The high-resolution crystal structure of human LCAT.

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/ß hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.

Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH).

Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.

Genetic transformation of the codling moth, Cydia pomonella L., with piggyBac EGFP.

Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct resulted in identification of insect genomic sequences, not plasmid sequences, thus providing evidence that the piggyBac EGFP cassette had integrated into the codling moth genome. EGFP-positive moths were confirmed in the 28th and earlier generations post injection through PCR and Southern blot analyses, indicating heritability of the transgene.

Thermodynamic analysis of mRNA cap binding by the human initiation factor eIF4E via free energy perturbations.

Eukaryotic mRNAs are appended at the 5' end, with the 7-methylguanosine cap linked by a 5'-5'-triphosphate bridge to the first transcribed nucleoside (m7GpppX). Initiation of cap-dependent translation of mRNA requires direct interaction between the cap structure and the eukaryotic translation initiation factor eIF4E. Biophysical studies of the association between eIF4E and various cap analogs have demonstrated that m(7)GTP binds to the protein ca. -5.0 kcal/mol more favorably than unmethylated GTP. In this work, a thermodynamic analysis of the binding process between eIF4E and several cap analogs has been conducted using Monte Carlo (MC) simulations in conjunction with free energy perturbation (FEP) calculations. To address the role of the 7-methyl group in the eIF4E/m7GpppX cap interaction, binding free energies have been computed for m(7)GTP, GTP, protonated GTP at N(7), the 7-methyldeazaguanosine 5'-triphosphate (m(7)DTP), and 7-deazaguanosine 5'-triphosphate (DTP) cap analogs. The MC/FEP simulations for the GTP-->m(7)DTP transformation demonstrate that half of the binding free energy gain of m(7)GTP with respect to GTP can be attributed to favorable van der Waals interactions with Trp166 and reduced desolvation penalty due to the N(7) methyl group. The methyl group both eliminates the desolvation penalty of the N(7) atom upon binding and creates a larger cavity within the solvent that further facilitates the desolvation step. Analysis of the pair m(7)GTP-m(7)DTP suggests that the remaining gain in affinity is related to the positive charge created on the guanine moiety due to the N(7) methylation. The charge provides favorable cation-pi interactions with Trp56 and Trp102 and decreases the negative molecular charge, which helps the transfer from the solvent, a more polar environment, to the protein.

A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.

The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol.

Proprotein convertase subtilisin kexin type 9 (PCSK9) has been shown to be involved in the regulation of extracellular levels of the low-density lipoprotien receptor (LDLR). Although PCSK9 is a subtilase, it has not been shown to degrade the LDLR, and its LDLR-lowering mechanism remains uncertain. Here we report the crystal structure of human PCSK9 at 2.3 A resolution. PCSK9 has subtilisin-like pro- and catalytic domains, and the stable interaction between these domains prevents access to PCSK9's catalytic site. The C-terminal domain of PCSK9 has a novel protein fold and may mediate protein-protein interactions. The structure of PCSK9 provides insight into its biochemical characteristics and biological function.

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.