β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ(1-40), defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ(1-40) is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.     

Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.     

The Mechanism of Membrane Disruption by Cytotoxic Amyloid Oligomers Formed by Prion Protein(106–126) Is Dependent on Bilayer Composition

The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106–126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106–126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.     

Aggregation Modulators Interfere with Membrane Interactions of?β2-Microglobulin Fibrils

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer’s disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of β₂-microglobulin (β₂m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of β₂m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.     

Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK

β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET¹⁷⁸P), but not rat β-arrestin-1 (PER¹⁷⁷P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK¹⁷⁸P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species.     

Calcium Currents Are Enhanced by ��2��-1 Lacking Its Membrane Anchor

The accessory α₂δ subunits of voltage-gated calcium channels are membrane-anchored proteins, which are highly glycosylated, possess multiple disulfide bonds, and are post-translationally cleaved into α₂ and δ. All α₂δ subunits have a C-terminal hydrophobic, potentially trans-membrane domain and were described as type I transmembrane proteins, but we found evidence that they can be glycosylphosphatidylinositol-anchored. To probe further the function of membrane anchoring in α₂δ subunits, we have now examined the properties of α₂δ-1 constructs truncated at their putative glycosylphosphatidylinositol anchor site, located before the C-terminal hydrophobic domain (α₂δ-1ΔC-term). We find that the majority of α₂δ-1ΔC-term is soluble and secreted into the medium, but unexpectedly, some of the protein remains associated with detergent-resistant membranes, also termed lipid rafts, and is extrinsically bound to the plasma membrane. Furthermore, heterologous co-expression of α₂δ-1ΔC-term with Ca_V2.1/β1b results in a substantial enhancement of the calcium channel currents, albeit less than that produced by wild-type α₂δ-1. These results call into question the role of membrane anchoring of α₂δ subunits for calcium current enhancement.     

β-Alanine Release from the Adult and Developing Hippocampus Is Enhanced by Ionotropic Glutamate Receptor Agonists and Cell-Damaging Conditions

The release of the inhibitory amino acid -alanine was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The release was enhanced by -alanine itself and the structural analogs taurine and -aminobutyrate. It was dependent on Na⁺, but independent of Ca²⁺ in both mature and immature hippocampus, being thus mostly mediated by uptake carriers operating in an outward direction. The release was potentiated in the developing mice, but not affected in the adults, by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and tetrazolylglycine in a receptor-mediated manner. Cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals, greatly enhanced -alanine release at both ages, but more markedly in the adults. The great amounts of -alanine, together with the inhibitory amino acids taurine and -aminobutyrate, released simultaneously with the excitatory amino acids in the hippocampus may constitute an important protective mechanism against excitotoxicity, which leads to neuronal death.     

Stability of Transmembrane Amyloid β-Peptide and Membrane Integrity Tested by Molecular Modeling of Site-Specific Aβ 42 Mutations

Interactions of the amyloid β-protein (Aβ) with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer’s disease. Natural mutations in Aβ₄₂, such as the Arctic mutation (E22G) have been shown to increase Aβ₄₂ aggregation and neurotoxicity, leading to the early-onset of Alzheimer’s disease. A correlation between the propensity of Aβ₄₂ to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ₄₂ peptide it was further revealed that the aggregation of different Aβ₄₂ mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ₄₂ mutation and peptide–membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ₄₂ variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants) as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ₄₂ stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ₄₂ peptide and a decreased stability for D23G compared to wild-type Aβ₄₂, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.     

Stacked Sets of Parallel, In-register ��-Strands of ��2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

β₂-microglobulin (β₂m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β₂m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β₂m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β₂m consist of about six β₂m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.     

The Monomer-Seed Interaction Mechanism in the Formation of the β2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques

Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.     

