Effects of the English (H6R) and Tottori (D7N) Familial Alzheimer Disease Mutations on Amyloid β-Protein Assembly and Toxicity

Mutations in the amyloid β-protein (Aβ) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid β-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Aβ40 and Aβ42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil → α/β → β and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial β-strand (H6R) or α/β (D7N) structure, in contrast to the predominately statistical coil structure of wild type Aβ oligomers. Mutant oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Aβ assembly at its earliest stages, monomer folding and oligomerization, and produce oligomers that are more toxic to cultured neuronal cells than are wild type oligomers.     

Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL₂ and HDL₃ were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed across the surface. The wild-type (WT) human and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the helix bundle domain. The results of fluorescence experiments with pyrene-labeled apoA-I are consistent with the N-terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants (K_d) measured for WT human apoA-I interactions with HDL₂ and HDL₃ are about 10 µM, indicating that the binding is low affinity. This K_d value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool.Understanding the structure and function of HDL is significant because of the beneficial cardioprotective properties of this lipoprotein (1). The anti-atherogenic effects of HDL arise, in part, from its participation in the reverse cholesterol transport pathway where the principal HDL protein, apolipoprotein A-I (apoA-I), plays a central role (2). As a result, the structure-function relationships of apoA-I have been studied extensively (for reviews, see Refs. 3–5). Perhaps the most important characteristic of the apoA-I molecule is its ability to bind lipids; this interaction is mediated by the amphipathic α-helices present in the protein molecule (6). ApoA-I binds well to phospholipid (PL)-water interfaces and, under appropriate conditions, can solubilize the PL to create discoidal HDL particles (7, 8). The binding of apoA-I to a PL surface involves a two-step mechanism. First, α-helices in the C-terminal domain of the protein interact with the surface, and, second, the N-terminal helix bundle domain opens to allow more helix-lipid interactions to occur (5, 9). Although the binding of apoA-I to model PL particles has been studied extensively, the binding of apoA-I to HDL particles has not been investigated much because of the difficulty of separating free and bound apoA-I in this system. This lack of information about apoA-I/HDL interactions is significant because the cycling of apoA-I molecules on and off HDL particles occurs during the metabolism of HDL particles (10, 11), in particular to release apoA-I molecules into the preβ-HDL pool (10, 12). This recycling is consistent with the well-established ability of apolipoproteins, such as apoA-I, to exchange spontaneously between different populations of lipoprotein particles (13–16) and PL vesicles (17, 18). As a rule, any remodeling event that depletes HDL particles of PL induces particle fusion and dissociation of that fraction of the apoA-I molecules that is in a labile pool (19). At this stage, quantitative understanding of the kinetics of apoA-I interactions with HDL particles is unavailable.Here, we exploit surface plasmon resonance (SPR) to monitor in real time the association and dissociation reactions in the apoA-I/HDL system. SPR has been used to derive quantitative information about the binding of both lipoproteins (20) and apoE (21–23) to proteoglycans. As far as the application of SPR to the HDL system is concerned, the binding of several plasma remodeling factors to HDL immobilized on a sensor chip has been investigated successfully (24–26). Also, the conformation of apoA-I in HDL was explored by comparing the binding of HDL particles to anti-apoA-I monoclonal antibodies immobilized on an SPR chip (27). We have extended these approaches to study the binding of apoA-I to HDL particles. The results show that apoA-I can bind reversibly and with low affinity to HDL particles by a two-step mechanism.     

CTnDOT Integrase Interactions with Attachment Site DNA and Control of Directionality of the Recombination Reaction

