Internalization of exogenously added memapsin 2 (beta-secretase) ectodomain by cells is mediated by amyloid precursor protein

Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer's disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2(ED)) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2(ED) into endosomes with increased Abeta production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2(ED). The internalization of M2(ED) was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2(ED) internalization. These results suggest that exogenously added M2(ED) interacts with the ectodomain of APP on the cell surface leading to the internalization of M2(ED), supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the beta-secretase site of APPsw had an effect on the uptake of M2(ED). In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.     

Transgenic overexpression of protein-tyrosine phosphatase 1B in muscle causes insulin resistance, but overexpression with leukocyte antigen-related phosphatase does not additively impair insulin action

Previous studies implicate protein-tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related phosphatase (LAR) as negative regulators of insulin signaling. The expression and/or activity of PTP1B and LAR are increased in muscle of insulin-resistant rodents and humans. Overexpression of LAR selectively in muscle of transgenic mice causes whole body insulin resistance. To determine whether overexpression of PTP1B also causes insulin resistance, we generated transgenic mice overexpressing human PTP1B selectively in muscle at levels similar to those observed in insulin-resistant humans. Insulin-stimulated insulin receptor (IR) tyrosyl phosphorylation and phosphatidylinositol 3'-kinase activity were impaired by 35% and 40-60% in muscle of PTP1B-overexpressing mice compared with controls. Insulin stimulation of protein kinase C (PKC)lambda/zeta activity, which is required for glucose transport, was impaired in muscle of PTP1B-overexpressing mice compared with controls, showing that PTP1B overexpression impairs activation of these PKC isoforms. Furthermore, hyperinsulinemic-euglycemic clamp studies revealed that whole body glucose disposal and muscle glucose uptake were decreased by 40-50% in PTP1B-overexpressing mice. Overexpression of PTP1B or LAR alone in muscle caused similar impairments in insulin action; however, compound overexpression achieved by crossing PTP1B- and LAR-overexpressing mice was not additive. Antibodies against specific IR phosphotyrosines indicated overlapping sites of action of PTP1B and LAR. Thus, overexpression of PTP1B in vivo impairs insulin sensitivity, suggesting that overexpression of PTP1B in muscle of obese humans and rodents may contribute to their insulin resistance. Lack of additive impairment of insulin signaling by PTP1B and LAR suggests that these PTPs have overlapping actions in causing insulin resistance in vivo.     

Evidence for heterodimers of 2,4,5-trichlorophenol on planar lipid layers. A FTIR-ATR investigation

Trichlorophenols are weak acids of high hydrophobicity and are able to transport protons across the mitochondrial membrane. Thus the proton motive force is dissipated and the ATP production decreased. In situ Fourier Transform Infrared-Attenuated Total Reflection (FTIR-ATR) experiments with 2,4,5-trichlorophenol (TCP) adsorbed to model membranes resulted in good evidence for the formation of the TCP-heterodimer. Two surfaces were examined: a dipalmitoyl phosphatidic acid (DPPA) monolayer and a planar DPPA/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. TCP was adsorbed from 1 to 3 mM solutions at pH 6.0 to the lipid layers leading to surface layers at the water/lipid interface. Difference spectra showed an effect on DPPA acyl chains even when it was covered with POPC. Time-resolved measurements revealed two distinct adsorption processes, which were assigned to TCP and its deprotonated anion (phenoxide), respectively. For DPPA/POPC bilayers, the adsorption of TCP was faster than that of its phenoxide, whereas adsorption of both species to DPPA monolayers proceeded with similar velocity. In both cases, phenoxide formation at the membrane was found to be delayed with respect to phenol adsorption. Phenoxide and phenol were retained after replacing the TCP solution with buffer. For the retained species, we estimated a phenol/phenoxide molar ratio of 1 at pH 6.0 (pKa=6.94 for TCP), demonstrating strong evidence for heterodimer formation.     

In situ FTIR ATR spectroscopic study of the interaction of immobilized human tumor necrosis factor-alpha with a monoclonal antibody in aqueous environment

By in situ FTIR ATR measurements, the antibody (AB) recognition of human tumor necrosis factor-alpha (TNFalpha) immobilized on the Ge surface of a multiple internal reflection element (MIRE) was investigated. The experiments were performed in aqueous environment in a flow-through cell. After immobilization of TNFalpha on the Ge-MIRE by direct adsorption from aqueous solution, the immobilisate reached stability after about 1 h under flow-through conditions. The remaining sites of the Ge surface were saturated by bovine serum albumin (BSA) in order to prevent unspecific binding of anti-TNFalpha AB which was then added. The obtained FTIR ATR spectra were shown to result exclusively from AB specifically interacting with TNFalpha, since the absence of immunoglobulin binding to BSA adsorbed to the Ge MIRE was verified by a reference experiment. Finally, the stability of all adsorbed protein immobilisates was monitored under flow-through conditions for 10.5 h. The TNFalpha-AB complex showed a decrease of 7.4%, whereas the BSA adsorbate remained stable. IR measurements were performed with polarized light in order to study orientational effects of the immobilized proteins. The dichroic ratios and surface concentrations of all used proteins are available after quantitative analysis of the amide II bands.     

