The molecular basis underpinning the potency and specificity of MAIT cell antigens.

Mucosal-associated invariant T (MAIT) cells are activated by microbial riboflavin-based metabolite antigens when presented by MR1. How modifications to the potent antigen 5-OP-RU affect presentation by MR1 and MAIT cell activation remains unclear. Here we design 20 derivatives, termed altered metabolite ligands (AMLs), to dissect the impact of different antigen components on the human MAIT-MR1 axis. Analysis of 11 crystal structures of MAIT T cell antigen receptor (TCR)-MR1-AML ternary complexes, along with biochemical and functional assays, shows that MR1 cell-surface upregulation is influenced by ribityl and non-ribityl components of the ligand and the hydrophobicity of the MR1-AML interface. The polar ribityl chain of the AML strongly influences MAIT cell activation potency through dynamic compensatory interactions within a MAIT TCR-MR1-AML interaction triad. We define the basis by which the MAIT TCR can differentially recognize AMLs, thereby providing insight into MAIT cell antigen specificity and potency.

Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells.

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals.

Self-assembled copper-based nanoparticles for enzyme catalysis-enhanced chemodynamic/photodynamic/antiangiogenic tritherapy against hepatocellular carcinoma.

As an emerging cancer treatment strategy, reactive oxygen species-based tumor catalytic therapies face enormous challenges due to hypoxia and the overexpression of glutathione (GSH) in the tumor microenvironment. Herein, a self-assembled copper-based nanoplatform, TCCHA, was designed for enzyme-like catalysis-enhanced chemodynamic/photodynamic/antiangiogenic tritherapy against hepatocellular carcinoma. TCCHA was fabricated from Cu2+, 3,3'-dithiobis (propionohydrazide), and photosensitizer chlorine e6 via a facile one-pot self-assembly strategy, after which an aldehyde hyaluronic acid was coated, followed by loading of the antivascular drug AL3818. The obtained TCCHA nanoparticles exhibited pH/GSH dual-responsive drug release behaviors and multienzymatic activities, including Fenton, glutathione peroxidase-, and catalase-like activities. TCCHA, a redox homeostasis disruptor, promotes ⋅OH generation and GSH depletion, thus increasing the efficacy of chemodynamic therapy. TCCHA, which has catalase-like activity, can also reinforce the efficacy of photodynamic therapy by amplifying O2 production. In vivo, TCCHA efficiently inhibited tumor angiogenesis and suppressed tumor growth without apparent systemic toxicity. Overall, this study presents a facile strategy for the preparation of multienzyme-like nanoparticles, and TCCHA nanoparticles display great potential for enzyme catalysis-enhanced chemodynamic/photodynamic/antiangiogenic triple therapy against cancer.

Structural model of human PORCN illuminates disease-associated variants and drug-binding sites.

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins, we developed and validated a structural model of human PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis, as well as the inhibition of its enzymatic activity, and can facilitate the development of improved inhibitors and the understanding of disease-relevant PORCN mutants. This article has an associated First Person interview with the joint first authors of the paper.

Identification and characterization of inhibitors covalently modifying catalytic cysteine of UBE2T and blocking ubiquitin transfer.

UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified. Solution NMR study demonstrated the direct interactions between UBE2T and compounds in solution. Further co-crystal structures reveal the binding modes of these compounds. Both compound hydrolysation and formation of a hydrogen bond with the thiol group of the catalytic cysteine were observed. The formation of covalent complex was confirmed with mass spectrometry. As these two compounds inhibit ubiquitin transfer, our study provides a strategy to develop potent inhibitors of UBE2T.

Analyzing master regulators and scRNA-seq of COVID-19 patients reveals an underlying anti-SARS-CoV-2 mechanism of ZNF proteins.

Studies have demonstrated that both mortality and severe illness rates exist significant difference in different gender COVID-19 patients, but the reasons are still very mysterious to date. Here, we firstly find that the survival outcome of female patients is better to male patients through analyzing the 3044 COVID-19 cases. Secondly, we identify many important master regulators [e.g. STAT1/STAT2 and zinc finger (ZNF) proteins], in particular female patients can express more ZNF proteins and stronger transcriptional activities than male patients in response to SARS-CoV-2 infection. Thirdly, we discover that ZNF protein activity is significantly negative correlation with the SARS-CoV-2 load of COVID-19 patients, and ZNF proteins as transcription factors can also activate their target genes to participate in anti-SARS-CoV-2 infection. Fourthly, we demonstrate that ZNF protein activity is positive correlation with the abundance of multiple immune cells of COVID-19 patients, implying that the highly ZNF protein activity might promote the abundance and the antiviral activity of multiple immune cells to effectively suppress SARS-CoV-2 infection. Taken together, our study proposes an underlying anti-SARS-COV-2 role of ZNF proteins, and differences in the amount and activity of ZNF proteins might be responsible for the distinct prognosis of different gender COVID-19 patients.

