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Soluble amyloid-β peptide (Aβ) oligomers, known to accumulate in Alzheimer's disease brains, target excitatory post-synaptic terminals. This is thought to trigger synapse deterioration, a mechanism possibly underlying memory loss in early stage Alzheimer's disease. A major unknown is the identity of the receptor(s) targeted by oligomers at synapses. Because oligomers have been shown to interfere with N-methyl-d-aspartate receptor (NMDAR) function and trafficking, we hypothesized that NMDARs might be required for oligomer binding to synapses. An amplicon vector was used to knock-down NMDARs in mature hippocampal neurons in culture, yielding 90% reduction in dendritic NMDAR expression and blocking neuronal oxidative stress induced by Aβ oligomers, a pathological response that has been shown to be mediated by NMDARs. Remarkably, NMDAR knock-down abolished oligomer binding to dendrites, indicating that NMDARs are required for synaptic targeting of oligomers. Nevertheless, oligomers do not appear to bind directly to NMDARs as indicated by the fact that both oligomer-attacked and non-attacked neurons exhibit similar surface levels of NMDARs. Furthermore, pre-treatment of neurons with insulin down-regulates oligomer-binding sites in the absence of a parallel reduction in surface levels of NMDARs. Establishing that NMDARs are key components of the synaptic oligomer binding complex may illuminate the development of novel approaches to prevent synapse failure triggered by Aβ oligomers.

2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.

We have investigated the folding of DM43, a homodimeric metalloproteinase inhibitor isolated from the serum of the South American opossum Didelphis marsupialis. Denaturation of the protein induced by GdnHCl (guanidine hydrochloride) was monitored by extrinsic and intrinsic fluorescence spectroscopy. While the equilibrium (un)folding of DM43 followed by tryptophan fluorescence was well described by a cooperative two-state transition, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) fluorescence measurements revealed an intensity maximum at the midpoint of the unfolding transition (2 M GdnHCl), indicating a partially folded intermediate state. We further investigated the DM43 intermediate stabilized at 2 M GdnHCl using size exclusion chromatography. This analysis revealed that the folding intermediate can be best described as partially folded DM43 monomers. Thermodynamic analysis of the GdnHCl-induced denaturation of DM43 revealed Gibbs free-energy changes of 13.57 kcal/mol for dimer dissociation and 1.86 kcal/mol for monomer unfolding, pointing to a critical role of dimerization as a determinant of the structure and stability of this protein. In addition, by using hydrostatic pressure (up to 3.5 kbar) we were able to stabilize partially folded states different from those stabilized in the presence of GdnHCl. Taken together, these results indicate that the conformational plasticity of DM43 could provide this protein with the ability to adapt its conformation to a variety of different environments and biological partners during its biological lifetime.

Synapse deterioration underlying severe memory loss in early Alzheimer's disease (AD) is thought to be caused by soluble amyloid beta (Aβ) oligomers. Mechanistically, soluble Aβ oligomers, also referred to as Aβ-derived diffusible ligands (ADDLs), act as highly specific pathogenic ligands, binding to sites localized at particular synapses. This binding triggers oxidative stress, loss of synaptic spines, and ectopic redistribution of receptors critical to plasticity and memory. We report here the existence of a protective mechanism that naturally shields synapses against ADDL-induced deterioration. Synapse pathology was investigated in mature cultures of hippocampal neurons. Before spine loss, ADDLs caused major downregulation of plasma membrane insulin receptors (IRs), via a mechanism sensitive to calcium calmodulin-dependent kinase II (CaMKII) and casein kinase II (CK2) inhibition. Most significantly, this loss of surface IRs, and ADDL-induced oxidative stress and synaptic spine deterioration, could be completely prevented by insulin. At submaximal insulin doses, protection was potentiated by rosiglitazone, an insulin-sensitizing drug used to treat type 2 diabetes. The mechanism of insulin protection entailed a marked reduction in pathogenic ADDL binding. Surprisingly, insulin failed to block ADDL binding when IR tyrosine kinase activity was inhibited; in fact, a significant increase in binding was caused by IR inhibition. The protective role of insulin thus derives from IR signaling-dependent downregulation of ADDL binding sites rather than ligand competition. The finding that synapse vulnerability to ADDLs can be mitigated by insulin suggests that bolstering brain insulin signaling, which can decline with aging and diabetes, could have significant potential to slow or deter AD pathogenesis.

Glutathione reductase (GR) carries out the enzymatic reduction of glutathione disulfide (GSSG) to its reduced form (GSH) at the expense of the reducing power of NADPH. Previous studies have shown that GR from several species is progressively inactivated in the presence of NADPH, but that the mechanism of inactivation (especially in the presence of metals) has not been fully elucidated. We have investigated the involvement of iron ions in the inactivation of yeast (Saccharomyces cerevisiae) GR in the presence of NADPH. Even in the absence of added iron, inactivation of GR was partly blocked by the iron chelators, deferoxamine and ortho-phenanthroline, suggesting the involvement of trace amounts of contaminating iron in the mechanism of inhibition. Exogenously added antioxidants including ethanol, dimethylsulfoxide and 2-deoxyribose did not protect GR against NADPH-induced inactivation, whilst addition of exogenous Fe(II) (but not Fe(III)) potentiated the inactivation. Moreover, removal of oxygen from the medium led to increased inhibition of GR, whereas pre-incubation of the Fe(II)-containing medium for 30 min under normoxic conditions prior to the addition of GR abolished the enzyme inactivation by NADPH. Under these pre-incubation conditions, Fe(II) is fully oxidized to Fe(III) within 1 min. Furthermore, GR that had been previously inactivated in the presence of Fe(II) plus NADPH could be partially reactivated by treatment with ortho-phenanthroline and deferoxamine. In contrast, Fe(III) had no effect on GR reactivation. Together, these results indicate that yeast GR is inactivated by a reductive mechanism mediated by NADPH and Fe(II). According to this mechanism, GR is diverted from its normal redox cycling by the generation of an inactive reduced enzyme form in which both the FAD and thiol groups at the active site are likely in a reduced state.

