Membrane targeting, shedding and protein interactions of brain acetylcholinesterase

The early stages of Alzheimer's disease are characterized by cholinergic deficits and the preservation of cholinergic function through the use of acetylcholinesterase inhibitors is the basis for current treatments of the disease. Understanding the causes for the loss of basal forebrain cholinergic neurons in neurodegeneration is therefore a key to developing new therapeutics. In this study, we review novel aspects of cholinesterase membrane localization in brain and propose mechanisms for its lipid domain targeting, secretion and protein-protein interactions. In erythrocytes, acetylcholinesterase (AChE) is localized to lipid rafts through a GPI anchor. However, the main splice form of AChE in brain lacks a transmembrane peptide anchor region and is bound to the 'proline-rich membrane anchor', PRiMA, in lipid rafts. Furthermore, AChE is secreted ('shed') from membranes and this shedding is stimulated by cholinergic agonists. Immunocytochemical studies on rat brain have shown that membrane-associated PRiMA immunofluorescence is located selectively at cholinergic neurons of the basal forebrain and striatum. A strong association of AChE with the membrane via PRiMA seems therefore to be a specific requirement of forebrain cholinergic neurons. α7 nicotinic acetylcholine receptors are also associated with lipid rafts where they undergo rapid internalisation on stimulation. We are currently probing the mechanism(s) of AChE shedding, and whether this process and its apparent association with α7 nicotinic acetylcholine receptors and metabolism of the Alzheimer's amyloid precursor protein is determined by its association with lipid raft domains either in normal or pathological situations.     

Streptococcus pneumoniae: the evolution of antimicrobial resistance to beta-lactams, fluoroquinolones and macrolides

Multi drug resistant Streptococcus pneumoniae constitute a major public health concern worldwide. In this review we discuss how the transformable nature of the pneumococcus, in parallel with antimicrobial induced stress, contributes to the evolution of antimicrobial resistance; and how the introduction of the pneumococcal conjugate vaccine has affected the situation.     

Use of mass spectrometry with chemical ionization in screening antibacterial antibiotics with a broad spectrum of action (aminoglycosides and macrolides)

It is shown that mass-spectrometry with ammonia desorption chemical ionization (ADCI) can be used for identification of aminoglycosides and macrolides at the initial stages of screening. ADCI can also be used for selection of strains which form the lowest amounts of by-products, as well as for optimization of biosynthetic conditions.     

Resistance to macrolides and lincosamides in Streptomyces lividans and to aminoglycosides in Micromonospora purpurea.

Ribosomal (r) resistance to gentamicin in clones containing DNA from the producing organism Micromonospora purpurea is determined by grmA, and not by kgmA as originally reported. The kgmA gene originated in Streptomyces tenebrarius and is identical to kgmB. Both grmA and kgm encode enzymes that methylate single specific sites within 16S rRNA, although the site of action of the grmA product has not yet been determined. In either case, the methylated nucleoside is 7-methyl G. Inducible resistance to lincomycin (Ln) and macrolides in Streptomyces lividans TK21 results from expression of two genes: lrm, encoding an rRNA methyltransferase and mgt, encoding a glycosyl transferase (MGT), that specifically inactivates macrolides. The lrm product monomethylates residue A2058 within 23S rRNA (Escherichia coli numbering scheme) and confers high-level resistance to Ln with much lower levels of resistance to macrolides. Substrates for MGT, which utilises UDP-glucose as cofactor, include macrolides with 12-, 14-, 15- or 16-atom cyclic polyketide lactones (as in methymycin, erythromycin, azithromycin or tylosin, respectively) although spiramycin and carbomycin are not apparently modified. The enzyme is specific for the 2'-OH group of saccharide moieties attached to C5 of the 16-atom lactone ring (corresponding to C5 or C3 in 14- or 12-atom lactones, respectively). The lrm and mgt genes have been cloned and sequenced. The deduced lrm product is a 26-kDa protein, similar to other rRNA methyltransferases, such as the carB, tlrA and ermE products, whereas the mgt product (deduced to be 42 kDa) resembles a glycosyl transferase from barley.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative in vitro activity of moxalactam (LY127935), other beta-lactam antibiotics,and aminoglycosides