Internalization of Coxsackievirus A9 Is Mediated by β2-Microglobulin,Dynamin, and Arf6 but Not by Caveolin-1 or Clathrin

Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize αV integrins, particularly αVβ6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin β6 subunit inhibited virus proliferation, confirming that αVβ6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of β2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin αVβ6, β2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.Coxsackievirus A9 (CAV9), a member of the human enterovirus B species in the family Picornaviridae, is a significant human pathogen. It causes infections of the central nervous system, myocarditis, and respiratory diseases and may occasionally cause fatal generalized infections in newborns (6, 22, 26). The CAV9 particle is about 30 nm in diameter and consists of a naked capsid with an icosahedral symmetry, surrounding a positive-sense RNA genome of approximately 7,400 nucleotides (30). The capsid is made up of 60 copies of each of the four proteins VP1 to VP4 and interacts with cell surface integrins during the early stages of infection via arginine-glycine-aspartic acid (RGD) motif that resides in the C terminus of the VP1 protein (11). While CAV9 binds to both integrin αVβ3 and αVβ6 in vitro (53, 61), our recent data show that integrin αVβ6 is the primary receptor of the virus (29).Viruses can utilize several endocytic pathways to enter mammalian cells: macropinocytosis and clathrin-mediated, caveolin-mediated, and clathrin- and caveolin-independent routes (14, 40-41, 50). Recent studies have shown that some of these pathways differ only slightly from each other, and certain endocytic components can participate in more than just one pathway (35, 41, 55). Most of the research carried out on enterovirus endocytosis has been done with echovirus 1 (EV1), coxsackievirus B3 (CBV3), and poliovirus (PV). Recently, Karjalainen et al. showed that EV1 enters SAOS cells via tubulovesicular structures in a dynamin-independent manner that resembles fluid-phase endocytosis and macropinocytosis and that at later stages of infection is targeted to caveosomes (33). EV1 entry to CV-1 cells, on the other hand, was shown to be strictly dynamin dependent (49). PV is endocytosed to HeLa cells by a rapid clathrin- and caveolin-independent pathway, whereas in brain microvascular endothelial cells it uses slower, caveolin- and dynamin-dependent endocytosis (4, 7, 17). CBV3 enters HeLa cells by clathrin-mediated endocytosis (13) and polarized epithelial CaCo-2 cells by a process that combines features of caveolar endocytosis and macropinocytosis (16, 18). Foot-and-mouth-disease virus (FMDV), a member of the Aphthovirus genus of the family Picornaviridae, binds to several αV-integrins, including αVβ6, and is internalized through the clathrin-mediated pathway (5, 19, 31). In the light of these examples, it is evident that enterovirus internalization to human cells is a complex phenomenon wherein a virus may use different mechanisms to enter different cell types.In the CAV9 infection cycle, the steps that follow the initial attachment of the virus to the cell surface integrins are still poorly characterized. An early electron microscopic work by Hecker et al. has shown that single CAV9 particles enter monkey kidney cells in vesicles, which then occasionally fuse and form larger structures (28). Interestingly, they found that most internalized virus particles became eventually trapped in large vacuoles, presumably lysosomes, where they were confined without proceeding to capsid uncoating and RNA release. More recently, a number of cell surface molecules have been proposed to contribute to CAV9 internalization. A subunit of major histocompatibility complex class I (MHC-I) complex, β2-microglobulin (β2M), has been shown to be essential for the infection of several picornaviruses, including CAV9, and it is supposed to have a postattachment role (12, 59, 61). In addition, heat shock 70-kDa protein 5 (HSPA5 protein, also known as glucose-regulated protein 78-kDa, or GRP78) has been suggested to function as a coreceptor for the virus and to mediate CAV9 infection by its interaction with β2M on the cell surface (57). CAV9 entry has been proposed to occur through lipid microdomains, where a number of signaling events takes place (58).The aim of this study was to elucidate the internalization mechanism of CAV9 in A549 human lung carcinoma cells. We used chemical inhibitors, RNA interference (RNAi) silencing, and the expression of dominant negative constructs combined to virus infectivity assays and confocal imaging to examine which cellular molecules are involved in the entry process. The results indicate that CAV9 internalization is dependent on integrin αVβ6, β2M, dynamin 2, and Arf6 (ADP-ribosylation factor 6) but not clathrin or caveolin-1.     