The amyloid-beta peptide (Abeta) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Abeta-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Abeta binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Abeta and are homologous to neuronal receptors putatively involved in Abeta interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Abeta with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Abeta and Fz. The results show that Abeta binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Abeta binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Abeta-binding site in Fz is homologous to cSP5 and that this is a relevant target for Abeta-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Abeta with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease.

2,4-dinitrophenol (DNP) has long been known to be toxic at high concentrations, an effect related to uncoupling of mitochondrial oxidative phosphorylation. Five years ago, however, we reported that low concentrations of DNP protect neurons against the toxicity of the amyloid-b peptide. Since then, a number of other studies have provided evidence of beneficial actions of DNP (at low concentrations), including neuroprotection against different types of insult, blockade of amyloid aggregation, stimulation of neurite outgrowth and neuronal differentiation, and even extension of lifespan in certain organisms. Some of these effects appear due to mild mitochondrial uncoupling and prevention of oxidative stress, whereas other actions are related to activation of additional intracellular signaling pathways. This study discusses the evidence supporting beneficial neuroprotective actions of DNP. DNP and other compounds with similar biological activities may be of interest in the development of novel therapeutic approaches for neurodegenerative diseases and other neurological disorders. Resumo O 2,4-dinitrofenol (DNP) tem sido conhecido há bastante tempo como tóxico em altas concentrações, um efeito relacionado a desacoplamento da fosforilação oxidativa nas mitocôndrias. Há cinco anos, entretanto, nós relatamos que baixas concentrações do DNF protegem neurônios da toxicidade do peptídeo b-amilóide. Desde então, outros estudos trouxeram evidência adicional dos efeitos benéficos do DNP (em baixas concentrações), incluindo neuroproteção contra diferentes tipos de agressão, bloqueio da agregação do amilóide, estimulação de crescimento neurítico e diferenciação neuronal, e mesmo extensão da sobrevida em alguns organismos. Alguns desses efeitos parecem ser devidos a leve desacoplamento mitocondrial e prevenção do estresse oxidativo, enquanto outras ações são relacionadas à ativação de sistemas adicionais de sinalização intracelular. Este estudo discute a as evidências que dão suporte a ações neuroprotetoras benéficas do DNP. DNP e outros compostos com atividades biológicas similares podem ser de interesse no desenvolvimento de novas abordagens terapêuticas para doenças neurodegenerativas e outros transtornos neurológicos.

The normal function of the mammalian brain is regulated by complex networks of interactions between cells and molecules, which are to a con|siderable extent dependent on mechanisms of transcriptional regulation. Disruption of such interactions by neurotoxic stimuli may lead to severe forms of dementia and to other neuropsychiatric disorders. Therefore, critical insight into mechanisms of neuronal dysfunction may be obtained by examining global patterns of gene expression in mammalian models of neurotoxicity. In this regard, the combined use of rat neuronal cultures and serial analysis of gene expression (SAGE) can be viewed as a general platform to enable the search for molecular targets involved in neurotoxic processes. Here, we discuss potential advantages of this approach, highlighting the need for generation of robust SAGE libraries from rat neuronal cultures. The availability and current limitations of bioinformatics tools for SAGE data derived from rat samples is also discussed.

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.

Oxidative stress is a major aspect of Alzheimer disease (AD) pathology. We have investigated the relationship between oxidative stress and neuronal binding of Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in brain tissue of AD patients and are considered centrally related to pathogenesis. Using hippocampal neuronal cultures, we found that ADDLs stimulated excessive formation of reactive oxygen species (ROS) through a mechanism requiring N-methyl-d-aspartate receptor (NMDA-R) activation. ADDL binding to neurons was reduced and ROS formation was completely blocked by an antibody to the extracellular domain of the NR1 subunit of NMDA-Rs. In harmony with a steric inhibition of ADDL binding by NR1 antibodies, ADDLs that were bound to detergent-extracted synaptosomal membranes co-immunoprecipitated with NMDA-R subunits. The NR1 antibody did not affect ROS formation induced by NMDA, showing that NMDA-Rs themselves remained functional. Memantine, an open channel NMDA-R antagonist prescribed as a memory-preserving drug for AD patients, completely protected against ADDL-induced ROS formation, as did other NMDA-R antagonists. Memantine and the anti-NR1 antibody also attenuated a rapid ADDL-induced increase in intraneuronal calcium, which was essential for stimulated ROS formation. These results show that ADDLs bind to or in close proximity to NMDA-Rs, triggering neuronal damage through NMDA-R-dependent calcium flux. This response provides a pathologically specific mechanism for the therapeutic action of memantine, indicates a role for ROS dysregulation in ADDL-induced cognitive impairment, and supports the unifying hypothesis that ADDLs play a central role in AD pathogenesis.