Enhanced H2O2 formation and norfloxacin removal by electro-Fenton process using a surface-reconstructed graphite felt cathode: New insight into synergistic mechanism of defective active sites.

The efficient catalytic activity and strong durability possibility of carbon-based three-dimensional fiber materials remains an important challenge in Electro-Fenton advanced oxidation technology. Graphite felt (GF) is a promising electrode material for 2-electron oxygen reduction reaction but with higher catalytic inertia. Anodizing modification of GF has been proved to enhance it electro-catalytic property, but the disadvantages of excessive or insufficient oxidation of GF need further improved. Herein, the surface reconstituted graphite felt by anodizing and HNO3 ultrasonic integrated treatment was used as cathode to degrade norfloxacin (NOR) and the substantial role of different modification processes was essentially investigated. Compared with the single modification process, the synergistic interaction between these two methods can generate more defective active sites (DASs) on GF surface and greatly improved 2-electron ORR activity. The H2O2 can be further co-activated by Fe2+ and DASs into •OH(ads and free) and •O2- to efficiently degrade NOR. The treated GF with 20 min anodizing and 1 h HNO3 ultrasound had the highest electrocatalytic activity in a wide electric potential (-0.4 V to -0.8 V) and pH range (3-9) in system and the efficient removal rate of NOR was basically maintained after 5 cycles. Under optimal reaction conditions, 50 mg L-1 NOR achieved 93% degradation and almost 63% of NOR was completely mineralized within 120 min. The possible NOR degradation pathways and ecotoxicity of intermediates were analyzed by LC-MS and T.E.S.T. theoretical calculation. This paper provided the underlying insights into designing a high-efficiency carbon-based cathode materials for commercial antibiotic wastewater treatment.

Design, Synthesis and Biological Evaluation of N-phenylindole Derivatives as Pks13 Inhibitors againstMycobacterium tuberculosis.

Polyketide synthase 13 (Pks13), an essential enzyme for the survival of Mycobacterium tuberculosis (Mtb), is an attractive target for new anti-TB agents. In our previous work, we have identified 2-phenylindole derivatives against Mtb. The crystallography studies demonstrated that the two-position phenol was solvent-exposed in the Pks13-TE crystal structure and a crucial hydrogen bond was lost while introducing bulkier hydrophobic groups at indole N moieties. Thirty-six N-phenylindole derivatives were synthesized and evaluated for antitubercular activity using a structure-guided approach. The structure-activity relationship (SAR) studies resulted in the discovery of the potent Compounds 45 and 58 against Mtb H37Rv, with an MIC value of 0.0625 µg/mL and 0.125 µg/mL, respectively. The thermal stability analysis showed that they bind with high affinity to the Pks13-TE domain. Preliminary ADME evaluation showed that Compound 58 displayed modest human microsomal stability. This report further validates that targeting Pks13 is a valid strategy for the inhibition of Mtb and provides a novel scaffold for developing leading anti-TB compounds.

Prochlorococcus phage ferredoxin: structural characterization and electron transfer to cyanobacterial sulfite reductases.

Marine cyanobacteria are infected by phages whose genomes encode ferredoxin (Fd) electron carriers. These Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, but it is unclear how the biophysical properties and partner specificities of phage Fds relate to those of photosynthetic organisms. Here, results of a bioinformatics analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects the cyanobacterium Prochlorococcus marinus, revealed high levels of similarity to cyanobacterial Fds (root mean square deviations of ≤0.5 Å). Additionally, pssm2-Fd exhibited a low midpoint reduction potential (-336 mV versus a standard hydrogen electrode), similar to other photosynthetic Fds, although it had lower thermostability (Tm = 28 °C) than did many other Fds. When expressed in an Escherichia coli strain deficient in sulfite assimilation, pssm2-Fd complemented bacterial growth when coexpressed with a P. marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high levels of structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterially encoded oxidoreductases.

Covalent Capture of Collagen Triple Helices Using Lysine-Aspartate and Lysine-Glutamate Pairs.

