Phenothiazine‐Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin‐Mediated Endocytosis

Chlorpromazine is a phenothiazine‐derived antipsychotic drug (APD) that inhibits clathrin‐mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine‐derived APDs inhibited dynamin I (dynI) in the 2–12 µm range, the most potent being trifluoperazine (IC₅₀ 2.6 ± 0.7 µm ). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8‐ to 10‐fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine‐derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine‐derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin‐active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure–activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP‐2 recruitment in the formation of clathrin‐coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP‐2 interaction with amphiphysin or clathrin. Super‐resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP‐2, nor AP‐2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine‐derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in‐cell target in endocytosis.      

Social behavior in a genetic model of dopamine dysfunction at different neurodevelopmental time points

Impairments in social behavior characterize many neurodevelopmental psychiatric disorders. In fact, the temporal emergence and trajectory of these deficits can define the disorder, specify their treatment and signal their prognosis. The sophistication of mouse models with neurobiological endophenotypes of many aspects of psychiatric diseases has increased in recent years, with the necessity to evaluate social behavior in these models. We adapted an assay for the multimodal characterization of social behavior at different development time points (juvenile, adolescent and adult) in control mice in different social contexts (specifically, different sex pairings). Although social context did not affect social behavior in juvenile mice, it did have an effect on the quantity and type of social interaction as well as ultrasonic vocalizations in both adolescence and adulthood. We compared social development in control mice to a transgenic mouse model of the increase in postsynaptic striatal D2R activity observed in patients with schizophrenia (D2R‐OE mice). Genotypic differences in social interactions emerged in adolescence and appeared to become more pronounced in adulthood. That vocalizations emitted from dyads with a D2R‐OE subject were negatively correlated with active social behavior while vocalizations from control dyads were positively correlated with both active and passive social behavior also suggest social deficits. These data show that striatal dopamine dysfunction plays an important role in the development of social behavior and mouse models such as the one studied here provide an opportunity for screening potential therapeutics at different developmental time points.     

FunRich: An open access standalone functional enrichment and interaction network analysis tool

As high‐throughput techniques including proteomics become more accessible to individual laboratories, there is an urgent need for a user‐friendly bioinformatics analysis system. Here, we describe FunRich, an open access, standalone functional enrichment and network analysis tool. FunRich is designed to be used by biologists with minimal or no support from computational and database experts. Using FunRich, users can perform functional enrichment analysis on background databases that are integrated from heterogeneous genomic and proteomic resources (>1.5 million annotations). Besides default human specific FunRich database, users can download data from the UniProt database, which currently supports 20 different taxonomies against which enrichment analysis can be performed. Moreover, the users can build their own custom databases and perform the enrichment analysis irrespective of organism. In addition to proteomics datasets, the custom database allows for the tool to be used for genomics, lipidomics and metabolomics datasets. Thus, FunRich allows for complete database customization and thereby permits for the tool to be exploited as a skeleton for enrichment analysis irrespective of the data type or organism used. FunRich ( http://www.funrich.org ) is user‐friendly and provides graphical representation (Venn, pie charts, bar graphs, column, heatmap and doughnuts) of the data with customizable font, scale and color (publication quality).     

Does screw–bone interface modelling matter in finite element analyses?

The effect of screw-bone interface modelling strategies was evaluated in the setting of a tibial mid-shaft fracture stabilised using locking plates. Three interface models were examined: fully bonded interface; screw with sliding contact with bone; and screw with sliding contact with bone in an undersized pilot hole. For the simulation of the last interface condition we used a novel thermal expansion approach to generate the pre-stress that the bone would be exposed to during screw insertion. The study finds that the global load-deformation response is not influenced by the interface modelling approach employed; the deformation varied by less than 1% between different interaction models. However, interface modelling is found to have a considerable impact on the local stress-strain environment within the bone in the vicinity of the screws. Frictional and tied representations did not have significantly different peak strain values (<5% difference); the frictional interface had higher peak compressive strains while the tied interface had higher tensile strains. The undersized pilot hole simulation produced the largest strains. The peak minimum principal strains for the frictional interface were 26% of those for the undersized pilot hole simulation at a load of 770 N. It is concluded that the commonly used tie constraint can be used effectively when the only interest is the global load-deformation behaviour. Different contact interface models, however, alter the mechanical response around screw holes leading to different predictions for screw loosening, bone damage and stress shielding.     

Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: A critical junction between aerobic and anaerobic metabolism

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L₃) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L₃s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L₃ and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.     

Therapeutic potential of Liver Receptor Homolog-1 modulators

Liver Receptor Homolog-1 (LRH-1; NR5A2) belongs to the orphan nuclear receptor superfamily, and plays vital roles in early development, cholesterol homeostasis, steroidogenesis and certain diseases, including cancer. It is expressed in embryonic stem cells, adult liver, intestine, pancreas and ovary. It binds to DNA as a monomer and is regulated by various ligand-dependent and -independent mechanisms. Recent work identified synthetic ligands for LRH-1; such compounds may yield useful therapeutics for a range of pathologic conditions associated with aberrant expression and activity of LRH-1.     

Synthesis and in vitro evaluation of [18F](R)-FEPAQ: a potential PET ligand for VEGFR2

Synthesis and in vitro evaluation of [(18)F](R)-N-(4-bromo-2-fluorophenyl)-7-((1-(2-fluoroethyl)piperidin-3-yl)methoxy)-6-methoxyquinazolin-4-amine ((R)-[(18)F]FEPAQ or [(18)F]1), a potential imaging agent for the VEGFR2, using phosphor image autoradiography are described. Synthesis of 2, the desfluoroethyl precursor for (R)-FEPAQ was achieved from t-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3) in five steps and in 50% yield. [(18)F]1 was synthesized by reaction of sodium salt of compound 2 with [(18)F]fluoroethyl tosylate in DMSO. The yield of [(18)F]1 was 20% (EOS based on [(18)F]F(-)) with >99% radiochemical purity and specific activity of 1-2 Ci/μmol (n=10). The total synthesis time was 75 min. The radiotracer selectively labeled VEGFR2 in slide-mounted sections of human brain and higher binding was found in surgically removed human glioblastoma sections as demonstrated by in vitro phosphor imager studies. These findings suggest [(18)F]1 may be a promising radiotracer for imaging VEGFR2 in brain using PET.     

