Joel D. A. Tyndall

Cu(II) Potentiation of Alzheimer Aβ Neurotoxicity CORRELATION WITH CELL-FREE HYDROGEN PEROXIDE PRODUCTION AND METAL REDUCTION

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Aβ amyloid deposits in Alzheimers disease. The neurotoxicity of Aβ in cell culture has been linked to H2O2generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Aβ in cell culture. The potentiation of toxicity is greatest for Aβ1–42 > Aβ1–40 ≫ mouse/rat Aβ1–40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H2O2 in cell-free assays and to exhibit amyloid pathology. The copper complex of Aβ1–42 has a highly positive formal reduction potential (≈+500–550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Aβ-mediated oxidative damage in Alzheimers disease.

The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.

Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase/prokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A(2), sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., vampire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Significance The absence in the Protein Data Bank of full-length structures of bitopic membrane proteins with one transmembrane helix, probably because of difficulties with ordered crystallization, has limited understanding of how single-transmembrane helices orient enzymes and sensors at the bilayer surface. X-ray crystal structures of full-length yeast lanosterol 14α-demethylase, a cytochrome P450, show how a helix spanning a single transmembrane may lead to constraints on the orientation of the putative substrate entry portal from within the bilayer. The crystal structures also locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding the drug resistance associated with orthologs of the enzyme found in fungal pathogens. Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.

Update 1 of: Proteases Universally Recognize Beta Strands In Their Active Sites

3. Protease-Bound Substrate and Product Structures PR5 3.1. Protease-Bound Substrate Structures PR5 3.2. Protease-Bound Product Structures PR7 3.3. Cyclic Substrates and Products PR8 4. Aspartic Protease Inhibitors PR9 4.1. Viral Aspartic Proteases (Retropepsins) PR9 4.2. Human and Mammalian Aspartic Proteases PR11 4.3. Parasitic, Plant, and Fungal Aspartic ProteasesPR12 5. Metalloprotease Inhibitors PR12 5.1. Bacterial Metalloproteases PR12 5.2. Human and Mammalian Metalloproteases PR12 5.3. Other Metalloproteases PR15 6. Serine Protease Inhibitors PR15 6.1. Viral Serine Proteases PR16 6.2. Bacterial Serine Proteases PR17 6.3. Human and Mammalian Serine Proteases PR19 6.4. Other Serine Proteases PR22 7. Cysteine Protease Inhibitors PR22 7.1. Viral, Bacterial, and Parasitic Cysteine Proteases PR22

Crystal Structure of a Thermostable Lipase from Bacillus stearothermophilus P1

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.

Update 1 of: Beta-Strand Mimetics

Small molecules that mimic β-strands can be very useful enzyme inhibitors and antagonists with important potential applications in medicine. It is only in the last 10 years or so that small molecule β-strand mimetics have been reported. In addition to such specific recognition of discrete β-strands, combinations of two or more strands to form β-sheets not only act as important scaffolding elements to stabilize protein structure but are sometimes key recognition motifs that bind to other proteins or DNA.

GPCR agonists and antagonists in the clinic.

This review describes current and new therapeutic agonists and antagonists of G-protein-coupled receptors (GPCRs) currently used in the clinic. GPCRs are classified under the GRAFS system (Glutamate, Rhodopsin, Adhesion, Frizzled/taste2 and Secretin), with therapies having been developed for about 30 GPCRs from the glutamate, rhodopsin and secretin families. Most of these therapies target the biogenic amine receptors of the rhodopsin family. Advancing technology has assisted in the identification of an increasing number of GPCRs, as well as contributing to the understanding of function and potential as pharmaceutical targets. With this has come the development of new therapies that target specific GPCRs, including peptide activated GPCRs. Where possible, agonists and antagonists are described individually, focusing on new therapies and their corresponding target receptors. However, the large number of reported biogenic amine therapies precludes, discussion of individual compounds and instead, they are discussed in relation to the receptor pharmacophore. Despite the large number of significant physiological responses known to be mediated by GPCRs, only about 4% of known GPCRs are currently targeted by therapeutics. This provides a great number of promising new targets for pharmaceutical development.

Macrocycles mimic the extended peptide conformation recognized by aspartic, serine, cysteine and metallo proteases.

It has been previously demonstrated that aspartic, serine, metallo and cysteine proteases bind to their inhibitors and substrate analogues in a single conformation, the saw-tooth or extended beta-strand. Consequently a generic approach to the development of protease inhibitors is the use of constraints that conformationally restrict putative inhibitor molecules to an extended form. In this way the inhibitor is pre-organized for binding to a protease and does not need to rearrange its structure. One constraining device that has proven to be effective for such pre-organization is macrocyclization. This article illustrates the general principle that macrocycles, especially those composed of 3-4 amino acids and usually 13-17 ring atoms, can effectively mimic the extended conformation of short peptide sequences. Such structure-stabilising macrocycles are stable to degradation by proteases, valuable components of potent protease inhibitors, and in many cases they are also bioavailable.

D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A2 (IIa) with antiinflammatory activity.

Few reported inhibitors of secretory phospholipase A2 enzymes truly inhibit the IIa human isoform (hnpsPLA2‐IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D‐tyrosine was derivatised to give a series of potent new inhibitors of hnpsPLA2‐IIa. A 2.2‐Å crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca2+ ion through carboxylate and amide oxygen atoms, H‐bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T‐shaped aromatic‐group–His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.

Honey Bee Dopamine and Octopamine Receptors Linked to Intracellular Calcium Signaling Have a Close Phylogenetic and Pharmacological Relationship

Background Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. Methodology/Principal Findings Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z)-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. Conclusions/Significance Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor function. Taken together, these results strongly support phylogenetic analyses indicating that the AmDOP2 and AmOA1 receptor genes are immediate paralogs.