Amyloid diseases are a group of degenerative disorders characterized by cell/tissue damage caused by toxic protein aggregates. Abnormal production, processing and/or clearance of misfolded proteins or peptides may lead to their accumulation and to the formation of amyloid aggregates. Early histopathological investigation of affected organs in different amyloid diseases revealed the ubiquitous presence of fibrillar protein aggregates forming large deposits known as amyloid plaques. Further in vitro biochemical and cell biology studies, as well as studies using transgenic animal models, provided strong support to what initially seemed to be a solid concept, namely that amyloid fibrils played crucial roles in amyloid pathogenesis. However, recent studies describing tissue-specific accumulation of soluble protein oligomers and their strong impact on cell function have challenged the fibril hypothesis and led to the emergence of a new view: Fibrils are not the only toxins derived from amyloidogenic proteins and, quite possibly, not the most important ones with respect to disease etiology. Here, we review some of the recent findings and concepts in this rapidly developing field, with emphasis on the involvement of soluble oligomers of the amyloid-beta peptide in the pathogenesis of Alzheimer's disease. Recent studies suggesting that soluble oligomers from different proteins may share common mechanisms of cytotoxicity are also discussed. Increased understanding of the cellular toxic mechanisms triggered by protein oligomers may lead to the development of rational, effective treatments for amyloid disorders.

Alzheimer's disease (AD) and major depressive disorder (MDD) are highly prevalent neuropsychiatric conditions with intriguing epidemiological overlaps. Depressed patients are at increased risk of developing late-onset AD, and around one in four AD patients are co-diagnosed with MDD. Microglia are the main cellular effectors of innate immunity in the brain, and their activation is central to neuroinflammation - a ubiquitous process in brain pathology, thought to be a causal factor of both AD and MDD. Microglia serve several physiological functions, including roles in synaptic plasticity and neurogenesis, which may be disrupted in neuroinflammation. Following early work on the 'sickness behavior' of humans and other animals, microglia-derived inflammatory cytokines have been shown to produce depressive-like symptoms when administered exogenously or released in response to infection. MDD patients consistently show increased circulating levels of pro-inflammatory cytokines, and anti-inflammatory drugs show promise for treating depression. Activated microglia are abundant in the AD brain, and concentrate around senile plaques, hallmark lesions composed of aggregated amyloid-β peptide (Aβ). The Aβ burden in affected brains is regulated largely by microglial clearance, and the complex activation state of microglia may be crucial for AD progression. Intriguingly, recent reports have linked soluble Aβ oligomers, toxins that accumulate in AD brains and are thought to cause memory impairment, to increased brain cytokine production and depressive-like behavior in mice. Here, we review recent findings supporting the inflammatory hypotheses of AD and MDD, focusing on microglia as a common player and therapeutic target linking these devastating disorders.

Alzheimer's disease (AD) is a severe neurodegenerative disorder still in search of effective methods of diagnosis. Altered levels of the NMDA receptor co-agonist, d-serine, have been associated with neurological disorders, including schizophrenia and epilepsy. However, whether d-serine levels are deregulated in AD remains elusive. Here, we first measured D-serine levels in post-mortem hippocampal and cortical samples from nondemented subjects (n=8) and AD patients (n=14). We next determined d-serine levels in experimental models of AD, including wild-type rats and mice that received intracerebroventricular injections of amyloid-β oligomers, and APP/PS1 transgenic mice. Finally, we assessed d-serine levels in the cerebrospinal fluid (CSF) of 21 patients with a diagnosis of probable AD, as compared with patients with normal pressure hydrocephalus (n=9), major depression (n=9) and healthy controls (n=10), and results were contrasted with CSF amyloid-β/tau AD biomarkers. d-serine levels were higher in the hippocampus and parietal cortex of AD patients than in control subjects. Levels of both d-serine and serine racemase, the enzyme responsible for d-serine production, were elevated in experimental models of AD. Significantly, d-serine levels were higher in the CSF of probable AD patients than in non-cognitively impaired subject groups. Combining d-serine levels to the amyloid/tau index remarkably increased the sensitivity and specificity of diagnosis of probable AD in our cohort. Our results show that increased brain and CSF d-serine levels are associated with AD. CSF d-serine levels discriminated between nondemented and AD patients in our cohort and might constitute a novel candidate biomarker for early AD diagnosis.

Parkinson’s disease (PD) is an incurable progressive neurodegenerative disorder. Clinical presentation of PD stems largely from the loss of dopaminergic neurons in the nigrostriatal dopaminergic pathway, motivating experimental strategies aimed at replacing dopaminergic innervation by cellular therapy. Transplantation of dopaminergic neurons derived from embryonic stem cells significantly improves motor functions in rodent and non-human primate models of PD. However, protocols to generate dopaminergic neurons from embryonic stem cells generally meet with low efficacy and high risk of teratoma development upon transplantation. To address these issues, we have pre-treated undifferentiated mouse embryonic stem cells (mESCs) with the DNA alkylating agent mitomycin C (MMC) before transplantation. MMC treatment of cultures prevented tumor formation in a 12-week follow-up after mESCs were injected in nude mice. In 6-OH-dopamine-lesioned mice, intrastriatal injection of MMC-treated mESCs markedly improved motor function without tumor formation for as long as 15 months. Furthermore, we show that halting mitotic activity of undifferentiated mESCs induces a four-fold increase in dopamine release following in vitro differentiation. Our findings indicate that treating mESCs with mitomycin C prior to intrastriatal transplant is an effective strategy that could be further investigated as a novel alternative for treatment of Parkinson's disease.

