Role of hippocalcin in mediating Aβ toxicity

Alzheimer's disease (AD) is the most common cause of dementia, and amyloid-β (Aβ) plaques and tau-containing tangles are its histopathological hallmark lesions. These do not occur at random; rather, the neurodegenerative process is stereotyped in that it is initiated in the entorhinal cortex and hippocampal formation. Interestingly, it is the latter brain area where the calcium-sensing enzyme hippocalcin is highly expressed. Because calcium deregulation is a well-established pathomechanism in AD, we aimed to address the putative role of hippocalcin in human AD brain and transgenic mouse models. We found that hippocalcin levels are increased in human AD brain and in Aβ plaque-forming APP23 transgenic mice compared to controls. To determine the role of hippocalcin in Aβ toxicity, we treated primary cultures derived from hippocalcin knockout (HC KO) mice with Aβ and found them to be more susceptible to Aβ toxicity than controls. Likewise, treatment with either thapsigargin or ionomycin, both known to deregulate intracellular calcium levels, caused an increased toxicity in hippocampal neurons from HC KO mice compared to wild-type. We found further that mitochondrial complex I activity increased from 3 to 6months in hippocampal mitochondria from wild-type and HC KO mice, but that the latter exhibited a significantly stronger aging phenotype than wild-type. Aβ treatment induced significant toxicity on hippocampal mitochondria from HC KO mice already at 3months of age, while wild-type mitochondria were spared. Our data suggest that hippocalcin has a neuroprotective role in AD, presenting it as a putative biomarker.     

Construction and characterization of two versions of bifunctional EGFP–sTRAIL fusion proteins

The extracellular portion (amino acids 95–281 or 114–281) of the human tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) was genetically linked to the C terminus of the fluoresce-enhanced green fluorescent protein variant (EGFP) to generate two versions of EGFP–sTRAIL fusion proteins, designated EGFP–sTR95 and EGFP–sTR114, respectively. The two versions of EGFP–sTRAIL fusion proteins both induce extensive apoptosis in lymphoid as well as nonlymphoid tumor cell lines. In addition, the two versions of fusion proteins retain similar fluorescence spectra to those of EGFP and have shown the specific binding to TRAIL receptor-positive cells; thus, the stained cells could be analyzed with flow cytometry. Hence, the two versions of fusion proteins represent a readily obtainable source of biologically active sTRAIL that may prove useful in exploit fully the characteristics of both the soluble TRAIL and its receptor system.     

Suppression of Aβ toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity

The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aβ toxicity.     

Molecular Plasticity Regulates Oligomerization and Cytotoxicity of the Multipeptide-length Amyloid-β Peptide Pool

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aβ_1–38 and Aβ_1–40 at the cost of longer Aβ peptides. Here, we consider the aggregation of Aβ_1–38 and Aβ_1–43 in addition to Aβ_1–40 and Aβ_1–42, in particular their behavior in mixtures representing the complex in vivo Aβ pool. We demonstrate that Aβ_1–38 and Aβ_1–43 aggregate similar to Aβ_1–40 and Aβ_1–42, respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aβ, Aβ_1–38 and Aβ_1–43 significantly affect the behaviors of Aβ_1–40 and Aβ_1–42. The short timescale conformational flexibility of Aβ_1–38 is suggested to be responsible for enhancing toxicity of Aβ_1–40 while exerting a cyto-protective effect on Aβ_1–42. Our results indicate that the complex in vivo Aβ peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.     

A recombinant human interleukin-1β protects adriamycin-induced toxicity

Pretreatment with recombinant human interleukin-1β (IL-1) protected normal BALB/c mice from the lethal effect adriamycin (ADM) of related to dose and frequency of administration. Posttreatment with IL-1 failed to protect. Neutrophil and platelet counts after the administration of ADM (16mg/kg) did not differ between the group with and that without IL-1 pretreatment, whereas lipid peroxide levels in the heart were reduced in the group pretreated with IL-1. It appears that the chemoprotection mechanism of IL-1 lies in the prevention of cardiotoxicity due to ADM-induced free radicals.     

Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ～2% of the productive human TCRβ CDR3 sequences.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

Construction and characterization of different fusion proteins between cellulases and β-glucosidase to improve glucose production and thermostability

A β-glucosidase from Clostridium cellulovorans (CcBG) was fused with one of three different types of cellulases from Clostridium thermocellum, including a cellulosomal endoglucanase CelD (CtCD), a cellulosomal exoglucanase CBHA (CtCA) and a non-cellulosomal endoglucanase Cel9I (CtC9I). Six bifunctional enzymes were constructed with either β-glucosidase or cellulase in the upstream. CtCD-CcBG showed the favorable specific activities on phosphoric acid swollen cellulose (PASC), an amorphous cellulose, with more glucose production (2 folds) and less cellobiose accumulation (3 folds) when compared with mixture of the single enzymes. Moreover, CtCD-CcBG had significantly improved thermal stability with a melting temperature (T_m) of 10.9 °C higher than that of CcBG (54.5 °C) based on the CD unfolding experiments. This bifunctional enzyme is thus useful in industrial application to convert cellulose to glucose.     

Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP

The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and β2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and β2M that are suitable for further studies.     

An optimized ultrasonication protocol for bacterial cell disruption and recovery of β-galactosidase fusion proteins

Summary In this work, we have developed and optimized an ultrasonication protocol for Escherichia coli recombinant cells, adapted to laboratory-scale release of -galactosidase fusion proteins. After a single sonication treatment of 15 minutes, about 30% of recombinant protein present in the sample remains still associated to cellular debris, and it can not be removed by increasing the sonication time. After a clarification step a second sonication treatment of the resuspended cell debris again releases only a 70% of the remaining product. Therefore, the application of two short, consecutive sonication treatments permits a global recovery yield of about 90%. The use of a new disruption buffer to stabilize -galactosidase allows the fusion proteins to maintain the active form throughout the process.     

A complex interplay of Gβ and Gγ proteins regulates plant growth and defence traits in the allotetraploid Brassica juncea

Plant Molecular Biology - Gene expression analysis coupled with in-planta studies showed that specific Gβγ combination regulates plant growth and defence traits in the allotetraploid...     

Copper and Zinc Mediated Oligomerisation of Aβ Peptides

The accumulation of senile plaques composed primarily of aggregated amyloid β-peptide (Aβ), is the major characteristic of Alzheimer’s disease. Many studies correlate plaque accumulation and the presence of metal ions, particularly copper and zinc. The metal binding sites of the amyloid Aβ peptide of Alzheimer’s disease are located in the N-terminal region of the full-length peptide. In this work, the interactions with metals of a model peptide comprising the first 16 amino acid residues of the amyloid Aβ peptide, Aβ(1–16), were studied. The effect of Cu²⁺ and Zn²⁺ binding to Aβ(1–16) on peptide structure and oligomerisation are reported. The results of ESI-MS, gel filtration chromatography and NMR spectroscopy demonstrated formation of oligomeric complexes of the peptide in the presence of the metal ions and revealed the stoichiometry of Cu²⁺ and Zn²⁺ binding to Aβ(1–16), with Cu²⁺ showing a higher affinity for binding the peptide than Zn²⁺.     

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide

Background

The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer''s disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species.

Methodology/Principal Findings

To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species.

Conclusions

Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.     

Recognition of nuclear targeting signals by Karyopherin-β proteins

The Karyopherin-β family of nuclear transport factors mediates the majority of nucleocytoplasmic transport. Although each of the 19 Karyopherin-βs transports unique sets of cargos, only three classes of nuclear localization and export signals, or NLSs and NESs, have been characterized. The short basic classical-NLS was first discovered in the 1980s and their karyopherin-bound structures were first reported more than 10 years ago. More recently, structural and biophysical studies of Karyopherin-β2-cargo complexes led to definition of the complex and diverse PY-NLS. Structural knowledge of the leucine-rich NES is finally available more than 10 years after the discovery of its recognition by the exportin CRM1. We review recent findings relating to how these three classes of nuclear targeting signals are recognized by their Karyopherin-β nuclear transport factors.     

Identification and partial characterization of Rhizopus nigricans Gβ proteins and their expression in the presence of progesterone

The mammalian steroid hormone progesterone actuates a signalling pathway in the zygomycete Rhizopus nigricans which includes heterotrimeric G proteins. To investigate the possibility that the Gβ subunit of these proteins is involved in the signalling, a cDNA library from R. nigricans exposed to progesterone was prepared and a sequence coding for a Gβ subunit was searched for. Using degenerate primers, two sequences, RnGPB1 and RnGPB2, were identified that exhibited a high degree of identity with those for Gβ from other filamentous fungi, but not from yeast. The presence of more than one Gβ subunit is very rare among the fungi, and it has been to date reported only for Rhizopus oryzae. We have shown that progesterone increases the expression of RnGPB1, but has no influence on the expression of RnGPB2. Therefore, our studies imply the involvement of Gβ subunit 1 in the response of R. nigricans to progesterone. Moreover, the Gβ subunit is subjected to endogenous ADP-ribosylation in the presence of NAD, which could be important in some, as yet unknown, cell process. Article from a special issue on steroids and microorganisms.     

Interaction with functional membrane proteins—A common mechanism of toxicity for lipophilic environmental chemicals?

• 1.1. The effects of several phenols, anilines and aliphatic alcohols on yeast plasma membrane H⁺-ATPase and purine transport system as well as on Na⁺, K⁺-ATPase and adenosine uptake by Chinese hamster ovary cells (CHO) were investigated.
• 2.2. In all cases an inhibition was observed, which could be correlated with the octanol/water partition coefficients of the substances tested, thus making quantitative structure-activity predictions possible.
• 3.3. The observed effects correlated well with the influence of the chemicals on cell growth.
• 4.4. The results suggest a common mechanism of toxicity by the action of hydrophobic xenobiotics on biomembranes.

     

What is the role of SNARE proteins in membrane fusion?

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion "machine." Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.