Moxalactam (LY127935), a novel beta-lactam antibiotic, was compared with semisynthetic penicillins, cephalosporins, and aminoglycosides by the agar dilution method against 5,317 recent clinical isolates of facultative and anaerobic bactria. At 0.5 μg/ml, moxalactam inhibited 90% of all Gram-negative bacilli tested except forPseudomonas aeruginosa (81% inhibited by 32 μg/ml) andAcinetobacter calcoaceticus (88% inhibited by 32 μg/ml). More than 90% ofBacteroides fragilis andStaphylococcus aureus were inhibited by 4 μg/ml and 8 μg/ml, respectively. Moxalactam was at least 16-fold more active by weight than cephalothin, cefamandole, and cefoxitin forEscherichia coli, Klebsiella pneumoniae, andEnterobacter species, and 2- to 4-fold more active than cefoxitin forB. fragilis. Moxalactam was 4-fold less active than cefamandole and cephalothin forS. aureus and 2- to 4-fold less active than piperacillin forP. aeruginosa. Moxalactam was as active or more active than the aminoglycosides for all facultative Gram-negative bacilli except forP. aeruginosa. Moxalactam was inhibitory (minimal inhibitory concentration <16 μg/ml) for 20/27 gentamicin-resistant isolates and 8/13 amikacin-resistant organisms. Moxalactam’s in vitro activity against Gram-negative bacilli is markedly superior to presently available cephalosporins and, except forP. aeruginosa, is comparable to the aminoglycosides.     

The electrochemical interactions of heparin and the aminoglycoside antibiotics

Various aminoglycoside antibiotics—tobramycin "analytical standard"; tobramycin, gentamycin, kanamycin, and amikacin, all containing preservatives added in pharmaceutical preparations; and streptomycin—without preservatives—yield in aqueous solutions a conductance peak when titrated against a heparin aqueous solution, indicating the formation of a charge transfer complex, which subsequently dissociates. At the volume ratio corresponding to the peak, a colloidal suspensoid forms which is possibly a micellar complex stabilized by the ions produced by the dissociation of the complex. The clinical significance of this interaction is suggested by preliminary microbiological tests. The possible effects of plasma protein binding and dissociation of the complexes here reported in tissues, however, were not studied.In the interaction with heparin, the aminoglycoside antibiotics appear to act as electron acceptors, though heparin is reported to act as an acceptor against chlorpromazine, which is well known to be a strong electron donor. There appears to be an interaction between nonpreserved tobramycin and the preservative added for the pharmaceutical preparation, which would explain the difference in titration behavior between the antibiotic alone and the antibiotic with preservative.Conductance titrations of heparin against penicillin and cloxacillin, which are not aminoglycosides, show no evidence of complexation or any other interaction. No suspensoid forms.     

Membrane interactions of intrinsically disordered proteins: The example of alpha-synuclein

Peripheral membrane proteins associate reversibly with biological membranes that, compared to protein binding partners, are structurally labile and devoid of specific binding pockets. Membranes in different subcellular compartments vary primarily in their chemical composition and physical properties, and recognition of these features is therefore critical for allowing such proteins to engage their proper membrane targets. Intrinsically disordered proteins (IDPs) are well-suited to accomplish this task using highly specific and low- to moderate-affinity interactions governed by recognition principles that are both similar to and different from those that mediate the membrane interactions of rigid proteins. IDPs have also evolved multiple mechanisms to regulate membrane (and other) interactions and achieve their impressive functional diversity. Moreover, IDP-membrane interactions may have a kinetic advantage in fast processes requiring rapid control of such interactions, such as synaptic transmission or signaling. Herein we review the biophysics, regulation and functional implications of IDP-membrane interactions and include a brief overview of some of the methods that can be used to study such interactions. At each step, we use the example of alpha-synuclein, a protein involved in the pathogenesis of Parkinson’s disease and one of the best characterized membrane-binding IDP, to illustrate some of the principles discussed.     