Collagen mimetic peptides (CMPs) self-assemble into a triple helix reproducing the most fundamental aspect of the collagen structural hierarchy. They are therefore important for both further understanding this complex family of proteins and use in a wide range of biomaterials and biomedical applications. CMP self-assembly is complicated by a number of factors which limit the use of CMPs including their slow rate of folding, relatively poor monomer-trimer equilibrium, and the large number of competing species possible in heterotrimeric helices. All of these problems can be solved through the formation of isopeptide bonds between lysine and either aspartate or glutamate. These amino acids serve two purposes: they first direct self-assemble, allowing for composition and register control within the triple helix, and subsequently can be covalently linked, fixing the composition and register of the assembled structure without perturbing the triple helical conformation. This self-assembly and covalent capture are demonstrated here with four different triple helices. The formation of an isopeptide bond between lysine and glutamate (K-E) is shown to be a faster and higher yielding reaction than lysine with aspartate (K-D). Additionally, K-E amide bonds increase the thermal stability, improve the refolding capabilities, and enhance the triple helical structure as compared to K-E supramolecular interactions, observed by circular dichroism. In contrast, covalent capture of triple helices with K-D amide bonds occurs slower, and the captured triple helices do not have enhanced helical structure. The crystal structure of a triple helix captured through the formation of three K-E isopeptide bonds unequivocally demonstrates the connectivity of the amide bonds formed while also confirming the preservation of the canonical triple helix. The rate of reaction and yield for covalently captured K-E triple helices along with the excellent preservation of triple helical structure demonstrate that this approach can be used to effectively capture and stabilize this important biological motif for biological and biomedical applications.

Computer Modelling and Synthesis of Deoxy and Monohydroxy Analogues of a Ribitylaminouracil Bacterial Metabolite that Potently Activates Human T Cells.

5-(2-Oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) is a natural product formed during bacterial synthesis of vitamin B2. It potently activates mucosal associated invariant T (MAIT) cells and has immunomodulatory, inflammatory, and anticancer properties. This highly polar and unstable compound forms a remarkably stable Schiff base with a lysine residue in major histocompatibility complex class I-related protein (MR1) expressed in antigen-presenting cells. Inspired by the importance of the ribityl moiety of 5-OP-RU for binding to both MR1 and the T cell receptor (TCR) on MAIT cells, each OH was removed in silico. DFT calculations and MD simulations revealed a very stable hydrogen bond between the C3'-OH and uracil N1H, which profoundly restricts flexibility and positioning of each ribityl-OH, potentially impacting their interactions with MR1 and TCR. By using deoxygenation strategies and kinetically controlled imine formation, four monodeoxyribityl and four monohydroxyalkyl analogues of 5-OP-RU were synthesised as new tools for probing T cell activation mechanisms.

Orally Absorbed Cyclic Peptides.

Peptides and proteins are not orally bioavailable in mammals, although a few peptides are intestinally absorbed in small amounts. Polypeptides are generally too large and polar to passively diffuse through lipid membranes, while most known active transport mechanisms facilitate cell uptake of only very small peptides. Systematic evaluations of peptides with molecular weights above 500 Da are needed to identify parameters that influence oral bioavailability. Here we describe 125 cyclic peptides containing four to thirty-seven amino acids that are orally absorbed by mammals. Cyclization minimizes degradation in the gut, blood, and tissues by removing cleavable N- and C-termini and by shielding components from metabolic enzymes. Cyclization also folds peptides into bioactive conformations that determine exposure of polar atoms to solvation by water and lipids and therefore can influence oral bioavailability. Key chemical properties thought to influence oral absorption and bioavailability are analyzed, including molecular weight, octanol-water partitioning, hydrogen bond donors/acceptors, rotatable bonds, and polar surface area. The cyclic peptides violated to different degrees all of the limits traditionally considered to be important for oral bioavailability of drug-like small molecules, although fewer hydrogen bond donors and reduced flexibility generally favored oral absorption.

A Potent Antagonist of Protease-Activated Receptor 2 That Inhibits Multiple Signaling Functions in Human Cancer Cells.