A routine for histogram analysis of images has been written in the object-oriented, graphical development environment LabVIEW. The program converts an RGB bitmap image into an intensity-linear greyscale image according to selectable conversion coefficients. This greyscale image is subsequently analysed by plots of the intensity histogram and probability distribution of brightness, and by calculation of various parameters, including average brightness, standard deviation, variance, minimal and maximal brightness, mode, skewness and kurtosis of the histogram and the median of the probability distribution. The program allows interactive selection of specific regions of interest (ROI) in the image and definition of lower and upper threshold levels (e.g., to permit the removal of a constant background signal). The results of the analysis of multiple images can be conveniently saved and exported for plotting in other programs, which allows fast analysis of relatively large sets of image data. The program file accompanies this manuscript together with a detailed description of two application examples: The analysis of fluorescence microscopy images, specifically of tau-immunofluorescence in primary cultures of rat cortical and hippocampal neurons, and the quantification of protein bands by Western-blot. The possibilities and limitations of this kind of analysis are discussed. Program summary Title of program: HAWGC Catalogue identifier: ADXG_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/ADXG_v1_0 Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Computers: Mobile Intel Pentium III, AMD Duron Installations: No installation necessary—Executable file together with necessary files for LabVIEW Run-time engine Operating systems or monitors under which the program has been tested: WindowsME/2000/XP Programming language used: LabVIEW 7.0 Memory required to execute with typical data: ∼ 16 MB for starting and ∼ 160 MB used for loading of an image No. of bits in a word: 32 No. of processors used: 1 Has the code been vectorized or parallelized?: No No of lines in distributed program, including test data, etc.: 138 946 No. of bytes in distributed program, including test data, etc.: 15 166 675 Distribution format: tar.gz Nature of physical problem: Quantification of image data (e.g., for discrimination of molecular species in gels or fluorescent molecular probes in cell cultures) requires proprietary or complex software packages, which might not include the relevant statistical parameters or make the analysis of multiple images a tedious procedure for the general user. Method of solution: Tool for conversion of RGB bitmap image into luminance-linear image and extraction of luminance histogram, probability distribution, and statistical parameters (average brightness, standard deviation, variance, minimal and maximal brightness, mode, skewness and kurtosis of histogram and median of probability distribution) with possible selection of region of interest (ROI) and lower and upper threshold levels. Restrictions on the complexity of the problem: Does not incorporate application-specific functions (e.g., morphometric analysis) Typical running time: Seconds (depending on image size and processor speed) Unusual features of the program: None

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP(C), into a misfolded, protease-resistant form, PrP(Sc). Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106-120 from the so-called amyloidogenic core of PrP (residues 106-126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 degrees C. Under partially denaturing conditions (65 degrees C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 degrees C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 degrees C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 degrees C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP(Sc) can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106-120 of PrP and does not require other cellular factors or a PrP(Sc) template.

Proteolytic cleavage of the amyloid precursor protein (APP) by beta and gamma-secretases gives rise to the beta-amyloid peptide, considered to be a causal factor in Alzheimer's disease. Conversely, the soluble extracellular domain of APP (sAPPalpha), released upon its cleavage by alpha-secretase, plays a number of important physiological functions. Several APP fragments have been structurally characterized at atomic resolution, but the structures of intact APP and of full-length sAPPalpha have not been determined. Here, ab initio reconstruction of molecular models from high-resolution solution X-ray scattering (SAXS) data for the two main isoforms of sAPPalpha (sAPPalpha(695) and sAPPalpha(770)) provided models of sufficiently high resolution to identify distinct structural domains of APP. The fragments for which structures are known at atomic resolution were fitted within the solution models of full-length sAPPalpha, allowing localization of important functional sites (i.e. glycosylation, protease inhibitory and heparin-binding sites). Furthermore, combined results from SAXS, analytical ultracentrifugation (AUC) and size-exclusion chromatography (SEC) analysis indicate that both sAPPalpha isoforms are monomeric in solution. On the other hand, SEC, bis-ANS fluorescence, AUC and SAXS measurements showed that sAPPalpha forms a 2:1 complex with heparin. A conformational model for the sAPPalpha:heparin complex was also derived from the SAXS data. Possible implications of such complex formation for the physiological dimerization of APP and biological signaling are discussed in terms of the structural models proposed.