Enhanced α-ketoglutaric acid production and recovery in Yarrowia lipolytica yeast by effective pH controlling

The replacement of chemical synthesis by environmentally friendly energy-efficient technologies for production of valuable metabolites is a principal strategy of developing biotechnological industry all over the world. In the present study, we develop a method for α-ketoglutaric acid (KGA) production from rapeseed oil with the use of Yarrowia lipolytica yeast. Sixty strains of Y. lipolytica yeasts were tested for their ability to produce KGA, and the strain Y. lipolytica 212 (Y. lipolytica VKM Y-2412) was selected as a promising KGA producer. Using a three-stage pH controlling, in which pH was 4.5 in the growth phase, then since 72 to 144 h, pH was maintained at 3.5 and in the later phase of acid production, the titration by KOH was switch off, selected strain produced 106.5 g l^?1 of KGA with mass yield of 0.95 g g^?1. KGA in the form of monopotassium salt was isolated from the culture broth and purified. The isolation procedure involved separation of biomass, extraction of residual triglycerides, filtrate bleaching, and acidification with mineral acid (to pH 2.8–3.4), concentration, precipitation of mineral salts, and crystallization of the product. The purity of KGA isolated from the culture filtrate reached 99.1 %.     

αvβ3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors

We report that αvβ3 integrin strongly affects the innate immune response in epithelial cells. αvβ3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvβ3 integrin. The boosting was exerted specifically by αvβ3 integrin but not by αvβ6 or αvβ8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response.     

Cholesterol Depletion by MβCD Enhances Cell Membrane Tension and Its Variations-Reducing Integrity

Cholesterol depletion by methyl-β-cyclodextrin (MβCD) remodels the plasma membrane’s mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it’s unclear if MβCD alters membrane tension or only enhances membrane-cytoskeleton interactions—and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MβCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations’ frequency spectrum. This helps us conclude that MβCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MβCD also has a strong impact on the cell membrane’s tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters—an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

Hepatic Gluconeogenesis Is Enhanced by Phosphatidic Acid Which Remains Uninhibited by Insulin in Lipodystrophic Agpat2?/? Mice

In this study we examined the role of phosphatidic acid (PA) in hepatic glucose production (HGP) and development of hepatic insulin resistance in mice that lack 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2). Liver lysophosphatidic acid and PA levels were increased ∼2- and ∼5-fold, respectively, in male Agpat2^−/− mice compared with wild type mice. In the absence of AGPAT2, the liver can synthesize PAs by activating diacylglycerol kinase or phospholipase D, both of which were elevated in the livers of Agpat2^−/− mice. We found that PAs C16:0/18:1 and C18:1/20:4 enhanced HGP in primary WT hepatocytes, an effect that was further enhanced in primary hepatocytes from Agpat2^−/− mice. Lysophosphatidic acids C16:0 and C18:1 failed to increase HGP in primary hepatocytes. The activation of HGP was accompanied by an up-regulation of the key gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This activation was suppressed by insulin in the WT primary hepatocytes but not in the Agpat2^−/− primary hepatocytes. Thus, the lack of normal insulin signaling in Agpat2^−/− livers allows unrestricted PA-induced gluconeogenesis significantly contributing to the development of hyperglycemia in these mice.     

The Heat Shock Response Is Modulated by and Interferes with Toxic Effects of Scrapie Prion Protein and Amyloid β

The heat shock response (HSR) is an evolutionarily conserved pathway designed to maintain proteostasis and to ameliorate toxic effects of aberrant protein folding. We have studied the modulation of the HSR by the scrapie prion protein (PrP^Sc) and amyloid β peptide (Aβ) and investigated whether an activated HSR or the ectopic expression of individual chaperones can interfere with PrP^Sc- or Aβ-induced toxicity. First, we observed different effects on the HSR under acute or chronic exposure of cells to PrP^Sc or Aβ. In chronically exposed cells the threshold to mount a stress response was significantly increased, evidenced by a decreased expression of Hsp72 after stress, whereas an acute exposure lowered the threshold for stress-induced expression of Hsp72. Next, we employed models of PrP^Sc- and Aβ-induced toxicity to demonstrate that the induction of the HSR ameliorates the toxic effects of both PrP^Sc and Aβ. Similarly, the ectopic expression of cytosolic Hsp72 or the extracellular chaperone clusterin protected against PrP^Sc- or Aβ-induced toxicity. However, toxic signaling induced by a pathogenic PrP mutant located at the plasma membrane was prevented by an activated HSR or Hsp72 but not by clusterin, indicating a distinct mode of action of this extracellular chaperone. Our study supports the notion that different pathological protein conformers mediate toxic effects via similar cellular pathways and emphasizes the possibility to exploit the heat shock response therapeutically.