Protease-activated receptor 2 (PAR2) is a cell surface protein linked to G-protein dependent and independent intracellular signaling pathways that produce a wide range of physiological responses, including those related to metabolism, inflammation, pain, and cancer. Certain proteases, peptides, and nonpeptides are known to potently activate PAR2. However, no effective potent PAR2 antagonists have been reported yet despite their anticipated therapeutic potential. This study investigates antagonism of key PAR2-dependent signaling properties and functions by the imidazopyridazine compound I-191 (4-(8-(tert-butyl)-6-(4-fluorophenyl)imidazo[1,2-b]pyridazine-2-carbonyl)-3,3-dimethylpiperazin-2-one) in cancer cells. At nanomolar concentrations, I-191 inhibited PAR2 binding of and activation by structurally distinct PAR2 agonists (trypsin, peptide, nonpeptide) in a concentration-dependent manner in cells of the human colon adenocarcinoma grade II cell line (HT29). I-191 potently attenuated multiple PAR2-mediated intracellular signaling pathways leading to Ca2+ release, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, Ras homologue gene family, member A (RhoA) activation, and inhibition of forskolin-induced cAMP accumulation. The mechanism of action of I-191 was investigated using binding and calcium mobilization studies in HT29 cells where I-191 was shown to be noncompetitive and a negative allosteric modulator of the agonist 2f-LIGRL-NH2 The compound alone did not activate these PAR2-mediated pathways, even at high micromolar concentrations, indicating no bias in these signaling properties. I-191 also potently inhibited PAR2-mediated downstream functional responses, including expression and secretion of inflammatory cytokines and cell apoptosis and migration, in human colon adenocarcinoma grade II cell line (HT29) and human breast adenocarcinoma cells (MDA-MB-231). These findings indicate that I-191 is a potent PAR2 antagonist that inhibits multiple PAR2-induced signaling pathways and functional responses. I-191 may be a valuable tool for characterizing PAR2 functions in cancer and in other cellular, physiological, and disease settings.

Growth of MCF-7 breast cancer cells and efficacy of anti-angiogenic agents in a hydroxyethyl chitosan/glycidyl methacrylate hydrogel.

BACKGROUND: Breast cancer negatively affects women's health worldwide. The tumour microenvironment plays a critical role in tumour initiation, proliferation, and metastasis. Cancer cells are traditionally grown in two-dimensional (2D) cultures as monolayers on a flat solid surface lacking cell-cell and cell-matrix interactions. These experimental conditions deviate from the clinical situation. Improved experimental systems that can mimic the in vivo situation are required to discover new therapies, particularly for anti-angiogenic agents that mainly target intercellular factors and play an essential role in treating some cancers. METHODS: Chitosan can be modified to construct three-dimensional (3D) tumour models. Here, we report an in vitro 3D tumour model using a hydroxyethyl chitosan/glycidyl methacrylate (HECS-GMA) hydrogel produced by a series of chitosan modifications. Parameters relating to cell morphology, viability, proliferation, and migration were analysed using breast cancer MCF-7 cells. In a xenograft model, secretion of angiogenesis-related growth factors and the anti-angiogenic efficacy of Endostar and Bevacizumab in cells grown in HECS-GMA hydrogels were assessed by immunohistochemistry. RESULTS: Hydroxyethyl chitosan/glycidyl methacrylate hydrogels had a highly porous microstructure, mechanical properties, swelling ratio, and morphology consistent with a 3D tumour model. Compared with a 2D monolayer culture, breast cancer MCF-7 cells residing in the HECS-GMA hydrogels grew as tumour-like clusters in a 3D formation. In a xenograft model, MCF-7 cells cultured in the HECS-GMA hydrogels had increased secretion of angiogenesis-related growth factors. Recombinant human endostatin (Endostar), but not Bevacizumab (Avastin), was an effective anti-angiogenic agent in HECS-GMA hydrogels. CONCLUSIONS: The HECS-GMA hydrogel provided a 3D tumour model that mimicked the in vivo cancer microenvironment and supported the growth of MCF7 cells better than traditional tissue culture plates. The HECS-GMA hydrogel may offer an improved platform to minimize the gap between traditional tissue culture plates and clinical applicability. In addition, the anti-angiogenic efficacy of drugs such as Endostar and Bevacizumab can be more comprehensively studied and assessed in HECS-GMA hydrogels.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus.

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-ß-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-ß-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 Å resolution, respectively. SgcE6 shares a similar ß-barrel fold with the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few other known FADH2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. The crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.

Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted.

Repurposing Registered Drugs as Antagonists for Protease-Activated Receptor 2.

