A differential association of Apolipoprotein E isoforms with the amyloid-β oligomer in solution

The molecular pathogenesis of disorders arising from protein misfolding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates, whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex misfolding and aggregation that initiates the toxic cascade resulting in Alzheimer's disease (AD), we have developed a 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin-labeled amyloid-β (Aβ) peptide to observe its isoform-dependent interaction with the apoE protein. Although most individuals carry the E3 isoform of apoE, ～15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimer's. ApoE is consistently associated with the amyloid plaque marker for AD. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aβ peptide processing and hence Aβ toxicity. We used electron paramagnetic resonance (EPR) spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aβ in solution. EPR spectra of the spin-labeled side chain report on side chain and backbone dynamics as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C-terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aβ oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimer's risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aβ.     

Human bocavirus NP1 inhibits IFN-β production by blocking association of IFN regulatory factor 3 with IFNB promoter

Human bocavirus (HBoV) mainly infects young children. Although many infected children suffer from respiratory or gastroenteric tract diseases, an association between HBoV and these diseases is not definite. Because modulation of type I IFN is crucial for viruses to establish efficient replication, in this study, we tested whether HBoV modulates type I IFN production. We observed that a nearly full-length HBoV clone significantly reduced both Sendai virus (SeV)- and poly(deoxyadenylic-thymidylic) acid-induced IFN-β production. Further study showed that NP1 blocked IFN-β activation in response to SeV, poly(deoxyadenylic-thymidylic) acid, and IFN-β pathway inducers, including retinoic acid-inducible protein I, mitochondrial antiviral signaling protein, inhibitor of κB kinase ε, and TANK-binding kinase 1. In addition, NP1 interfered with IRF-3-responsive PRD(III-I) promoter activated by SeV and a constitutively active mutant of IRF-3 (IRF-3/5D). Although NP1 suppressed the IRF-3 pathway, it did not affect IRF-3 activation processes, including phosphorylation, dimerization, and nuclear translocation. Coimmunoprecipitation assays confirmed the interaction between NP1 and IRF-3. Additional deletion mutagenesis and coimmunoprecipitation assays revealed that NP1 bound to the DNA-binding domain of IRF-3, resulting in the interruption of an association between IRF-3 and IFNB promoter. Altogether, our results indicate that HBoV NP1 blocks IFN production through a unique mechanism. To our knowledge, this is the first study to investigate the modulation of innate immunity by HBoV. Our findings suggest a potential immune-evasion mechanism used by HBoV and provide a basis for better understanding HBoV pathogenesis.     

Prominent expression of FRS2β protein in neural cells and its association with intracellular vesicles

The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2α, a well-known central mediator for fibroblast growth factor signaling, and FRS2β, whose endogenous expression pattern and function are not yet defined. Immunohistochemical analysis revealed that expression of FRS2β was restricted to neural tissues and it colocalized with Tuj1, a neuronal marker. There are two distinct patterns of FRS2β expression in neural cells: punctate and cup/ring-shaped; moreover, some particles colocalized with lysosomes. Stimulation with brain-derived neurotrophic factor (BDNF) enhanced FRS2β phosphorylation and the cup/ring-shaped pattern. These results suggest a probable role of FRS2β in the intracellular degradation systems of neural cells, which involves lysosomes.     

Genetic association of IDE,POU2F1, PON1, IL1α and IL1β with type 2 diabetes in Pakistani population