Protein amyloid aggregation is associated with a number of important human pathologies, but the precise mechanisms underlying the toxicity of amyloid aggregates are still incompletely understood. In this context, drugs capable of blocking or interfering with the aggregation of amyloidogenic proteins should be considered in strategies aimed at the development of novel therapeutic agents. Human lysozyme variants have been shown to form massive amyloid deposits in the livers and kidneys of individuals affected by hereditary systemic amyloidosis. Currently, there are no clinical treatments available to prevent or reverse formation of such amyloid deposits. We have recently described a number of di- and trisubstituted aromatic compounds that block the formation of soluble oligomers and amyloid fibrils of the beta-amyloid peptide (Abeta) and protect hippocampal neurons in culture from Abeta-induced toxicity. Here, we show that some of those compounds inhibit the formation and disrupt preformed amyloid fibrils from both human and hen egg white lysozyme. These results suggest that these small molecule compounds may serve as prototypes for the development of drugs for the prevention or treatment of different types of amyloidoses.

The beta-amyloid peptide (Abeta) is centrally related to the pathogenesis of Alzheimer's disease (AD) and is potently neurotoxic to central nervous system neurons. The neurotoxicity of Abeta has been partially related to the over activation of glutamatergic transmission and excitotoxicity. Taurine is a naturally occurring beta-amino acid present in the mammalian brain. Due to its safety and tolerability, taurine has been clinically used in humans in the treatment of a number of non-neurological disorders. Here, we show that micromolar doses of taurine block the neurotoxicity of Abeta to rat hippocampal and cortical neurons in culture. Moreover, taurine also rescues central neurons from the excitotoxicity induced by high concentrations of extracellular glutamate. Neuroprotection by taurine is abrogated by picrotoxin, a GABA(A) receptor antagonist. GABA and muscimol, an agonist of the GABA(A) receptor, also block neuronal death induced by Abeta in rat hippocampal and cortical neurons. These results suggest that activation of GABA(A) receptors protects neurons against Abeta toxicity in AD-affected regions of the mammalian brain and that taurine should be investigated as a novel therapeutic tool in the treatment of AD and of other neurological disorders in which excitotoxicity plays a relevant role.

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic beta-hematin (betaH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to betaH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.

Neurite outgrowth is a critical event in neuronal development, formation, and remodeling of synapses, response to injury, and regeneration. We examined the effects of 2,4-dinitrophenol (DNP), a recently described blocker of the aggregation and neurotoxicity of the beta-amyloid peptide, on neurite elongation of central neurons. Morphometric analysis of rat embryo hippocampal and cortical neuronal cultures showed that neurite outgrowth was stimulated by DNP. This effect was accompanied by increases in the neuronal levels of the microtubule-associated protein tau and of cyclic adenosine 3',5' monophosphate (cAMP). DNP also promoted cAMP accumulation, increased tau level, neurite outgrowth, and neuronal differentiation in the mouse neuroblastoma cell line N2A. We show that DNP-induced differentiation requires activation of the extracellular signal-regulated kinase (ERK). The finding that DNP promotes neuritogenesis and neuronal differentiation suggests that, in addition to its anti-amyloidogenic actions, it may be a useful lead compound in the development of novel therapeutic approaches targeting neurite dystrophy and synaptic dysfunction in neurodegenerative pathologies such as Alzheimer's disease.

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four α-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.

alpha-Tropomyosin (Tm) is a two-stranded alpha-helical coiled-coil protein, which participates in the regulation of muscle contraction. Unlike Tm purified from vertebrate muscle, recombinant Tm expressed in Escherichia coli is not acetylated at the N-terminal residue and loses the capacity to undergo head-to-tail polymerization, to bind actin and to inhibit actomyosin ATPase activity. These functions are restored by fusion of an N-terminal Ala-Ser (AS) dipeptide tail to recombinant Tm. Here, we have employed chemical (guanidine hydrochloride and urea) and physical (elevated hydrostatic pressures and low temperatures) denaturing agents to compare the structural stabilities of polymeric alanine-serine-tropomyosin (ASTm, containing the AS dipeptide) and dimeric "non-fusion" Tm (nfTm, i.e., not containing the AS dipeptide). Binding of the hydrophobic fluorescent dye bis-ANS, circular dichroism and size-exclusion chromatography were used to monitor the stabilities and state of association of both proteins under different solution conditions. Bis-ANS binding was markedly decreased at low concentrations (

Pancreatic amyloid plaques formed by the pancreatic islet amyloid polypeptide (IAPP) are present in more than 95% of type II diabetes mellitus patients, and their abundance correlates with the severity of the disease. IAPP is currently considered the most amyloidogenic peptide known, but the molecular bases of its aggregation are still incompletely understood. Detailed characterization of the mechanisms of amyloid formation requires large quantities of pure material. Thus, availability of recombinant IAPP in sufficient amounts for such studies constitutes an important step toward elucidation of the mechanisms of amyloidogenicity. Here, we report, for the first time, the successful expression, purification and characterization of the amyloidogenicity and cytotoxicity of recombinant human mature IAPP. This approach is likely to be useful for the production of other amyloidogenic peptides or proteins that are difficult to obtain by chemical synthesis.

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined “open” and “closed” conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5 A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.