Virtual screening of a drug database identified Carvedilol, Loratadine, Nefazodone and Astemizole as PAR2 antagonists, after ligand docking and molecular dynamics simulations using a PAR2 homology model and a putative binding mode of a known PAR2 ligand. The drugs demonstrated competitive binding and antagonism of calcium mobilization and ERK1/2 phosphorylation in CHO-hPAR2 transfected cells, while inhibiting IL-6 secretion in PAR2 expressing MDA-MB-231 breast cancer cells. This research highlights opportunities for GPCR hit-finding from FDA-approved drugs.

Three Homology Models of PAR2 Derived from Different Templates: Application to Antagonist Discovery.

Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor (GPCR) involved in inflammation and metabolism. It is activated through cleavage of its N-terminus by proteases. The new N-terminus functions as a tethered ligand that folds back and intramolecularly activates PAR2, initiating multiple downstream signaling pathways. The only compounds reported to date to inhibit PAR2 activation are of moderate potency. Three structural models for PAR2 have been constructed based on sequence homology with known crystal structures for bovine rhodopsin, human ORL-1 (also called nociceptin/orphanin FQ receptor), and human PAR1. The three PAR2 model structures were compared and used to predict potential interactions with ligands. Virtual screening for ligands using the Chembridge database, and either ORL-1 or PAR1 derived PAR2 models led to identification of eight new small molecule PAR2 antagonists (IC50 10-100 µM). Notably, the most potent compound 1 (IC50 11 µM) was derived from the less homologous template protein, human ORL-1. The results suggest that virtual screening against multiple homology models of the same GPCR can produce structurally diverse antagonists and that this may be desirable even when some models have less sequence homology with the target protein.

A novel NKX2.6 mutation associated with congenital ventricular septal defect.

Congenital heart disease (CHD) is the most common birth defect and is the most prevalent non-infectious cause of infant death. Aggregating evidence demonstrates that genetic defects are involved in the pathogenesis of CHD. However, CHD is genetically heterogeneous and the genetic determinants for CHD in an overwhelming majority of patients remain unknown. In this study, the coding regions and splice junctions of the NKX2.6 gene, which encodes a homeodomain transcription factor crucial for cardiovascular development, were sequenced in 210 unrelated CHD patients. As a result, a novel heterozygous NKX2.6 mutation, p.K152Q, was identified in an index patient with ventricular septal defect (VSD). Genetic analysis of the proband's available family members showed that the mutation cosegregated with VSD transmitted as an autosomal dominant trait with complete penetrance. The missense mutation was absent in 400 control chromosomes and the altered amino acid was completely conserved evolutionarily across species. Due to unknown transcriptional targets of NKX2.6, the functional characteristics of the identified mutation at transcriptional activity were analyzed by using NKX2.5 as a surrogate. Alignment between human NKX2.6 and NKX2.5 proteins displayed that K152Q-mutant NKX2.6 was equivalent to K158Q-mutant NKX2.5, and introduction of K158Q into NKX2.5 significantly reduced its transcriptional activating function when compared with its wild-type counterpart. This study firstly links NKX2.6 loss-of-function mutation with increased susceptibility to isolated VSD, providing novel insight into the molecular mechanism underpinning VSD and contributing to the development of new preventive and therapeutic strategies for this common form of CHD.

Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8.

Degradation of nuclear proteins by the 26S proteasome is essential for cell viability. In yeast, the nuclear envelope protein Cut8 mediates nuclear proteasomal sequestration by an uncharacterized mechanism. Here we describe structures of Schizosaccharomyces pombe Cut8, which shows that it contains a unique, modular fold composed of an extended N-terminal, lysine-rich segment that when ubiquitinated binds the proteasome, a dimer domain followed by a six-helix bundle connected to a flexible C tail. The Cut8 six-helix bundle shows structural similarity to 14-3-3 phosphoprotein-binding domains, and binding assays show that this domain is necessary and sufficient for liposome and cholesterol binding. Moreover, specific mutations in the 14-3-3 regions corresponding to putative cholesterol recognition/interaction amino acid consensus motifs abrogate cholesterol binding. In vivo studies confirmed that the 14-3-3 region is necessary for Cut8 membrane localization and that dimerization is critical for its function. Thus, the data reveal the Cut8 organization at the nuclear envelope. Reconstruction of Cut8 evolution suggests that it was present in the last common ancestor of extant eukaryotes and accordingly that nuclear proteasomal sequestration is an ancestral eukaryotic feature. The importance of Cut8 for cell viability and its absence in humans suggests it as a possible target for the development of specific chemotherapeutics against invasive fungal infections.