A number of genes are known to be involved in glucose homeostasis. Mutations and polymorphisms in candidate genes may effect insulin production, action or resistance. This study was designed to report the association of genetic polymorphism with the type 2 diabetes (T2D) in Pakistani population. A total of 458 subjects (case n = 288, control n = 170) participated in the study. Nine single nucleotide polymorphisms were investigated in genes IDE (rs6583813 C>T, rs7910977 C>T), POU2F1 (rs3767434 A>T, rs10918682 A>T, rs2146727 A>G), WFS1 (rs734312 A>G), PON1 (rs854560 T>A), IL1α (rs1800587 C>T) and IL1β (rs1143634 C>T). Genotyping was performed by DNA sequencing after nested polymerase chain reaction of targeted regions. Results indicated that rs7910977 in IDE showed significant association with the development of T2D [P = 0.012, OR 1.677 (95 % CI 1.112–2.438)]. The rs10918682 in POU2F1 was associated with T2D [P < 0.001, OR 3.606 (95 % CI 2.165–6.005)]. The rs854560 in PON1was associated with incidences of T2D and increased the risk of cardiovascular complications [P = 0.031, OR 0.663 (95 % CI 0.455–0.965)] in diabetics. The rs734312 from WFS1 gene was associated with diabetes at genotype level (P < 0.01). Haplotype analysis of rs1800587–rs1143634 depicted CC haplotype increased the susceptibility to diabetes (P < 0.05). Haplotype GAA from rs2146727–10918682–rs3767434 was protective against diabetes (P < 0.01) and GGA exhibited the association with T2D (P < 0.01). Haplotype CT from rs6583813–rs7910977 was protective against diabetes (P = 0.02). Our study provided evidence to IDE, PON1, WFS1, POU2F1, IL1α and IL1β associated with T2D in Pakistanis.     

HP1-β is required for development of the cerebral neocortex and neuromuscular junctions

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-β isotype, and show that the Cbx1^−/−-null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1^−/− mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1^−/− mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.     

Investigation of interleukin-1β release from cryopreserved blood stimulated with endotoxin

The feasibility of an indigenously developed ELISA method to determine cytokine response to wide spectrum of pyrogenic stimuli utilizing fresh human whole blood is limited by the availability of healthy donors. The possibility of using cryopreservation of pooled human blood for detection of cytokine response to lipopolysaccharide is explored in this study. The effect of cryopreservation on blood parameters, cellular morphology and cytokine response were compared with that of the pooled fresh blood and cryopreserved blood from single and multiple donors. In vitro pyrogenic stimulation with 0.5 and 5 EU of LPS was monitored on fresh and cryopreserved pooled blood from single and multiple donors. The release of IL-1β was quantitated by Sandwich ELISA (1, 10, 25, 45 and 75 days) after storage. The results indicated that the cryopreserved blood displayed enhanced IL-1β release on stimulation with LPS, when compared to fresh blood. The maximum release of IL-1β level was observed at 2 h when 5 EU of LPS was treated with pooled fresh blood which is similar to that of fresh blood. After 75 days storage of pooled cryopreserved blood the IL-1β release was maximum at 9 and 15 h when treated with 5 and 0.5 EU of LPS. Observations of the study suggest that cryopreserved pooled blood is an economically and experimentally viable alternative to fresh blood. This investigative study promises short term storage and regular supply of non-allergic, pathogen free human blood for the detection of interleukin-1β for the evaluation of in vitro pyrogenicity.     

Identification of LRP1B-interacting proteins and inhibition of protein kinase Cα-phosphorylation of LRP1B by association with PICK1

Recent studies show LDL receptor-related protein 1B, LRP1B as a transducer of extracellular signals. Here, we identify six interacting partners of the LRP1B cytoplasmic region by yeast two-hybrid screen and confirmed their in vivo binding by immunoprecipitation. One of the partners, PICK1 recognizes the C-terminus of LRP1B and LRP1. The cytoplasmic domains of LRP1B are phosphorylated by PKCα about 100 times more efficiently than LRP1. Binding of PICK1 inhibits phosphorylation of LRP1B, but does not affect LRP1 phosphorylation.This study presents the possibility that LRP1B participates in signal transduction which PICK1 may regulate by inhibiting PKCα phosphorylation of LRP1B.