The beta-amyloid peptide (A beta), the major component of the senile plaques found in the brains of Alzheimer's disease patients, is derived from proteolytic processing of a transmembrane glycoprotein known as the amyloid precursor protein (APP). Human APP exists in various isoforms, of which the major ones contain 695, 751, and 770 amino acids. Proteolytic cleavage of APP by alpha- or beta-secretases releases the extracellular soluble fragments sAPP alpha or sAPP beta, respectively. Despite the fact that sAPP alpha plays important roles in both physiological and pathological processes in the brain, very little is known about its structure and stability. We have recently presented a structural model of sAPP alpha 695 obtained from small-angle x-ray scattering measurements (Gralle, M., Botelho, M. M., Oliveira, C. L. P., Torriani, I., and Ferreira, S. T. (2002) Biophys. J. 83, 3513-3524). We now report studies on the folding and stabilities of sAPP alpha 695 and sAPP alpha 770. The combined use of intrinsic fluorescence, 4-4'-Dianilino-1,1'binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, circular dichroism, differential ultraviolet absorption, and small-angle x-ray scattering measurements of the equilibrium unfolding of sAPP alpha 695 and sAPP alpha 770 by GdnHCl and urea revealed multistep folding pathways for both sAPP alpha isoforms. Such stepwise folding processes may be related to the identification of distinct structural domains in the three-dimensional model of sAPP alpha. Furthermore, the relatively low stability of the native state of sAPP alpha suggests that conformational plasticity may play a role in allowing APP to interact with a number of distinct physiological ligands.

The major storage globulins (vicilins) of cowpea (Vigna unguiculata) and pea (Pisum sativum) seeds were purified by ammonium sulfate precipitation, and a semipurified cowpea protein isolate (CPI) was prepared by isoelectric precipitation. Some of the functional properties of these proteins, including solubility, foaming, and emulsifying capacities, were investigated and compared. The solubility of purified cowpea vicilin was reduced at pH 5.0, increasing markedly below and above this value. Pea vicilin exhibited poor solubility between pH 5.0 and pH 6.0, and CPI was little soluble in the pH range from 4.0 to 6.0. At neutral pH, the emulsifying activity indexes (EAI) of purified pea vicilin and CPI were 194 and 291 m(2)/g, respectively, which compare quite favorably to EAIs of 110 and 133 m(2)/g for casein and albumin, respectively. Remarkably, purified cowpea vicilin exhibited an EAI of 490 m(2)/g, indicating a very high emulsifying activity. Purified cowpea and pea vicilins exhibited lower foaming capacities and foam stablity indexes (FSI) than CPI. FSI values of 80 and 260 min were obtained for purified pea and cowpea vicilin, respectively, whereas a FSI value of 380 min was obtained for CPI. These results are discussed in terms of the possible utilization of purified vicilins or protein isolates from pea and cowpea in the food processing industry.

Mounting evidence from clinical, epidemiological, neuropathology and preclinical studies indicates that mechanisms similar to those leading to peripheral metabolic deregulation in metabolic disorders, such as diabetes and obesity, take place in the brains of Alzheimer's disease (AD) patients. These include pro-inflammatory mechanisms, brain metabolic stress and neuronal insulin resistance. From a molecular and cellular perspective, recent progress has been made in unveiling novel pathways that act in an orchestrated way to cause neuronal damage and cognitive decline in AD. These pathways converge to the activation of neuronal stress-related protein kinases and excessive phosphorylation of eukaryotic translation initiation factor 2α (eIF2α-P), which plays a key role in control of protein translation, culminating in synapse dysfunction and memory loss. eIF2α-P signaling thus links multiple neuronal stress pathways to impaired neuronal function and neurodegeneration. Here, we present a critical analysis of recently discovered molecular mechanisms underlying impaired brain insulin signaling and metabolic stress, with emphasis on the role of stress kinase/eIF2α-P signaling as a hub that promotes brain and behavioral impairments in AD. Because very similar mechanisms appear to operate in peripheral metabolic deregulation in T2D and in brain defects in AD, we discuss the concept that targeting defective brain insulin signaling and neuronal stress mechanisms with anti-diabetes agents may be an attractive approach to fight memory decline in AD. We conclude by raising core questions that remain to be addressed toward the development of much needed therapeutic approaches for AD.

© 2015 The Authors. Published under the terms of the CC BY 4.0 license. Alzheimer's disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD-associated Aβ oligomers (AβOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AβOs failed to induce glucose intolerance, suggesting AβOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AβOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α-P). AβOs further induced eIF2α-P and activated pro-inflammatory IKKβ/NF-κB signaling in the hypothalamus of mice and macaques. AβOs failed to trigger peripheral glucose intolerance in tumor necrosis factor-α (TNF-α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AβOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AβOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.

The amyloid precursor protein (APP) is the precursor of the beta-amyloid peptide (Abeta), which is centrally related to the genesis of Alzheimer's disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89% of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 A calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 A and 130 A, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30% of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed.

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.

1. Despite major efforts aimed at elucidating the molecular basis and physiopathology of Alzheimer's disease (AD), there is still no effective treatment available for this devastating disorder. The biological mechanisms underlying the development of AD are complex, as multiple factors appear to modulate (either positively or negatively) the progression of neurodegeneration in the brains of AD patients. Not surprisingly, a number of different therapeutic approaches aimed at distinct aspects of the disease are currently being pursued. Given its central role in the neuropathology of AD, the beta-amyloid peptide (Abeta) is the focus of many such approaches. 2. In this review, we discuss recent developments along three major lines of investigation: (i) identification and characterization of inhibitors of the enzymes involved in proteolytic processing of the amyloid precursor protein and production of Abeta; (ii) identification of the pathways involved in cerebral degradation and clearance of Abeta; and (iii) characterization of small-molecule inhibitors of amyloid aggregation that prevent cerebral amyloid deposition and neurotoxicity. 3. Significant progress has been achieved in these directions, opening up new perspectives toward the development of effective approaches for the treatment or prevention of AD.