Membrane targeting and translocation of bacterial hydrogenases

Periplasmic or membrane-bound bacterial hydrogenases are generally composed of a small subunit and a large subunit. The small subunit contains a peculiar N-terminal twin-arginine signal peptide, whereas the large subunit lacks any known targeting signal for export. Genetic and biochemistry data support the assumption that the large subunit is cotranslocated with the small subunit across the cytoplasmic membrane. Indeed, the signal peptide carried by the small subunit directs both the small and the large subunits to the recently identified Mtt/Tat pathway, independently of the Sec machinery. In addition, the twin-arginine signal peptide of hydrogenase is capable of directing protein import into the thylakoidal lumen of chloroplasts via the homologous deltapH-driven pathway, which is independent of the Sec machinery. Therefore, the translocation of hydrogenase shares characteristics with the deltapH-driven import pathway in terms of Sec-independence and requirement for the twin-arginine signal peptide, and with protein import into peroxisomes in a "piggyback" fashion.     

Membrane and protein interactions of oxysterols

PURPOSE OF REVIEW: Oxysterols, oxidation products of cholesterol, mediate numerous and diverse biological processes. The objective of this review is to explain some of the biochemical and cell biological properties of oxysterols based on their membrane biophysical properties and their interaction with integral and peripheral membrane proteins. RECENT FINDINGS: According to their biophysical properties, which can be distinct from those of cholesterol, oxysterols can promote or inhibit the formation of membrane microdomains or lipid rafts. Oxysterols that inhibit raft formation are cytotoxic. The stereo-specific binding of cholesterol to sterol-sensing domains in cholesterol homeostatic pathways is not duplicated by oxysterols, and some oxysterols are poor substrates for the pathways that detoxify cells of excess cholesterol. The cytotoxic roles of oxysterols are, at least partly, due to a direct physical effect on membranes involved in cholesterol-induced cell apoptosis and raft mediated cell signaling. Oxysterols regulate cellular functions by binding to oxysterol binding protein and oxysterol binding protein-related proteins. Oxysterol binding protein is a sterol-dependent scaffolding protein that regulates the extracellular signal-regulated kinase signaling pathway. According to a recently solved structure for a yeast oxysterol binding protein-related protein, Osh4, some members of this large family of proteins are likely sterol transporters. SUMMARY: Given the association of some oxysterols with atherosclerosis, it is important to identify the mechanisms by which their association with cell membranes and intracellular proteins controls membrane structure and properties and intracellular signaling and metabolism. Studies on oxysterol binding protein and oxysterol binding protein-related proteins should lead to new understandings about sterol-regulated signal transduction and membrane trafficking pathways in cells.     

Membrane targeting and remodeling through phosphoinositide-binding domains

In mammals, there are seven inositolphospholipids, collectively called phosphoinositides that serve as versatile molecules not only in receptor-mediated signal transduction but also in a variety of cellular events such as cytoskeletal reorganization, membrane trafficking, cell proliferation and cell death. Recent studies have revealed that the latter functions are mediated by direct interactions between phosphoinositides and proteins. Such proteins contain two types of phosphoinositide-binding regions; basic amino acid stretch and globular structural domain. Furthermore, spatially restricted compartment of phosphoinositides and their concentration are finely regulated by a large number of phosphoinositide kinases and -phosphatases, controlling localization-specific metabolism of this simple lipid whose aberrations cause various diseases such as cancer and diabetes.     

The signal recognition particle and its interactions during protein targeting

The synthesis of secretory or integral membrane proteins can be directly coupled to their translocation across or insertion into membranes. In co-translational targeting, the translation machine, the ribosome, is transferred to the respective membrane by the signal recognition particle (SRP) and its receptor (SR) as soon as a signal sequence emerges. Protein synthesis can continue at the membrane, with the nascent peptide chain directly inserting into the ribosome-bound protein-conducting channel, the Sec61 complex. During the past two years, several structures have been solved by crystallography and cryo-electron microscopy that represent distinct functional states of the SRP cycle. On this basis, the first structure-based models can be suggested that explain important aspects of protein targeting, such as the SRP-ribosome and SRP-SR interactions.     

Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide capreomycin antibiotics in Korea

Aminoglycosides are key drugs for the treatment of multidrug-resistant tuberculosis. A total of 97 extensively drug-resistant (XDR) and 29 pan-susceptible Mycobacterium tuberculosis isolates from Korean tuberculosis patients were analyzed to characterize mutations within the rrs, rpsL, gidB, eis and tlyA genes. Thirty (56.6 %) of the 53 streptomycin (STR)-resistant strains had a rpsL mutation and eight strains (15.1 %) had a rrs (514 or 908 site) mutation, whereas 11 (20.8 %) of the 53 STR-resistant strains had a gidB mutation without rpsL or either rrs mutation. Most of the gidB mutations conferred low-level STR resistance, and 22 of these mutations were novel. Mutation at position 1401 in rrs lead to resistance to kanamycin (80/95 = 84.2 %; KAN), amikacin (80/87 = 92.0 %; AMK), and capreomycin (74/86 = 86.0 %; CAP). In this study, 13.7 % (13/95) of KAN-resistant strains showed eis mutations, including 4 kinds of novel mutations. Isolates with eis structural gene mutations were cross-resistant to STR, KAN, CAP, and AMK. Here, 5.8 % (5/86) of the CAP-resistant strains harbored a tlyA mutation that included 3 different novel point mutations. Detection of the A1401G mutation appeared to be 100 % specific for the detection of resistance to KAN and AMK. These data establish the presence of phenotypic XDR strains using molecular profiling and are helpful to understanding of aminoglycoside resistance at the molecular level.     

Membrane targeting of endocytic adaptors: cargo and lipid do it together

The adaptor complex AP-2 plays an important role in cargo selection and clathrin lattice formation during clathrin-mediated endocytosis. In a recent issue of Molecular Cell, Honing et al. demonstrate that high-affinity AP-2 membrane association is achieved through a combination of low-affinity interactions with membrane phosphoinositides and cargo proteins, regulated by phosphorylation.     

Membrane binding and subcellular targeting of C2 domains

C2 domains are a ubiquitous structural module and many of them function in Ca2+ -dependent membrane binding and thereby serve as Ca2+ effectors for divergent Ca2+ -mediated cellular processes. Extensive structural, biochemical, biophysical, and cellular studies of C2 domains and host proteins in the past decade have shown that due to their structural diversity C2 domains have disparate Ca2+ sensitivity, lipid selectivity and membrane binding mechanisms. This review summarizes the basic structural and functional properties of C2 domains as well as recent findings on Ca2+ and membrane binding, lipid selectivity, and subcellular localization of C2 domains and their host proteins.     

Membrane targeting of a folded and cofactor-containing protein.

Targeting of proteins to and translocation across the membranes is a fundamental biological process in all organisms. In bacteria, the twin arginine translocation (Tat) system can transport folded proteins. Here, we demonstrate in vivo that the high potential iron-sulfur protein (HiPIP) from Allochromatium vinosum is translocated into the periplasmic space by the Tat system of Escherichia coli. In vitro, reconstituted HiPIP precursor (preHoloHiPIP) was targeted to inverted membrane vesicles from E. coli by a process requiring ATP when the Tat substrate was properly folded. During membrane targeting, the protein retained its cofactor, indicating that it was targeted in a folded state. Membrane targeting did not require a twin arginine motif and known Tat system components. On the basis of these findings, we propose that a pathway exists for the insertion of folded cofactor-containing proteins such as HiPIP into the bacterial cytoplasmic membrane.