaloid composed of six rings. In the molecule, two 6-membered rings have stable chair conformation, but other two 6-membered rings have unstable boat conformation because of their bonding strain and hydrogen bonds. Based on the three-dimensional structure of tuguaconitine, we suggest another 2D-diagram for the compound which is far better in imagination of the spatial shape of the molecule than the ordinary one.

Concise synthesis of Obovatol: Chemoselective ortho-bromination of phenol and survey of Cu-catalyzed diaryl ether couplings.

Concise total synthesis of obovatol (1) was achieved from the commercially available eugenol (5) via linear 4 steps in 40% overall yield. The key features of the synthesis involve the chemoselective orthobromination of phenol in the presence of isolated double bond and the efficient Cu-catalyzed Ullmann coupling of two aromatic moieties for the diaryl ether skeleton.

Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant hepatoma cells to TRAIL-induced apoptosis through reactive oxygen species-mediated up-regulation of DR5.

Sulforaphane is a chemopreventive agent present in various cruciferous vegetables, including broccoli. Here, we show that treatment with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in combination with subtoxic doses of sulforaphane significantly induces rapid apoptosis in TRAIL-resistant hepatoma cells. Neither TNF-alpha- nor Fas-mediated apoptosis was sensitized in hepatoma cells by cotreatment with sulforaphane, suggesting that sulforaphane can selectively sensitize cells to TRAIL-induced apoptosis but not to apoptosis mediated by other death receptors. We found that sulforaphane treatment significantly up-regulated mRNA and protein levels of DR5, a death receptor of TRAIL. This was accompanied by an increase in the generation of reactive oxygen species (ROS). Pretreatment with N-acetyl-l-cysteine and overexpression of catalase inhibited sulforaphane-induced up-regulation of DR5 and almost completely blocked the cotreatment-induced apoptosis. Furthermore, the sulforaphane-mediated sensitization to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs for DR5. These results collectively indicate that sulforaphane-induced generation of ROS and the subsequent up-regulation of DR5 are critical for triggering and amplifying TRAIL-induced apoptotic signaling. We also found that sulforaphane can sensitize both Bcl-xL- and Bcl-2-overexpressing hepatoma cells to TRAIL-induced apoptosis, indicating that treatment with a combination of TRAIL and sulforaphane may be a safe strategy for treating resistant hepatomas.

Structural Basis for Selective Binding of Export Cargoes by Exportin-5.

In the nucleus, RanGTP binding to importin dissociates the cargo. On the other hand, RanGTP enables exportin to bind export cargo and form the export complex by each exportin's own cargo selection mechanism. Here, we present two X-ray structures for Exportin-5 (Exp-5) alone and Exp-5:RanGTP intermediate complex. The structure of Exp-5 adopts a ring-shaped closed conformation by C-terminal anchor residues 1,167-1,179, interacting with N-terminal heat repeats 4-9. The closed form of Exp-5 is important for the stability of the cargo-free state. Interaction between Exp-5 and RanGTP induces elimination of intramolecular contacts of the C-terminal anchor. A large movement of N-terminal 1-9th heat repeats and C-terminal 19-20th heat repeats creates an open space for RanGTP accommodation. Exp-5 in Exp-5:RanGTP and Exp-5:RanGTP:pre-miRNA adopts the same conformation. RanGTP binding to Exp-5 creates a selective molecular cage area for accepting its cargoes, such as small double-stranded RNAs, without conformational change in Exp-5:RanGTP.