Proteins exhibit a variety of motions ranging from amino acid side-chain rotations to the motions of large domains. Recognition of their conformational flexibility has led to the view that protein molecules undergo fast dynamic interconversion between different conformational substates. This proposal has received support from a wide variety of experimental techniques and from computer simulations of protein dynamics. More recently, studies of the subunit dissociation of oligomeric proteins induced by hydrostatic pressure have shown that the characteristic times for subunit exchange between oligomers and for interconversion between different conformations may be rather slow (hours or days). In such cases, proteins cannot be treated as an ensemble of rapidly interconverting conformational substates, but rather as a persistently heterogeneous population of different long-lived conformers. This is reminiscent of the deterministic behavior exhibited by macroscopic bodies, and may have important implications for our understanding of protein folding and biological functions. Here, we propose that the deterministic behavior of proteins may be closely related to the genesis of conformational diseases, a class of pathological conditions that includes transmissible spongiform encephalopathies, Alzheimer’s disease and other amyloidosis.

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.

Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232–239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramirez-Silva, L., Tuena de Gomez-Puyou, M., & Gomez-Puyou, A. (1993) Biochemistry32, 5332–5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 µmol·min−1·mg−1 in 40% dimethylsulfoxide, compared with 280 and 0.023 µmol· min1·mg−1 observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg2+, phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30–40% dimethylsulfoxide coincided with that of the enzyme–Mg2+–phosphenolpyruvate–K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase–Mn2+–(CH3)4N+ complex in 30–40% dimethylsulfoxide were similar to those of the pyruvate kinase–Mn2+–K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30–40% dimethylsulfoxide induces its active conformation.

The effects of glutamate receptor antagonists on the toxicity of the b-amyloid peptide (Ab1‐42) in embryonic chick retina were investigated. When used alone or in combination, the N-methyl-d-asparate antagonist, MK-801, the (^)-aamino-3hydroxyl-5-methylisoxazole-4-propinic acid/kainate antagonist, DNQX, and the metabotropic receptor antagonist, (RS)1-aminoindan-1,5-dicarboxylic acid, blocked the neurotoxicity of Ab1‐42. Aggregation of Ab1‐42 was significantly increased in the presence of acidic glutamate solutions, but not in the presence of other neurotransmitters. These results point to a dual role of glutamatergic transmission in Alzheimer’s disease (AD): (i) Ab neurotoxicity requires activation of glutamate receptors and its blockade prevents cell death; (ii) high concentrations of glutamate in the synaptic cleft indirectly enhance Ab aggregation through acidification of the medium, resulting in increased amounts of neurotoxic amyloid fibrils. These results suggest that glutamatergic neurotransmission may represent a novel target for therapeutic approaches in AD. q 2001 Elsevier Science Ireland Ltd. All rights reserved.

The crystal structures of beta-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C222(1) (lattice Y) by X-ray diffraction at 2.0 A and 1.95 A resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N.Jameson, G.B. (1999) Protein Sci. 8, 75-83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.

The stability and equilibrium unfolding of a model three-helix bundle protein, α3-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of α3-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid). The overall free-energy change for complete unfolding of α3-1, determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native state is stabilized by −1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I1). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I2), suggesting a significant contribution of hydrophobic interactions to the stability of α3-1. The free energy of stabilization of the native-like state relative to I2 is evaluated to be −2.5 kcal/mol. Bis-ANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of α3-1. The demonstration of folding intermediates of α3-1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.

a Departamento de Bioquimica Medica, Uni6ersidade Federal do Rio de Janeiro, Cidade Uni6ersitaria, 21941-590, Rio de Janeiro, RJ, Brazil b Di6isao de Quimica, Setor de Quimica Orgânica, CENPES, Petrobras, Cidade Uni6ersitaria, 21949-900, Rio de Janeiro, RJ, Brazil c Centro de Biociencias e Biotecnologia, Uni6ersidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, 28015-620, Brazil d Instituto de Biofisica Carlos Chagas Filho, Uni6ersidade Federal do Rio de Janeiro, Cidade Uni6ersitaria, 21941-590, Rio de Janeiro, RJ, Brazil

The effects of glycosylation on the stability and subunit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine epsilon-amino groups with various carbohydrates. Average modification levels of 13.4 +/- 3.0, 11.1 +/- 3.6, 7.5 +/- 4.2, and 4.7 +/- 0.3 moles of carbohydrate/mol of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046-4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (deltaG(diss)) and molar volume change (deltaV(diss)) of dissociation of vicilin subunits. For unmodified vicilin, deltaG(diss) = 18.2 kcal/mol and deltaV(diss) = -102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with deltaG(diss) of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in deltaV(diss) were found for the glucose and galactose derivatives, whereas deltaV(diss) of -128 and -135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.

ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable of hydrolyzing nucleoside di- and triphosphates. Although a number of possible physiological roles have been proposed for ATPDases, detailed studies on structure-function relationships have generally been hampered by the lack of specific inhibitors of these enzymes. We have previously characterized a Ca2+-activated ATPDase on the external surface of the tegument of Schistosoma mansoni, the etiologic agent of human schistosomiasis. In the present work, we have examined the effects of thapsigargin, a sesquiterpene lactone known as a high affinity inhibitor of sarco-endoplasmic reticulum calcium transport (SERCA) ATPase, on ATPDase activity. Whereas other lactones tested had little or no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (K(i) approximately 20 microM). Interestingly, hydrolysis of ADP was not inhibited by thapsigargin. The lack of inhibition of ATPase activity by orthovanadate, a specific inhibitor of P-type ATPases, and the inhibition of the Mg2+-stimulated ATP hydrolysis by thapsigargin ruled out the possibility that the observed inhibition of the ATPDase by thapsigargin could be due to the presence of contaminating SERCA ATPases in our preparation. Kinetic analysis indicated that a single active site in the ATPDase is responsible for hydrolysis of both ATP and ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigargin was little or not affected by changes in free Ca2+ or Mg2+ concentrations. These results suggest that interaction of thapsigargin with the S. mansoni ATPDase prevents binding of ATP or its hydrolysis at the active site, while ADP can still undergo catalysis.

The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size-exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure-induced subunit dissociation. Size-exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure-dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far-UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K+, Mg2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls growth and differentiation of hematopoietic cells. Previous reports have indicated that the mitogenic activity of GM-CSF may be modulated by the glycosidic moiety of proteoglycans associated with the membrane of stromal cells. In this work, we have performed in vitro studies of the interaction between GM-CSF and glycosaminoglycans. The addition of heparin promoted a marked blue shift in the fluorescence emission spectrum of GM-CSF as well as a 30-fold increase in the intensity of light scattering, which indicates formation of large molecular weight complexes between the two molecules. Interestingly, heparin-induced changes in the spectral properties of GM-CSF were only observed at acidic pH. The dependence on acidic pH, together with a strict dependence on glycosaminoglycan sulfation and the fact that high ionic strength destabilized the interaction, indicates that the association between GM-CSF and glycosaminoglycans is mediated by electrostatic interactions. These interactions probably involve sulfate groups in the glycosaminoglycans and positively charged histidine residues in GM-CSF. We propose that negatively charged glycolipids present on the plasma membrane of the hematopoietic and/or the stromal cell could promote an acidic microenvironment capable of triggering interaction between GM-CSF and membrane-bound proteoglycans in vivo.

Kinetics of unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM) were measured as a function of guanidine hydrochloride (GdnHCl) concentration. From the rate constants of these processes, the activation free-energy barriers (delta G++) were calculated using the Arrhenius equation. Assuming a linear dependence of delta G++ on the concentration of GdnHCl, activation energies in the absence of GdnHCl were estimated. The Gibbs free-energy change of dissociation/unfolding (delta G) was determined from GdnHCl unfolding curves in equilibrium. Using these data and the literature value for the bimolecular association rate constant of folded TIM monomers [Zabori, S., Rudolph, R., and Jaenicke, R. (1980) Z. Naturforsch. 35C, 999-1004], a model was developed that fully describes both kinetics and energetics of subunit dissociation/unfolding of TIM. Unfolded TIM monomers are susceptible to proteolytic digestion and thiol oxidation, while native TIM is resistant to both. The present model explains how the dimeric nature of TIM decreases the frequency of subunit unfolding by several orders of magnitude, thus increasing the chemical stability of the protein. Furthermore, the model also explains the recently demonstrated persistence (on a time scale of hours to days) of conformational heterogeneity of native TIM dimers [Rietveld, A. W. M., and Ferreira, S. T. (1996) Biochemistry 35, 7743-7751]. Again, it appears that the dimeric nature of TIM is essential for this behavior.

Hydrolysis of ATP or ADP catalyzed by the ATP diphosphohydrolase of Schistosoma mansoni tegument was measured in the presence of different cations. ATP diphosphohydrolase was stimulated by micromolar concentrations of either Ca2+ or Mg2+, Ca2+ producing threefold higher maximal activities than Mg2+. Kinetic studies indicated that Ca2+ and Mg2+ compete for the same binding site on the enzyme. The effect of covalent labeling of ATP diphosphohydrolase with the ATP analog fluorosulfonylbenzoyl adenosine (FSO2BzAdo) was studied. Schistosome tegument was passed through with Sephadex G-50 filtration centrifugation columns to remove endogenous nucleotides, and this was followed by labeling with FSO2BzAdo. Incubation of ATP diphosphohydrolase with 1 mM FSO2BzAdo for 1 h inhibited ATPase or ADPase activities by 60% and 50%, respectively. Addition of ATP together with FSO2BzAdo provided greater than 90% protection against FSO2BzAdo inactivation, indicating that FSO2BzAdo binds to an ATP-binding site on the ATP diphosphohydrolase. Furthermore, addition of FSO2BzAdo to a medium containing intact worms caused 30% and 50% inhibition of ATPase and ADPase activities, respectively, indicating that the ATP-binding site of diphosphohydrolase is accessible to FSO2BzAdo from the external surface of S. mansoni worms.

International audience; In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrP(c)) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APP(LDN)) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APP(LDN)-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease.