A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.     

A Genetic Screen for Suppressors of a Mutated 5′ Splice Site Identifies Factors Associated With Later Steps of Spliceosome Assembly

Many alleles of human disease genes have mutations within splicing consensus sequences that activate cryptic splice sites. In Caenorhabditis elegans, the unc-73(e936) allele has a G-to-U mutation at the first base of the intron downstream of exon 15, which results in an uncoordinated phenotype. This mutation triggers cryptic splicing at the −1 and +23 positions and retains some residual splicing at the mutated wild-type (wt) position. We previously demonstrated that a mutation in sup-39, a U1 snRNA gene, suppresses e936 by increasing splicing at the wt splice site. We report here the results of a suppressor screen in which we identify three proteins that function in cryptic splice site choice. Loss-of-function mutations in the nonessential splicing factor smu-2 suppress e936 uncoordination through changes in splicing. SMU-2 binds SMU-1, and smu-1(RNAi) also leads to suppression of e936. A dominant mutation in the conserved C-terminal domain of the C. elegans homolog of the human tri-snRNP 27K protein, which we have named SNRP-27, suppresses e936 uncoordination through changes in splicing. We propose that SMU-2, SMU-1, and SNRP-27 contribute to the fidelity of splice site choice after the initial identification of 5′ splice sites by U1 snRNP.PRE-mRNA splicing takes place in a large ribonucleoprotein complex called the spliceosome (Burge et al. 1999). Components of this splicing machinery assemble at conserved signal sequences within the pre-mRNA. The 5′ splice site consensus sequence M₋₃A₋₂G₋₁ | G₊₁U₊₂R₊₃A₊₄G₊₅U₊₆ and the 3′ splice site consensus sequence Y₋₃A₋₂G₋₁ | R₊₁ (M is either A or C; R is a purine, and Y is a pyrimidine) define the limits of the intron. Base-pairing interactions between the 5′ end of the U1 snRNA and the 5′ splice site consensus sequence occur early in spliceosome assembly. It is the nearly invariable GU dinucleotide at the first two positions of the 5′ end of the intron that defines the beginning of the intron. The 5′ consensus sequence is essential but insufficient for splice site selection, as 5′ splice sites with weaker consensus matches may require additional determinants for proper activation (Sanford et al. 2005).Mutations that disrupt the 5′ consensus splice signal can lead to genetic disease in humans (Nelson and Green 1990; Cohen et al. 1994). Approximately 15% of point mutations that cause genetic diseases affect pre-mRNA splicing consensus sequences (Krawczak et al. 1992). For some specific disease genes, as many as 50% of the known heritable alleles alter splicing (Teraoka et al. 1999; Ars et al. 2000; Roca et al. 2003; Pagenstecher et al. 2006). Among all the positions of the 5′ splice site consensus sequence, the highest proportion of human disease mutations occur at the +1G position (Buratti et al. 2007). The fidelity of pre-mRNA splice site choice is largely disrupted by this defect, since this mutation causes splicing at this site to be either abolished or outcompeted by the activation of nearby cryptic 5′ splice sites (Nelson and Green 1990; Cohen et al. 1994). Cryptic splice sites are used only when the wild-type splice donor is disrupted by mutation, as they tend to have very weak splice donor consensus sequences outside of a 5′-GU dinucleotide that defines the beginning of the intron (Roca et al. 2003). Suppression of mutations to the 5′ splice site consensus sequence in vivo has been achieved through the expression of U1 snRNAs containing compensatory base substitutions (Zhuang and Weiner 1986); however, suppression of mutations to the +1 position of the intron using reverse genetic approaches has not been successful (Newman et al. 1985; Nelson and Green 1990; Cohen et al. 1994).We have used a specific allele of the Caenorhabditis elegans unc-73 gene, e936, which contains a G-to-U mutation at the first nucleotide of intron 16 (Steven et al. 1998), as a model for studying cryptic splice site choice (Roller et al. 2000; Zahler et al. 2004). unc-73 encodes a RAC guanine nucleotide exchange factor that is expressed in neurons and is important for axon guidance (Steven et al. 1998). The e936 allele induces the use of three different cryptic 5′ splice sites (Figure 1A). Two of these 5′ splice sites, located at the −1 and +23 positions, define introns beginning with GU. The third 5′ splice site used is at the mutated wild-type (wt) position and is referred to as “wt” since splicing at this site still produces wild-type unc-73 mRNA and protein, even though the intron begins with UU (Roller et al. 2000). Use of either the −1 or the +23 cryptic site causes a shift in the reading frame and loss of gene function. In e936 animals, 90% of the stable messages of unc-73 are out-of-frame, yet the phenotype is not as severe as for other alleles in this gene. This indicates that the 10% of steady-state messages that are in frame have some functional role.Open in a separate windowFigure 1.—(A) Diagram of the unc-73 gene between exons 15 and 16. The positions of the −1 and +23 cryptic 5′ splice sites are indicated by arrows. The intronic e936 (+1G → U) point mutation is highlighted. (B) γ-³²P-labeled RT–PCR results across the cryptic splicing region of unc-73(e936) for different strains. Lanes 1, 2, and 3 are loaded with RT–PCR reactions from wild type (N2), unc-73(e936);sup-39(je5), and unc-73(e936) RNA, respectively. The lines carrying the suppressor alleles and e936 follow in lanes 4–10 as indicated. (C) The unc-73 genomic sequence from exon 15 (uppercase letters) and intron 15 (lowercase letters). The locations of the az23 and e936 mutational substitutions are indicated below. The position of the −9 cryptic splice donor activated in e936az23 is indicated by an arrow above.In a previous genetic screen for extragenic suppressors of e936 movement defects, Way and colleagues identified sup-39 (Run et al. 1996). It was subsequently shown that mutations in sup-39 alter cryptic splice site choice of e936 (Roller et al. 2000). sup-39 encodes a U1 snRNA gene with a compensatory mutation at the position that normally base pairs with the +1G. This allows sup-39 to base pair with an intron with a +1U (Zahler et al. 2004). This dominant suppressor increases usage of the mutated splice site and improves the fraction of in-frame messages from e936 from 10 to 33%, with a dramatic improvement in coordination. A similar mutant U1 snRNA suppressor with a different compensatory substitution, sup-6(st19), was found to suppress the intronic +1G to A transition of unc-13(e309) to allow for splicing at the mutated wild-type site, even though the intron begins with AU instead of GU (Zahler et al. 2004).We are interested in identifying additional factors that play a role in cryptic 5′ splice site choice. To do this, we took advantage of unc-73(e936), in which modest increases in the use of the wt splice site lead to dramatic increases in coordination, as a sensitive screen for changes in cryptic splice site choice. In this article we report that the proteins SMU-1 and SMU-2, which are nonessential factors previously shown to have a role in alternative splicing (Spartz et al. 2004), have a role in selection of cryptic 5′ splice sites. We also report the identification of a new dominant suppressor of cryptic splicing, snrp-27, which encodes a C. elegans homolog of the human tri-snRNP 27K protein.     

IL-28 Supplants Requirement for Treg Cells in Protein σ1-Mediated Protection against Murine Experimental Autoimmune Encephalomyelitis (EAE)

Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen uptake using reovirus adhesin, protein sigma 1 (pσ1), may circumvent these shortcomings based upon the recent finding that when reovirus pσ1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP_130–151) was genetically fused to OVA to pσ1 (PLP:OVA-pσ1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10⁺ forkhead box P3 (FoxP3)⁺ CD25⁺CD4⁺ T_reg and IL-4⁺CD25⁻CD4⁺ Th2 cells. IL-10R or IL-4 neutralization reversed protection to EAE conferred by PLP:OVA-pσ1, and adoptive transfer of Ag-specific T_reg or Th2 cells restored protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired PLP:OVA-pσ1''s protective capacity, triggering TGF-β-mediated inflammation; however, concomitant inactivation of TGF-β and CD25 reestablished PLP:OVA-pσ1-mediated protection by IL-28-producing FoxP3⁺CD25⁻CD4⁺ T cells. Thus, pσ1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.     

Transgene Rescue Identifies an Essential Function for Drosophila β Spectrin in the Nervous System and a Selective Requirement for Ankyrin-2–binding Activity

The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential β spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a β spectrin transgene or to knock down β spectrin expression with dsRNA. The results show that 1) overexpression of β spectrin in most of the cell types studied was lethal; 2) knockdown of β spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-β spectrin transgene was sufficient to overcome the lethality of a loss-of-function β spectrin mutation. Thus β spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for β spectrin function in vivo.     

A Broad Phenotypic Screen Identifies Novel Phenotypes Driven by a Single Mutant Allele in Huntington’s Disease CAG Knock-In Mice

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.     

RNAi Screen of Endoplasmic Reticulum–Associated Host Factors Reveals a Role for IRE1α in Supporting Brucella Replication

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α^−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.     

Casein Kinases I and 2α Phosphorylate Oryza Sativa Pseudo-Response Regulator 37 (OsPRR37) in Photoperiodic Flowering in Rice

Flowering time (or heading date) is controlled by intrinsic genetic programs in response to environmental cues, such as photoperiod and temperature. Rice, a facultative short-day (SD) plant, flowers early in SD and late in long-day (LD) conditions. Casein kinases (CKs) generally act as positive regulators in many signaling pathways in plants. In rice, Heading date 6 (Hd6) and Hd16 encode CK2α and CKI, respectively, and mainly function to delay flowering time. Additionally, the major LD-dependent floral repressors Hd2/Oryza sativa Pseudo-Response Regulator 37 (OsPRR37; hereafter PRR37) and Ghd7 also confer strong photoperiod sensitivity. In floral induction, Hd16 acts upstream of Ghd7 and CKI interacts with and phosphorylates Ghd7. In addition, Hd6 and Hd16 also act upstream of Hd2. However, whether CKI and CK2α directly regulate the function of PRR37 remains unclear. Here, we use in vitro pull-down and in vivo bimolecular fluorescence complementation assays to show that CKI and CK2α interact with PRR37. We further use in vitro kinase assays to show that CKI and CK2α phosphorylate different regions of PRR37. Our results indicate that direct posttranslational modification of PRR37 mediates the genetic interactions between these two protein kinases and PRR37. The significance of CK-mediated phosphorylation for PRR37 and Ghd7 function is discussed.     

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.     

Gangliosides smelt nanostructured amyloid Aβ(1–40) fibrils in a membrane lipid environment

Gangliosides induced a smelting process in nanostructured amyloid fibril-like films throughout the surface properties contributed by glycosphingolipids when mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/Aβ(1–40) amyloid peptide. We observed a dynamical smelting process when pre-formed amyloid/phospholipid mixture is laterally mixed with gangliosides. This particular environment, gangliosides/phospholipid/Aβ(1–40) peptide mixed interfaces, showed complex miscibility behavior depending on gangliosides content. At 0% of ganglioside covered surface respect to POPC, Aβ(1–40) peptide forms fibril-like structure. In between 5 and 15% of gangliosides, the fibrils dissolve into irregular domains and they disappear when the proportion of gangliosides reach the 20%. The amyloid interfacial dissolving effect of gangliosides is taken place at lateral pressure equivalent to the organization of biological membranes.Domains formed at the interface are clearly evidenced by Brewster Angle Microscopy and Atomic Force Microscopy when the films are transferred onto a mica support. The domains are thioflavin T (ThT) positive when observed by fluorescence microscopy.We postulated that the smelting process of amyloids fibrils-like structure at the membrane surface provoked by gangliosides is a direct result of a new interfacial environment imposed by the complex glycosphingolipids. We add experimental evidence, for the first time, how a change in the lipid environment (increase in ganglioside proportion) induces a rapid loss of the asymmetric structure of amyloid fibrils by a simple modification of the membrane condition (a more physiological situation).     

Genetic Heterogeneity of Bardet–Biedl Syndrome in a Distinct Canadian Population: Evidence for a Fifth Locus

Bardet–Biedl syndrome (BBS) is a rare, autosomal recessive disease characterized by retinal dystrophy, renal structural abnormalities, obesity, dysmorphic extremities, and hypogenitalism in males. BBS is genetically heterogeneous with four known loci: BBS1 (11q), BBS2 (16q), BBS3 (3p), and BBS4 (15q). The prevalence of BBS in Newfoundland is approximately 10-fold greater than in Switzerland (1:160,000) and similar to the prevalence among the Bedouin of Kuwait (1:13,500). A population-based genetic survey was performed on 17 BBS families from the island portion of the province of Newfoundland, a comparatively isolated region of Canada. The families in the study had a total of 36 well-documented, affected individuals with 12 families having 2 or more affected individuals. Linkage at each of the four known loci was tested with two-point linkage and haplotype analysis. Three of the 17 kindreds showed linkage to 11q, 1 to 16q, and 1 to 3p. The latter is the first BBS3 family identified in a population of northern European descent. Six families remain undetermined because of poor pedigree structure or inconclusive haplotype analyses. Six families were excluded from all four known BBS loci, indicating that there is at least a fifth BBS locus (BBS5).     

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.     

Innate Immunity and Non-Hodgkin’s Lymphoma (NHL) Related Genes in a Nested Case-Control Study for Gastric Cancer Risk

Objective

Genetic variants regulating the host immune system may contribute to the susceptibility for the development of gastric cancer. Little is known about the role of the innate immunity- and non-Hodgkin’s lymphoma (NHL)-related genes for gastric cancer risk. This nested case-control study was conducted to identify candidate genes for gastric cancer risk for future studies.

Methods

In the Discovery phase, 3,072 SNPs in 203 innate immunity- and 264 NHL-related genes using the Illumine GoldenGateTM OPA Panel were analyzed in 42 matched case-control sets selected from the Korean Multi-center Cancer Cohort (KMCC). Six significant SNPs in four innate immunity (DEFA6, DEFB1, JAK3, and ACAA1) and 11 SNPs in nine NHL-related genes (INSL3, CHMP7, BCL2L11, TNFRSF8, RAD50, CASP7, CHUK, CD79B, and CLDN9) with a permutated p-value <0.01 were re-genotyped in the Replication phase among 386 cases and 348 controls. Odds ratios (ORs) for gastric cancer risk were estimated adjusting for age, smoking status, and H. pylori and CagA sero-positivity. Summarized ORs in the total study population (428 cases and 390 controls) are presented using pooled- and meta-analyses.

Results

Four SNPs had no heterogeneity across the phases: in the meta-analysis, DEFA6 rs13275170 and DEFB1 rs2738169 had both a 1.3-fold increased odds ratio (OR) for gastric cancer (95% CIs = 1.1–1.6; and 1.1–1.5, respectively). INSL3 rs10421916 and rs11088680 had both a 0.8-fold decreased OR for gastric cancer (95% CIs = 0.7–0.97; and 0.7–0.9, respectively).

Conclusions

Our findings suggest that certain variants in the innate immunity and NHL-related genes affect the gastric cancer risk, perhaps by modulating infection-inflammation-immunity mechanisms that remain to be defined.     

Accumulation of amyloid-like Aβ1–42 in AEL (autophagy–endosomal–lysosomal) vesicles: potential implications for plaque biogenesis

Abnormal accumulation of Aβ (amyloid β) within AEL (autophagy–endosomal–lysosomal) vesicles is a prominent neuropathological feature of AD (Alzheimer''s disease), but the mechanism of accumulation within vesicles is not clear. We express secretory forms of human Aβ_1–40 or Aβ_1–42 in Drosophila neurons and observe preferential localization of Aβ_1–42 within AEL vesicles. In young animals, Aβ_1–42 appears to associate with plasma membrane, whereas Aβ_1–40 does not, suggesting that recycling endocytosis may underlie its routing to AEL vesicles. Aβ_1–40, in contrast, appears to partially localize in extracellular spaces in whole brain and is preferentially secreted by cultured neurons. As animals become older, AEL vesicles become dysfunctional, enlarge and their turnover appears delayed. Genetic inhibition of AEL function results in decreased Aβ_1–42 accumulation. In samples from older animals, Aβ_1–42 is broadly distributed within neurons, but only the Aβ_1–42 within dysfunctional AEL vesicles appears to be in an amyloid-like state. Moreover, the Aβ_1–42-containing AEL vesicles share properties with AD-like extracellular plaques. They appear to be able to relocate to extracellular spaces either as a consequence of age-dependent neurodegeneration or a non-neurodegenerative separation from host neurons by plasma membrane infolding. We propose that dysfunctional AEL vesicles may thus be the source of amyloid-like plaque accumulation in Aβ_1–42-expressing Drosophila with potential relevance for AD.     

A routine method for cloning,expressing and purifying Aβ(1–42) for structural NMR studies

Nuclear magnetic resonance (NMR) is a key technology in the biophysicist’s toolbox for gaining atomic-level insight into structure and dynamics of biomolecules. Investigation of the amyloid-β peptide (Aβ) of Alzheimer’s disease is one area where NMR has proven useful, and holds even more potential. A barrier to realizing this potential, however, is the expense of the isotopically enriched peptide required for most NMR work. Whereas most biomolecular NMR studies employ biosynthetic methods as a very cost-effective means to obtain isotopically enriched biomolecules, this approach has proven less than straightforward for Aβ. Furthermore, the notorious propensity of Aβ to aggregate during purification and handling reduces yields and increases the already relatively high costs of solid phase synthesis methods. Here we report our biosynthetic and purification developments that yield pure, uniformly enriched ¹⁵N and ¹³C¹⁵N Aβ(1–42), in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods, which we characterize by solution-state NMR, thioflavin T assays, circular dichroism, electrospray mass spectrometry, and dynamic light scattering. These developments should facilitate further investigations into Alzheimer’s disease, and perhaps misfolding diseases in general.     

Molecular Dynamics Simulations of Adrenocorticotropin (1–24) Peptide in a Solvated Dodecylphosphocholine (DPC) Micelle and in a Dimyistoylphosphatidylcholine (DMPC) Bilayer

Abstract

The structure and interactions of the 1–24 fragment of the adrenocorticotropin hormone, ACTH (1–24), with membrane have been studied by molecular dynamics (MD) simulation in an NPT ensembles in two explicit membrane mimics, a dodecylphosphocholine (DPC) micelle and a dimyristoylphosphatidylcholine (DMPC) bilayer. The starting configuration of the peptide/lipid systems had the 1–10 segment of the peptide lying on the surface of the model membrane, the same as the equilibrated structure (by MD) of ACTH (1–10) in a DPC micelle. The simulations showed that the peptide adopts the surface-binding mode and essentially the same structure in both systems. Thus the results of this work lend support to the assumption that micelles are reasonable mimics for biological membranes for the study of peptide binding. The 1–10 segment is slightly tilted from the parallel orientation to the interface and interacts strongly with the membrane surface while the more polar 11–24 segment shows little tendency to interact with the membrane surface, preferring to reside primarily in the aqueous phase. Furthermore, the 1–10 segment of the peptide binds to the DPC micelle in essentially the same way as ACTH (1–10). Thus the MD results are in excellent agreement with the model of interaction of ACTH (1–24) with membrane derived from NMR experiments. The secondary structure and the hydration of the peptide and the interactions of specific residues with the lipid head groups have also been analyzed.