Discovery and SAR of novel mGluR5 non-competitive antagonists not based on an MPEP chemotype

This Letter describes the discovery and SAR of three novel series of mGluR5 non-competitive antagonists/negative allosteric modulators (NAMs) not based on manipulation of an MPEP/MTEP chemotype. This work demonstrates fundamentally new mGluR5 NAM chemotypes with submicromolar potencies, and the first example of a mode of pharmacology ‘switch’ to provide PAMs with a non-MPEP scaffold.     

Camelid nanobodies raised against an integral membrane enzyme,nitric oxide reductase

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N₂O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies™). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.     

Differential protection against oxidative stress and nitric oxide overproduction in cardiovascular and pulmonary systems by propofol during endotoxemia

Background  

Both overproduction of nitric oxide (NO) and oxidative injury of cardiovascular and pulmonary systems contribute to fatal cardiovascular depression during endotoxemia. We investigated in the present study the relative contribution of oxidative stress and NO to cardiovascular depression during different stages of endotoxemia, and delineated their roles in cardiovascular protective effects of a commonly used anesthetic propofol during endotoxemia.     

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon × Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0–34.4 and 28.9–31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14–70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.     

Structure of α6β3δ GABAA receptors and their lack of ethanol sensitivity

Delta (δ) subunit containing GABA_A receptors are expressed extra‐synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with α₆ subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA_A receptor pentamers by subunit concatenation. These receptors (composed of α₆, β₃ and δ subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one and to 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. α₆‐β₃‐α₆/δ receptors showed a substantial response to GABA alone. Three receptors, β₃‐α₆‐δ/α₆‐β₃, α₆‐β₃‐α₆/β₃‐δ and β₃‐δ‐β₃/α₆‐β₃, were only uncovered in the combined presence of the neurosteroid 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one with GABA. All four receptors were activated by 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the δ subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the δ subunit can assume multiple positions in a receptor pentamer composed of α₆, β₃ and δ subunits.     

The MalF P2 Loop of the ATP-Binding Cassette Transporter MalFGK2 from Escherichia coli and Salmonella enterica Serovar Typhimurium Interacts with Maltose Binding Protein (MalE) throughout the Catalytic Cycle