IntDOT is a tyrosine recombinase encoded by the conjugative transposon CTnDOT. The core binding (CB) and catalytic (CAT) domains of IntDOT interact with core-type sites adjacent to the regions of strand exchange, while the N-terminal arm binding (N) domain interacts with arm-type sites distal to the core. Previous footprinting experiments identified five arm-type sites, but how the arm-type sites participate in the integration and excision of CTnDOT was not known. In vitro integration assays with substrates containing arm-type site mutants demonstrated that attDOT sequences containing mutations in the L1 arm-type site or in the R1 and R2 or R1 and R2′ arm-type sites were dramatically defective in integration. Substrates containing mutations in the L1 and R1 arm-type sites showed a 10- to 20-fold decrease in detectable in vitro excision, but introduction of multiple arm-type site mutations in attR did not have an effect on the excision frequency. A sixth arm-type site, the R1′ site, was also identified and shown to be required for integration and important for efficient excision. These results suggest that intramolecular IntDOT interactions are required for integration, while the actions of accessory factors are more important for excision. Gel shift assays performed in the presence of core- and arm-type site DNAs showed that IntDOT affinity for the attDOT core was enhanced when IntDOT was simultaneously bound to arm-type site DNA.Conjugative transposons (CTns), also known as integrative and conjugative elements (ICEs), are mobile genetic elements that are widespread in Bacteroides spp. and are implicated in the spread of antibiotic resistance. These elements are normally integrated into the host chromosome but can excise, replicate, and transfer to a recipient cell by conjugation (34). Since CTns commonly carry antibiotic resistance genes, it is likely that the increase in antibiotic-resistant Bacteroides strains has been mediated through the lateral transfer of these elements (36). One of the best-studied ICEs in Bacteroides is the conjugative transposon CTnDOT. CTnDOT is 65 kb in size and carries genes encoding resistance to tetracycline and erythromycin. Over the past 30 years, the incidence of tetracycline resistance has increased to 80% of Bacteroides isolates due to the presence of CTnDOT-type elements (36).Integration and excision of CTnDOT results from site-specific recombination between regions of DNA known as attachment (att) sites. During integration, the joined ends of the closed circular intermediate (attDOT) recombine with the bacterial target sequence (attB) to form the recombinant sites (attL and attR). The integration reaction requires IntDOT, a CTnDOT-encoded protein that has been identified as a member of the tyrosine recombinase family, as well as a host factor encoded by Bacteroides (8, 21). Site-specific recombination between the attL and attR attachment sites results in excision of CTnDOT from the host chromosome. IntDOT is also required for excision, as are three element-encoded proteins: Orf2c, Orf2d, and Exc, as well a Bacteroides host factor (8, 38). The roles of these accessory proteins are not well understood, although Orf2c and Orf2d have been shown to bind DNA (unpublished results).One of the best-studied tyrosine recombinases is the integrase (Int) of the lambda system. The C terminus of Int includes the core binding (CB) and catalytic (CAT) domains that bind to core-type sites, which flank the sites of cleavage and strand exchange (2, 24). The N-terminal arm-binding (N) domain binds to arm-type sites that are distal to the core-type sites. In the presence of the appropriate host and accessory factors, Int binding to arm-type sites is required for the formation of higher-order protein/DNA complexes known as intasomes, which are required for integration and excision (15, 18, 22). Int is capable of making intramolecular interactions (interactions between Int monomers on the same attachment site) and intermolecular interactions (interactions between Int monomers on different attachment sites) during recombination (15, 16). In the lambda system, the directionality of the reaction is regulated by Int interactions with arm-type sites in conjunction with the integration host factor (IHF) during the formation of an integrative intasome, or IHF, Xis, and FIS during the formation of the two excisive intasomes (1, 4, 42).Presumably, IntDOT occupancy of specific arm-type sites in conjunction with interactions of accessory factors with att sites leads to the assembly of integrative or excisive intasomes and thus contributes to the directionality of IntDOT-mediated recombination. Previous DNase I footprinting experiments identified five arm-type binding sites on attDOT (11). In this study, mutations were constructed in the five sites to determine their roles in the integration and excision of CTnDOT. In addition, a sixth arm-type site was discovered that is important for both integrative and excisive recombination. The results of gel shift assays also show that the interaction of IntDOT with core-type sites and arm-type sites involves cooperative interactions.     

Testosterone may increase selective attention to threat in young male macaques

Animal studies indicate that sex hormones have widespread effects on the brain, cognition and emotion, but findings in humans are inconsistent. Well-controlled studies in nonhuman primates are crucial to resolve these discrepancies. In this study, we examined the effects of testosterone (T) on emotion in male rhesus monkeys. Six young adult males were tested on two emotional tasks during three hormonal conditions in a crossover design: when intact at baseline and when pharmacologically hypogonadal with add-back of T or placebo. The emotional tasks were the Approach–Avoidance task, which tested behavioral responses to three categories of objects (familiar, novel, and negative) and a Social Playback task which tested behavioral responses to scenes of unfamiliar conspecifics engaged in three types of social activities (neutral, positive, or negative). Following a 4-week baseline period, monkeys were treated with Depot Lupron, 200 μg/kg before being randomly assigned to one of two treatment groups: Depot Lupron + Testosterone Enanthate (TE, 20 mg/kg) or Depot Lupron + oil vehicle. In each treatment group, monkeys received one injection of Lupron and one injection of TE or one injection of Lupron and one injection of oil at the onset of a 4-week testing period, before crossing over to the alternate treatment for an additional 4 weeks of testing. TE treatment had no effect on behavioral measures in the Approach–Avoidance task. For the Social Playback task, however, TE significantly increased watching time of video clips which depicted fights between unfamiliar conspecifics. The enhancing effect of T on watching time for negative social scenes is consistent with human data suggesting that T decreases aversion or facilitates approach to threatening social stimuli. Further studies are needed to understand the mechanisms by which T may mediate responsiveness to social threat in male primates.     

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.     

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the α/β-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the α/β-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the α/β-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.