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.

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.

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.

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.

The p21-activated kinase 4-Slug transcription factor axis promotes epithelial-mesenchymal transition and worsens prognosis in prostate cancer.

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and metastasis and thus accelerates cancer progression. p21-activated kinase 4 (PAK4) is a critical regulator of prostate cancer (PC) progression. Here, we report that PAK4 activation promotes PC progression through the EMT regulator Slug. We find that phosphorylated PAK4S474 (pPAK4) levels, an index of PAK4 activation, were tightly associated with Gleason score (p < 0.001), a clinical indicator of PC progression, but not with prostate serum antigen levels or tumor stage. Stable silencing of PAK4 in PC cells reduced their potential for EMT, cellular invasion, and metastasis in vivo. PAK4 bound and directly phosphorylated Slug at two previously unknown sites, S158 and S254, which resulted in its stabilization. The non-phosphorylatable form SlugS158A/S254A upregulated transcription of CDH1, which encodes E-cadherin, and thus suppressed EMT and invasion, to a greater extent than did wild-type Slug. The strong EMT inducer TGF-ß elevated pPAK4 and pSlugS158 levels; PAK4 knockdown or introduction of a dominant-negative form of PAK4 inhibited both TGF-ß-stimulated EMT and an increase in pSlugS158 levels. Finally, immunohistochemistry revealed a positive correlation between pPAK4 and pSlugS158 but an inverse correlation between pSlugS158 and E-cadherin. The results suggest that the PAK4-Slug axis represents a novel pathway that promotes PC progression.

Pyrazole derived ultra-short antimicrobial peptidomimetics with potent anti-biofilm activity.

In this study, we report on the first chemical synthesis of ultra-short pyrazole-arginine based antimicrobial peptidomimetics derived from the newly synthesized N-alkyl/aryl pyrazole amino acids. Through the systematic tuning of hydrophobicity, charge, and peptide length, we identified the shortest peptide Py11 with the most potent antimicrobial activity. Py11 displayed greater antimicrobial activity against antibiotic-resistant bacteria, including MRSA, MDRPA, and VREF, which was approximately 2-4 times higher than that of melittin. Besides its higher selectivity (therapeutic index) toward bacterial cells than LL-37, Py11 showed highly increased proteolytic stability against trypsin digestion and maintained its antimicrobial activity in the presence of physiological salts. Interestingly, Py11 exhibited higher anti-biofilm activity against MDRPA compared to LL-37. The results from fluorescence spectroscopy and transmission electron microscopy (TEM) suggested that Py11 kills bacterial cells possibly by integrity disruption damaging the cell membrane, leading to the cytosol leakage and eventual cell lysis. Furthermore, Py11 displayed significant anti-inflammatory (endotoxin-neutralizing) activity by inhibiting LPS-induced production of nitric oxide (NO) and TNF-α. Collectively, our results suggest that Py11 may serve as a model compound for the design of antimicrobial and antisepsis agents.

Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization.

In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.

Low pH-driven folding of WW45-SARAH domain leads to stabilization of the WW45-Mst2 complex.

The scaffolding protein Salvador (Sav) plays a key role in the Hippo (Hpo) signalling pathway, which controls tissue growth by inhibiting cell proliferation and promoting apoptosis. Dysregulation of the Hippo pathway contributes to cancer development. Since the identification of the first Sav gene in 2002, very little is known regarding the molecular basis of Sav-SARAH mediating interactions due to its insolubility. In this study, refolding of the first Sav (known as WW45)-SARAH provided insight into the biochemical and biophysical properties, indicating that WW45-SARAH exhibits properties of a disordered protein, when the domain was refolded at a neutral pH. Interestingly, WW45-SARAH shows folded and rigid conformations relative to the decrease in pH. Further, diffracting crystals were obtained from protein refolded under acidic pH, suggesting that the refolded WW45 protein at low pH has a homogeneous and stable conformation. A comparative analysis of molecular properties found that the acidic-stable fold of WW45-SARAH enhances a heterotypic interaction with Mst2-SARAH. In addition, using an Mst2 mutation that disrupts homotypic dimerization, we showed that the monomeric Mst2-SARAH domain could form a stable complex of 1:1 stoichiometric ratio with WW45 refolded under acidic pH.

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.

A new class of peptidomimetics targeting the polo-box domain of Polo-like kinase 1.

Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.

Crystallization and preliminary X-ray crystallographic study of human Hikeshi, a new nuclear transport receptor for Hsp70.

Hikeshi is a new nuclear transport receptor that plays an important role in the nuclear import of Hsp70 heat-shock proteins under thermal stress. Wild-type human Hikeshi and its Phe97Ala mutant were overproduced and purified using an Escherichia coli expression system. The purified proteins were crystallized using the hanging-drop vapour-diffusion technique. Wild-type crystals grew in space group C2221, with unit-cell parameters a = 61.1, b = 137.8, c = 97.9 Å, α = 90.0, ß = 90.0, γ = 90.0°. Phe97Ala mutant crystals were obtained in space group P32, with unit-cell parameters a = 85.7, b = 85.7, c = 69.1 Å, α = 90.0, ß = 90.0, γ = 120.0°. These crystals diffracted to 1.8 and 2.5 Šresolution, respectively. This study is the first to yield structural insight into this highly unusual fourth import receptor after importins, NTF2 and TAP.

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.

X-ray structure of prephenate dehydratase from Streptococcus mutans.

Prephenate dehydratase is a key enzyme of the biosynthesis of L-phenylalanine in the organisms that utilize shikimate pathway. Since this enzymatic pathway does not exist in mammals, prephenate dehydratase can provide a new drug targets for antibiotics or herbicide. Prephenate dehydratase is an allosteric enzyme regulated by its end product. The enzyme composed of two domains, catalytic PDT domain located near the N-terminal and regulatory ACT domain located near the C-terminal. The allosteric enzyme is suggested to have two different conformations. When the regulatory molecule, phenylalanine, is not bound to its ACT domain, the catalytic site of PDT domain maintain open (active) state conformation as Sa-PDT structure. And the open state of its catalytic site become closed (allosterically inhibited) state if the regulatory molecule is bound to its ACT domain as Ct-PDT structure. However, the X-ray structure of prephenate dehydratase from Streptococcus mutans (Sm-PDT) shows that the catalytic site of Sm-PDT has closed state conformation without phenylalanine molecule bound to its regulatory site. The structure suggests a possibility that the binding of phenylalanine in its regulatory site may not be the only prerequisite for the closed state conformation of Sm-PDT.

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.

Composition of the extracellular matrix of lymphatic novel threadlike structures: is it keratin?

Background. The lumen of novel threadlike structures (NTSs) is enclosed by a single layer of endothelial cells surrounded by extracellular matrix (ECM). We hypothesized that collagen may be a component of the ECM associated with lymphatic NTSs. Methods. Six female New Zealand white rabbits were anesthetized, and the NTS structures within lymphatic vessels were identified by contrast-enhanced stereomicroscopy or alcian blue staining. Isolated NTS specimens were stained with acridine orange, YOYO-1, and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The structural and molecular composition of the ECM was investigated using transmission electron microscopy (TEM), electrospray ionization-mass spectrometry, and proteomic analysis. Results. The lymph vessel wall was stained red by DiI, and rod-shaped nuclei were stained green by YOYO-1. The area surrounding the NTS was also stained red and contained green rod-shaped nuclei. TEM images showed that the NTS consisted of many ECM fibers and the ECM fibers appeared to be ~100 nm in diameter and had narrowly spaced striated bands. Proteomic analysis of the lymphatic NTS-associated ECM identified 4 proteins: keratin 10, cytokeratin 3, cytokeratin 12, and soluble adenylyl cyclase. Conclusion. The TEM study suggested that the lymphatic NTS-associated ECM did not contain collagen. This was confirmed by proteomic analysis, which showed that keratin was the major component of the ECM.

Biosynthesis of the tunicamycin antibiotics proceeds via unique exo-glycal intermediates.

The tunicamycins are archetypal nucleoside antibiotics targeting bacterial peptidoglycan biosynthesis and eukaryotic protein N-glycosylation. Understanding the biosynthesis of their unusual carbon framework may lead to variants with improved selectivity. Here, we demonstrate in vitro recapitulation of key sugar-manipulating enzymes from this pathway. TunA is found to exhibit unusual regioselectivity in the reduction of a key α,ß-unsaturated ketone. The product of this reaction is shown to be the preferred substrate for TunF--an epimerase that converts the glucose derivative to a galactose. In Streptomyces strains in which another gene (tunB) is deleted, the biosynthesis is shown to stall at this exo-glycal product. These investigations confirm the combined TunA/F activity and delineate the ordering of events in the metabolic pathway. This is the first time these surprising exo-glycal intermediates have been seen in biology. They suggest that construction of the aminodialdose core of tunicamycin exploits their enol ether motif in a mode of C-C bond formation not previously observed in nature, to create an 11-carbon chain.

Lipid MALDI profile classifies non-small cell lung cancers according to the histologic type.

We investigated whether direct tissue matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis on lipid may assist with the histopathologic diagnosis of non-small cell lung cancers (NSCLCs). Twenty-one pairs of frozen, resected NSCLCs and adjacent normal tissue samples were initially analyzed using histology-directed, MALDI MS. 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument. A lipid profile that could differentiate cancer and adjacent normal samples with a median accuracy of 92.9% was discovered. Several phospholipids including phosphatidylcholines (PC) {34:1} were overexpressed in lung cancer. Squamous cell carcinomas and adenocarcinomas were found to have different lipid profiles. Discriminatory lipids correctly classified the histology of 80.4% of independent NSCLC surgical tissue samples (41 out of 51) in validation set. MALDI MS image of 11 discriminatory lipids validated their differential expression according to the histologic type in cancer cells of bronchoscopic biopsy samples. PC {32:0} [M+Na](+) (m/z 756.68) and ST-OH {42:1} [M-H](-) (m/z 906.89) were overexpressed in adenocarcinomas. Thus, lipid profiles accurately distinguish tumor from adjacent normal tissue and classify non-small cell lung cancers according to the histologic type.

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.

Crystal structure of prephenate dehydrogenase from Streptococcus mutans.

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD(+) shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between ß6 and ß7, which may be a reason for the high K(m) values of PDH from Streptococcus mutans.

Discovery of the serum biomarker proteins in severe preeclampsia by proteomic analysis.

Preeclapsia (PE) is a severe disorder that occurs during pregnancy, leading to maternal and fetal morbidity and mortality. PE affects about 3-8% of all pregnancies. In this study, we conducted liquid chromatography- mass spectrometry/mass spectrometry (LC-MS/MS) to analyze serum samples depleted of the six most abundant proteins from normal and PE-affected pregnancies to profile serum proteins. A total of 237 proteins were confidently identified with <1% false discovery rate from the two groups of duplicate analysis. The expression levels of those identified proteins were compared semiquantitatively by spectral counting. To further validate the candidate proteins with a quantitative mass spectrometric method, selective reaction monitoring (SRM) and enzyme linked immune assay (ELISA) of serum samples collected from pregnant women with severe PE (n = 8) or normal pregnant women (n = 5) was conducted. α2- HS-glycoprotein (AHSG), retinol binding protein 4 (RBP4) and α-1-microglobulin/bikunin (AMBP) and Insulin like growth factor binding protein, acid labile subunit (IGFBP-ALS) were confirmed to be differentially expressed in PE using SRM (P<0.05). Among these proteins, AHSG was verified by ELISA and showed a statistically significant increase in PE samples when compared to controls.