Structured summary

MINT-6801075: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with SNTG2 (uniprotkb:Q925E0) by two hybrid (MI:0018)MINT-6801030, MINT-6801468: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by two hybrid (MI:0018)MINT-6801284: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by anti tag coimmunoprecipitation (MI:0007)MINT-6801108: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Grb7 (uniprotkb:Q03160) by two hybrid (MI:0018)MINT-6801090: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by two hybrid (MI:0018)MINT-6801008: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by two hybrid (MI:0018)MINT-6801052: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-2 (uniprotkb:Q9ERE9) by two hybrid (MI:0018)MINT-6801258, MINT-6801271: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by anti tag coimmunoprecipitation (MI:0007)MINT-6801244: RanBPM (uniprotkb:P69566) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801131, MINT-6801158: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by anti tag coimmunoprecipitation (MI:0007)MINT-6801231: PICK1 (uniprotkb:Q80VC8) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801173: Jip-1b (uniprotkb:Q9WVI9-1) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)     

Spatial association of the Cav1.2 calcium channel with α5β1-integrin

Engagement of α(5)β(1)-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca(L)) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing Ca(L). Using coimmunoprecipitation strategies, we show that coassociation of Ca(L) with α(5)- or β(1)-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between Ca(L) and α(5)β(1)-integrin and between Ca(L) and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca(L) heterologously expressed in HEK293-T cells, we identified two regions of Ca(L) required for these interactions: 1) COOH-terminal residues Ser(1901) and Tyr(2122), known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type Ca(L) colocalization with β(1)-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, Ca(L) associates with α(5)β(1)-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of Ca(L) at Y(2122) and/or S(1901) is required for association of Ca(L) with α(5)β(1)-integrin; and 3) c-Src, via binding to PRDs that reside in the II-III linker region and/or the COOH terminus of Ca(L), mediates current potentiation following α(5)β(1)-integrin engagement. These findings provide new evidence for how interactions between α(5)β(1)-integrin and FN can modulate Ca(L) entry and consequently alter the physiological function of multiple types of excitable cells.     

Distribution of HERV-LTR elements in the 5′-flanking region of HLA-DQB1 and association with autoimmunity

We established the detailed polymorphism of the 5'-flanking region and the first exon of the human leukocyte antigen (HLA)-DQB1 alleles. One hundred and forty-five Spanish rheumatoid arthritis (RA) patients and 200 healthy voluntary blood donors from southern Spain along with 42 B-cell lines were analyzed for the presence of the retrovirus-derived long terminal repeats (LTRs) LTR3, LTR5, and LTR13. LTR3 positivity was always associated with certain DQB1 alleles, i.e., *0302, *0402, *0601, *0202, and *0305. Sequencing analysis of the 5'-flanking region of DQB1*0301, *0303 and *0502 alleles in homozygous B-cell lines showed the absence of LTR3 and a massive deletion of 5635 base pairs. The undetected deletion in the flanking region of some DQB1 alleles and a lack of stratification for HLA typing explain previously reported associations of the LTR3 element with RA and type I diabetes (IDDM). LTR5 showed identical distribution to LTR3, consistent with a previously suggested LTR3-LTR5 tandem arrangement. LTR13 positivity was associated with DQB1*0302, *0303, and *0402 alleles. Distributions of the LTR elements in all B-cell lines, RA patients, and controls could be explained entirely by linkage disequilibrium with DQB1 alleles, independently of the haplotypes carrying them. LTR elements are known to regulate gene expression. Therefore, a possible involvement of LTR13 in the association of DQB1*0302, *0303, and *0402 with IDDM requires further investigation. The sequencing results of the DQB1 first exon demonstrated that DQB1*0601 was generated by a recombination event between a DR53 and a non-DR53 haplotype. Our results shed new light on the phylogeny of the HLA region and the possible contribution of DQB1 to susceptibility to autoimmunity.     

Regulation of Interferon-β by MAGI-1 and Its Interaction with Influenza A Virus NS1 Protein with ESEV PBM

The NS1 protein from avian influenza A viruses contains a PDZ binding motif (PBM) at its carboxyl terminus with the consensus sequence ESEV. The ESEV PBM confers binding to several cellular PDZ proteins, including Dlg1, MAGI-1 and Scribble. The interaction between NS1 and Scribble protects infected cells from apoptosis, while the interaction between NS1 and both Dlg1 and Scribble disrupts tight junctions. In this study, we examined the MAGI-1 protein. We made the unexpected observation that siRNA depletion of MAGI-1 activates IRF3 and induces the IFN-β promoter. We found that the ESEV NS1 protein sequesters MAGI-1 away from the plasma membrane in infected cells. Using plasmid vectors to express NS1 proteins, we observed that the ESEV PBM elicits an IFN-β induction signal as indicated by activation of IRF3 and a relative deficiency in NS1 inhibition of induction of the IFN-β promoter by dsRNA or RIG-I. Taken together, our data suggest that disruption of MAGI-1 by the ESEV PBM activates an IFN-β induction signal. During viral infection, however, induction of the IFN-β gene does not occur presumably because other anti-IFN functions dominate over the IFN-activation activity of the ESEV PBM. We postulate that the ESEV PBM's broad binding activity for PDZ proteins may allow NS1 to bind to some PDZ proteins such as MAGI-1 that confer no benefit or may even be detrimental to viral replication. However, the advantage of binding to key PDZ proteins such as Dlg1 and Scribble may dominate and therefore provide an overall benefit for the virus to encode the ESEV PBM.     

Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

α₁-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α₁-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α₁-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α₁-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α₁-antitrypsin augmentation therapy contains latent α₁-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10⁻¹⁴). We conclude that latent α₁-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α₁-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α₁-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.     

Expression of the VEGF receptor-3 in osteoarthritic chondrocytes: stimulation by interleukin-1β and association with β<Subscript>1</Subscript>-integrins

Recent studies have demonstrated enhanced expression of vascular endothelial growth factor and vascular endothelial growth factor receptor (VEGFR)-1 and -2 in chondrocytes of rheumatoid and osteoarthritic cartilage. Since expression of VEGFR-3 (Flt-4) in chondrocytes has not yet been investigated, we studied the distribution of VEGFR-3 in osteoarthritic cartilage samples by immunohistochemistry and immunoelectron microscopy. Furthermore, we looked for functional colocalization of VEGFR-3 with the signal transduction receptor ₁-integrin. Superficial osteoarthritic chondrocytes exhibited VEGFR-3 expression in the cytoplasm and on the cell membrane. Using western blotting we could demonstrate that interleukin-1 (IL-1) stimulates the expression of VEGFR-3 in chondrocytes in vitro in a dose-dependent manner. By coimmunoprecipitation assay we found a functional complex between the ₁-integrin and VEGFR-3 in IL-1-stimulated chondrocytes indicating that activated VEGFR-3 may interact with ₁-integrin and associated subcellular pathways in osteoarthritic chondrocytes. Taken together with results of previous studies showing that ₁-integrins were also associated with other surface receptors and proteins in chondrocytes that cause cartilage destruction in arthritis (for example, urokinase-type plasminogen activator receptor and matrix metalloproteinases), we can hypothesize that signal transduction by these receptor complexes via ₁-integrins may play a crucial role in pathogenesis of osteoarticular disorders. Further work needs to be done to elucidate downstream signaling events activated by these receptors.     

Genetic inhibition of solute-linked carrier 39 family transporter 1 ameliorates aβ pathology in a Drosophila model of Alzheimer's disease

The aggregation or oligomerization of amyloid-β (Aβ) peptide is thought to be the primary causative event in the pathogenesis of Alzheimer's disease (AD). Considerable in vitro evidence indicates that the aggregation/oligomerization of Aβ is promoted in the presence of Zn; however, the functional role of Zn in AD pathogenesis is still not well clarified in vivo. Zn is imported into the brain mainly through the solute-linked carrier (Slc) 39 family transporters. Using a genetically tractable Drosophila model, we found that the expression of dZip1, the orthologue of human Slc39 family transporter hZip1 in Drosophila, was altered in the brains of Aβ42-expressing flies, and Zn homeostasis could be modulated by forcible dZip1 expression changes. An array of phenotypes associated with Aβ expression could be modified by altering dZip1 expression. Importantly, Aβ42 fibril deposits as well as its SDS-soluble form were dramatically reduced upon dZip1 inhibition, resulting in less neurodegeneration, significantly improved cognitive performance, and prolonged lifespan of the Aβ42-transgenic flies. These findings suggest that zinc contributes significantly to the Aβ pathology, and manipulation of zinc transporters in AD brains may provide a novel therapeutic strategy.