Crystallographic analysis of the Pseudomonas aeruginosa strain K122-4 monomeric pilin reveals a conserved receptor-binding architecture

Adherence of pathogens to host cells is critical for the initiation of infection and is thus an attractive target for anti-infective therapeutics and vaccines. In the opportunistic human pathogen Pseudomonas aeruginosa, host-cell adherence is achieved predominantly by type IV pili. Analysis of several clinical strains of P. aeruginosa reveals poor sequence conservation between pilin genes, including the residues in the receptor-binding site. Interestingly, the receptor-binding sites appear to retain a conserved surface epitope because all Pseudomonas type IV pili recognize the same receptor on the host cell and cross-reactive antibodies specific for the receptor-binding site exist. Here, we present the crystallographic analysis of two crystal forms of truncated pilin from P. aeruginosa strain K122-4 (DeltaK122-4) at 1.54 and 1.8 A resolution, respectively. The DeltaK122-4 structure is compared to other crystallographically determined type IV pilin structures and an NMR structure of DeltaK122-4 pilin. A comparison with the structure of the highly divergent P. aeruginosa strain K (DeltaPAK) pilin indicates that the receptor-binding loop in both pilins forms a shallow depression with a surface that is formed by main-chain atoms. Conservation of this putative binding site is independent of the sequence as long as the main-chain conformation is conserved and could therefore explain the shared receptor specificity and antibody cross reactivity of highly divergent Pseudomonas type IV pilins.     

O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress

Myriad nuclear and cytoplasmic proteins in metazoans are modified on Ser and Thr residues by the monosaccharide O-linked beta-N-acetylglucosamine (O-GlcNAc). The rapid and dynamic change in O-GlcNAc levels in response to extracellular stimuli, morphogens, the cell cycle and development suggests a key role for O-GlcNAc in signal transduction pathways. Modulation of O-GlcNAc levels has profound effects on the functioning of cells, in part mediated through a complex interplay between O-GlcNAc and O-phosphate. In many well-studied proteins, the O-GlcNAc modification and phosphorylation are reciprocal. That is, they occur on different subsets of the protein population, as the site of attachment occurs on the same or adjacent Ser/Thr residues. Recently, O-GlcNAc has been implicated in the etiology of type II diabetes, the regulation of stress response pathways, and in the regulation of the proteasome.     

Role of protein kinase C isoforms in the regulation of interleukin-13-induced 15-lipoxygenase gene expression in human monocytes

We reported previously that interleukin-13 (IL-13) induces tyrosine phosphorylation/activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6 in primary human monocytes. We recently revealed that p38 MAPK-mediated serine phosphorylation of both Stat1 and Stat3 is required for the induction of 15-lipoxygenase (15-LO) expression by IL-13. In this study, we present data indicating that another serine/threonine kinase, PKCdelta, is also required for IL-13-induced 15-LO expression. PKCdelta, a member of the novel protein kinase C (PKC) subclass, was rapidly phosphorylated and activated upon exposure to IL-13. Treatment of cells with rottlerin, a PKCdelta inhibitor, blocked IL-13-induced 15-LO mRNA and protein expression, whereas Go6976, an inhibitor of the conventional PKC subclass, had no inhibitory effects. Down-regulation of cellular PKCdelta protein levels by PKCdelta-specific antisense oligodeoxyribonucleotides also inhibited 15-LO expression markedly. IL-13-induced 15-LO expression resulted in significant inhibition of synthesis of the potent chemotactic factor leukotriene B4, and that process was reversed by rottlerin, presumably through the blockage of PKCdelta-dependent 15-LO expression. Furthermore, our data demonstrate that IL-13-mediated activation of PKCdelta and p38 MAPK are independent pathways, because inhibition of one kinase activity had no effect on the other, suggesting that the two pathways act in parallel to regulate the downstream targets necessary for 15-LO expression. Inhibition of PKCdelta activation by rottlerin also markedly attenuated IL-13-induced Stat3 DNA binding activity. Our findings indicate that PKCdelta plays an important role in regulating IL-13-induced 15-LO expression in human monocytes and subsequently modulates the inflammatory responses mediated by 15-LO products.     

Interaction with transducin depletes metarhodopsin III: a regulated retinal storage in visual signal transduction?

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (G(t)). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of G(t) with Meta III, which was previously considered inactive. G(t) thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.     

Cyclic adenosine monophosphate regulates calcium channels in the plasma membrane of Arabidopsis leaf guard and mesophyll cells

The effect of cAMP on Ca(2+)-permeable channels from Arabidopsis thaliana leaf guard cell and mesophyll cell protoplasts was studied using the patch clamp technique. In the whole cell configuration, dibutyryl cAMP was found to increase a hyperpolarization-activated Ba(2+) conductance (I(Ba)). The increase of I(Ba) was blocked by the addition of GdCl(3). In excised outside-out patches, the addition of dibutyryl cAMP consistently activated a channel with particularly fast gating kinetics. Current/voltage analyses indicated a single channel conductance of approximately 13 picosiemens. In patches where we measured some channel activity prior to cAMP application, the data suggest that cAMP enhances channel activity without affecting the single channel conductance. The cAMP activation of these channels was reversible upon washout. The results obtained with excised patches indicate that the cAMP-activated I(Ba) seen in the whole cell configuration could be explained by a direct effect of cAMP on the Ca(2+) channel itself or a close entity to the channel. This work represents the first demonstration using patch clamp analysis of the presence in plant cell membranes of an ion channel directly activated by cAMP.