We have investigated the interaction of the uncommonly large periplasmic P2 loop of the MalF subunit of the maltose ATP-binding cassette transporter (MalFGK₂) from Escherichia coli and Salmonella enterica serovar Typhimurium with maltose binding protein (MalE) by site-specific chemical cross-linking in the assembled transport complex. We focused on possible distance changes between two pairs of residues of the P2 loop and MalE during the transport cycle. The distance between MalF(S205C) and MalE(T80C) (∼5 Å) remained unchanged under all conditions tested. Cross-linking did not affect the ATPase activity of the complex. The distance between MalF(T177C) and MalE(T31C) changed from ∼10 Å to ∼5 Å upon binding of ATP (or maltose, with a less pronounced result) and was reset to ∼10 Å after hydrolysis of one ATP. A cross-link (∼25 Å) between MalF(S205C) and MalE(T31C) was observed only when the transporter resided in a transition state-like conformation, as was the case after vanadate trapping or in a binding protein-independent mutant, both of which are characterized by tight binding of unliganded MalE to the transporter. Thus, we propose that the observed cross-link is indicative of catalytic intermediates of the transporter. Together, our results strengthen the notion that the MalF P2 loop plays an important role in intersubunit communication. In particular, this loop is involved in keeping MalE in close contact with the transporter. The data are discussed with respect to a crystal structure and current transport models.ATP-binding cassette (ABC) transporters utilize the free energy of ATP hydrolysis to translocate substrates across biological membranes and can function as import or export systems (17). ABC transporters are generally composed of two hydrophobic, pore-forming transmembrane subunits and transmembrane domains (TMDs) and two hydrophilic nucleotide-binding (or ABC) subunits and nucleotide-binding domains (NBDs) that hydrolyze ATP (9). The crystal structures of isolated NBDs (6, 23, 34, 43) revealed that NBDs can be divided into a RecA-like subdomain comprising both the Walker A and the Walker B motifs, which are involved in nucleotide binding, and a helical subdomain harboring the unique LSGGQ motif (35). Furthermore, in the physiologically relevant NBD dimer, the nucleotide is complexed between the Walker A and B sites of one monomer and the LSGGQ motif of the opposing monomer. Both subdomains are joined by the “Q loop” containing a conserved glutamine residue that binds to the Mg²⁺ ion and attacking water and is likely to be involved in communicating ATP binding to the TMDs (10, 20, 29). ATP-dependent closing of the NBD dimer is thought to provide one possibility of the power stroke of ABC transporters (38).ABC importers that are confined to prokaryotes mediate the uptake of a large variety of solutes, including inorganic ions, amino acids, sugars, vitamins, oligopeptides, and polyamines (5). They require an additional protein, the extracytoplasmic solute binding protein (SBP), in order to capture the substrate and to deliver it to the cognate ABC transporter (37). SBPs typically consist of two lobes that are connected by a linker region. The interface between the two lobes forms the substrate binding site. Upon binding of the ligand, the proteins undergo a conformational change from an open toward a closed state (33) which, by interaction with extracytoplasmic peptide regions of TMDs of the cognate ABC transporter, initiates the transport process (31). The molecular events by which binding of ATP to the NBDs and interaction of liganded binding proteins with the TMDs are communicated to eventually trigger substrate translocation are still poorly understood.The maltose ABC transporter of Escherichia coli and Salmonella enterica serovar Typhimurium is one of the best-characterized transporters and thus serves as a model system for studying the mechanism by which ABC transporters exert their functions in general (15). The transporter is composed of the extracytoplasmic (periplasmic) maltose binding protein (MalE), the membrane-spanning subunits MalF and MalG, and two copies of the ATP-hydrolyzing subunit (MalK) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.(A) Structure of the catalytic intermediate of the maltose transporter [MalFGK(E159Q)₂-E]. The complex is shown in a ribbon diagram. White horizontal bars mark the boundaries of the membrane. Color code: yellow, MalE; cyan, MalF; red, MalG; green and magenta, MalK dimer. (B) Close-up view of the contact site between MalF P2 and the N-terminal lobe of MalE. The color code is the same as that for panel A. Residues from regions I and II that were replaced by cysteines are indicated in pink (MalF) and green (MalE). Residue MalE-K179, which was used as a control, is shown in green. The figure was drawn with DS ViewerPro 6.0 (Accelrys, Cambridge, United Kingdom), using the coordinates from entry 2R6G in the Brookhaven Protein Data Bank.Recently, suppressor mutational analysis provided a first hint that substrate availability is communicated from MalE to the MalK dimer via periplasmic loop regions of MalFG (11). Moreover, by site-directed cross-linking based on previous genetic evidence (19, 40), we demonstrated a close proximity of MalE G13 to Pro-78 in the first periplasmic loop (P1) of MalG, independently of cofactors such as maltose or ATP. Interaction of both residues was also observed in intact cells (11). These findings led us to propose that a copy of MalE is permanently associated with the transporter throughout the catalytic cycle. Furthermore, we have found that a region of the large, periplasmic P2 loop of MalF around Ser-205 (Fig. ​(Fig.1)1) is in cross-linking distance from MalE in the presence of maltose and MgATP only or when the transporter resides in the vanadate-trapped transition state. These results were perfectly confirmed by the subsequently published crystal structure of the MalFGK(E159Q)₂-E complex, which represents a transport intermediate (32). Here, the MalK dimer is complexed with two ATP molecules, and MalE is tightly associated with MalFG, but maltose has already been released into a binding pocket formed by MalF only. In particular, the N-terminal lobe of MalE is in close contact with the P2 loop of MalF (Fig. ​(Fig.1A1A).In this communication, we have taken advantage of this structural information to gain further insight into the MalF P2-MalE interaction during the transport cycle. We demonstrate ATP- and maltose-dependent distance changes between selected pairs of residues of the loop and MalE in the assembled complex by site-specific cross-linking. Our data demonstrate for the first time that the MalF P2 loop is in close contact to MalE throughout the catalytic cycle.     

Pisonia grandis monocultures limit the spread of an invasive ant—a case of carbohydrate quality?

The mechanisms by which invasive species are able to spread into and dominate natural communities are poorly understood and remain a focus of invasion research. In this quest, studying invasions that are limited by a controlling factor will be more informative than will studies documenting unabated spread and impacts. Some ant species are very successful invaders, and research demonstrating abiotic and biotic factors limiting their success has aided the understanding of invasion ecology. We report here a study showing the highly invasive African big headed ant Pheidole megacephala having a novel distribution on coral cays within Australia’s Great Barrier Reef. These patterns displayed a clear limitation of its distribution with monocultures of the tree Pisonia grandis. This distribution was contrary to the known environmental limitations of the ant, and the limitation could not be associated with an underlying abiotic determinant of the vegetation type. We present these distributional patterns, and following consideration of all known biotic and abiotic limitations of ant invasions we discuss the potential that the peculiar ecophysiology of P. grandis is the causal factor. Specifically, we suggest that the quality of carbohydrate supply to ants is a limitation to invasive spread in much the same way that carbohydrate quantity is known to affect ant population densities in other ecosystems.     

NEDD4-2 associates with γc and regulates its degradation rate

Interleukin-2 (IL-2) is a cytokine that regulates proliferation, differentiation and survival of various lymphoid cell subsets. Its actions are mediated through its binding to the IL-2 receptor which is composed of three subunits (IL-2Rα, IL-2Rβ and γ_c). Only β and γ_c have been shown to transduce intra cellular signals. The γ_c chain is shared by the interleukin-2, 4, 7, 9, 15 and 21 receptors, and is essential for lymphocyte functions. The regulation of γ_c expression level is therefore critical for the ability of cells to respond to these cytokines. In the present work, we show that the IL-2R constitutively associates with the ubiquitin ligase NEDD4-2, and to a lesser extent NEDD4-1. We identified the specific binding site on γ_c. And we show that the loss of NEDD4 association on γ_c is accompanied by a dramatic increase of the half-life of the receptor subunit.