A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes.sn-Glycerol-3-phosphate 1-O-acyltransferase (GPAT; Enzyme Commission [EC] 2.3.1.15) is the first enzyme in the pathway for the de novo synthesis of membrane and storage lipids. It catalyzes the transfer of an acyl group from acyl-CoA or acyl-ACP to the sn-1 position of sn-glycerol-3-phosphate (G3P). This reaction has been extensively characterized in bacteria, fungi, animals, and plants (Murata and Tasaka, 1997; Zheng and Zou, 2001; Gimeno and Cao, 2008; Zhang and Rock, 2008; Wendel et al., 2009; Chen et al., 2011a). In the Arabidopsis (Arabidopsis thaliana) genome, there are 10 genes annotated as GPATs. One of these is the soluble, plastid-localized GPAT (At1g32200) that utilizes acyl-ACP substrates and exhibits sn-1 acyl transfer regiospecificity (Nishida et al., 1993). A second enzyme is GPAT9 (At5g60620), which is localized to the endoplasmic reticulum (Gidda et al., 2009) and may be an acyl-CoA-dependent sn-1 GPAT that enables nonplastid glycerolipid synthesis. The remaining eight GPATs cluster together in a family (Zheng et al., 2003; Beisson et al., 2007; Gidda et al., 2009) that is not required for membrane or storage lipid biosynthesis; instead, several members of the family clearly affect the composition and quantity of cutin or suberin (Beisson et al., 2012).Cutin and suberin are extracellular lipid barriers deposited by certain types of plant cells. These insoluble polymers, and associated waxes, function to control water, gas, and ion fluxes and serve as physical barriers to protect plants from pathogen invasion (Kolattukudy, 2001; Schreiber, 2010; Ranathunge et al., 2011). From an evolutionary perspective, the appearance of these lipid barriers was likely a requirement for the adaptation of plants to a terrestrial environment (Rensing et al., 2008). ω-oxidized fatty acids and glycerol are usually major constituents of both polymers (Bernards, 2002; Graça and Santos, 2007; Pollard et al., 2008). The detailed structures of cutin and suberin polymers are still largely unknown (Pollard et al., 2008), but direct esterification of fatty acids to glycerol and to each other has been demonstrated in a large number of partial depolymerization studies of cutin and suberin (Graça and Santos, 2007; Graça and Lamosa, 2010). In Arabidopsis, GPAT4 and GPAT8 are required for the accumulation of C16 and C18 ω-hydroxy fatty acid (ω-OHFA) and α,ω-dicarboxylic acid (DCA) cutin monomers in stems and leaves (Li et al., 2007a). GPAT6 is required for the incorporation of the following C16 monomers: 10,16-dihydroxypalmitate (10,16-diOH C16:0-FA), hexadecane-1,16-dioic acid (C16:0-DCA), and 16-hydroxypalmitate (16-OH C16:0-FA), into flower cutin (Li-Beisson et al., 2009). (For a fatty acid, the abbreviation used is Cm:n-FA, where m is the number of carbon atoms and n is the number of double bonds. The position and number of hydroxyl groups precedes this notation. The same nomenclature is used for DCAs.) GPAT5 controls the accumulation of C22:0- and C24:0-FA, ω-OHFA, and DCA monomers in the suberin of roots and seed coats (Beisson et al., 2007). Recently, we demonstrated that GPAT4 and -6 are bifunctional enzymes that possess sn-2 acyltransferase and phosphatase activities (Yang et al., 2010) and that therefore produce sn-2 monoacylglycerols (MAGs) as the major product. GPAT5 also exhibits strong preference for sn-2 acylation but lacks phosphatase activity; thus, sn-2 lysophosphatidic acids (LPAs) are its major product (Yang et al., 2010).These observations all attest to the fact that several members of the GPAT1 to -8 family are enzymatically very distinct from the GPATs required for membrane and storage lipid biosynthesis. Indeed, they represent a new acylglycerol biosynthesis pathway that provides precursors for cutin and suberin biosynthesis. To better understand the early steps in polyester synthesis and the roles contributed by GPAT4 to -8, and to determine whether the clade of GPAT1 to -3 has distinct or similar activity, we have characterized the regiochemistry and acyl substrate specificity of GPATs representing all three clades. We show that the cutin-associated GPAT8 is a bifunctional sn-2 acyltransferase/phosphatase, while GPAT1, an isozyme with uncertain function but important for tapetum and anther development (Zheng et al., 2003; Li et al., 2012), possesses sn-2 acyltransferase activity but not phosphatase activity. An important issue in defining the pathway of cutin/suberin biosynthesis is whether to place the P450 oxidation reactions before or after the G3P acylation reactions. As discussed (Pollard et al., 2008), previous evidence has not allowed definitive determination of the alternative pathways. However, conducting a GPAT substrate specificity study, particularly with a range of ω-oxidized and unmodified acyl-CoAs can help clarify the situation. Here, we show the acyl-CoA specificities of GPAT4, -5, -6, and -8 are concordant with the compositions of their respective cutins and suberins and the resulting changes in corresponding gpat mutants and overexpression lines. Furthermore, the results provide strong evidence that acyl transfer to glycerol by GPAT occurs after ω-oxidation of acyl chains, thus increasing our limited understanding of the biochemical pathway for cutin and suberin polymer assembly.A phylogenetic analysis of Arabidopsis GPATs with vascular and nonvascular land-plant homologs provides an evolutionary view of the expansion and divergence of the gene family into three distinct clades that are associated with morphological and functional evolution and with the loss of phosphatase activity.     

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinase-impaired alleles of RAF1 (～3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1(D486N). Raf1(D486N/+) (here D486N/+) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1(D486N)-expressing cells compared with controls. RAF1(D486N), as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.     

G-protein-coupled receptor dynamics: dimerization and activation models compared with experiment

Our previously derived models of the active state of the β2-adrenergic receptor are compared with recently published X-ray crystallographic structures of activated GPCRs (G-protein-coupled receptors). These molecular dynamics-based models using experimental data derived from biophysical experiments on activation were used to restrain the receptor to an active state that gave high enrichment for agonists in virtual screening. The β2-adrenergic receptor active model and X-ray structures are in good agreement over both the transmembrane region and the orthosteric binding site, although in some regions the active model is more similar to the active rhodopsin X-ray structures. The general features of the microswitches were well reproduced, but with minor differences, partly because of the unexpected X-ray results for the rotamer toggle switch. In addition, most of the interacting residues between the receptor and the G-protein were identified. This analysis of the modelling has also given important additional insight into GPCR dimerization: re-analysis of results on photoaffinity analogues of rhodopsin provided additional evidence that TM4 (transmembrane helix 4) resides at the dimer interface and that ligands such as bivalent ligands may pass between the mobile helices. A comparison, and discussion, is also carried out between the use of implicit and explicit solvent for active-state modelling.