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The cblC disease is an inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by methylmalonic aciduria and homocystinuria. The clinical consequences of this disease are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The illness is caused by mutations in the gene codifying for MMACHC, a 282aa protein that transports and transforms the different Cbl forms. Here we present data on the structural properties of the truncated protein p.R132X resulting from the c.394C > T mutation that, along with c.271dupA and c.331C > T, is among the most common mutations in cblC. Although missing part of the Cbl binding domain, p.R132X is associated to late-onset symptoms and, therefore, it is supposed to retain residual function. However, to our knowledge structuralfunctional studies on c.394C > T mutant aimed at verifying this hypothesis are still lacking. By using a biophysical approach including Circular Dichroism, fluorescence, Small Angle X-ray Scattering, and Molecular Dynamics, we show that the mutant protein MMACHC-R132X retains secondary structure elements and remains compact in solution, partly preserving its binding affinity for Cbl. Insights on the fragile stability of MMACHCR132X-Cbl are provided.

The understanding of amyloid beta-peptide (A beta) interactions with cellular membranes is a crucial molecular challenge against Alzheimer's disease. Indeed, A beta prefibrillar oligomeric intermediates are believed to be the most toxic species, able to induce cellular damages directly by membrane damage. We present a neutron-scattering study on the interaction of large unilamellar vesicles (LUV), as cell membrane models, with both freshly dissolved A beta and early toxic prefibrillar oligomers, intermediate states in the amyloid pathway. In addition, we explore the effect of coincubating the A beta peptide with the chaperonin Hsp60, which is known to strongly interact with it in its aggregation pattern. In fact, the interaction of the LUV with coincubated A beta/Hsp60, right after mixing and after following the aggregation protocol leading to the toxic intermediates in the absence of Hsp60, is studied. Neutron spin echo experiments show that the interaction with both freshly dissolved and aggregate A beta species brings about an increase in membrane stiffness, whereas the presence even very low amounts of Hsp60 (ratio A beta/Hsp60 = 25:1) maintains unaltered the elastic properties of the membrane bilayer. A coherent interpretation of these results, related to previous literature, can be based on the ability of the chaperonin to interfere with A beta aggregation, by the specific recognition of the A beta-reactive transient species. In this framework, our results strongly suggest that early in a freshly dissolved A beta solution are present some species able to modify the bilayer dynamics, and the chaperonin plays the role of an assistant in such stochastic "misfolding events", avoiding the insult on the membrane as well as the onset of the aggregation cascade.

Early impairments in Alzheimer’s disease are related to neuronal membrane damage. Aberrant Amyloid β (Aβ) species and specific membrane components both play a role in promoting aggregation, deposition and signal dysfunction. Ganglioside GM1, present with cholesterol and sphingomyelin in lipid rafts, seems to be able to interact with Aβ and initiate Aβ aggregation on membrane. In this chapter, several studies are reviewed, highlighting the role of different lipids and in particular GM1 in the interaction between Aβ and membrane systems. Results concerning GM1-Aβ interaction obtained by means of different experimental methods are reported and discussed. Small angle X-ray scattering is used to study the structure of the unilamellar vesicle bilayer. Mixing Aβ species with vesicles clearly showed an effect on the bilayer structure, but only for GM1-containing liposomes. The elastic properties of GM1-containing membrane have been studied by Neutron Spin Echo, showing that the presence of Aβ species brings about an increase in the membrane rigidity, as reflected by the measured elastic modulus. This effect can be hindered by the presence of molecular chaperones, like Hsp60. This chaperonin has proven to be a strong inhibitor of the amyloidogenesis process, due to its dose dependent ability to neutralize oligomeric metastable species needed to onset the fibril growth cascade. The fact that the incubation of Aβ in the presence of Hsp60 leads to a loss of the Aβ effects on the membrane dynamical properties suggests that the aberrant interaction between Aβ and membranes is important in these specific intermediate Aβ species.

Alzheimer's disease is a chronic neurodegenerative disease characterized by the accumulation of pathological aggregates of amyloid beta peptide. Many efforts have been focused on understanding peptide aggregation pathways and on identification of molecules able to inhibit aggregation in order to find an effective therapy. As a result, interest in neuroprotective proteins, such as molecular chaperones, has increased as their normal function is to assist in protein folding or to facilitate the disaggregation and/or clearance of abnormal aggregate proteins. Using biophysical techniques, we evaluated the effects of two chaperones, human Hsp60 and bacterial GroEL, on the fibrillogenesis of Aβ

Alzheimer's disease is a chronic neurodegenerative disease characterized by the accumulation of pathological aggregates of amyloid beta peptide. Many efforts have been focused on understanding peptide aggregation pathways and on identification of molecules able to inhibit aggregation in order to find an effective therapy. As a result, interest in neuroprotective proteins, such as molecular chaperones, has increased as their normal function is to assist in protein folding or to facilitate the disaggregation and/or clearance of abnormal aggregate proteins. Using biophysical techniques, we evaluated the effects of two chaperones, human Hsp60 and bacterial GroEL, on the fibrillogenesis of Aβ1-42. Both chaperonins interfere with Aβ1-42 aggregation, but the effect of Hsp60 is more significant and correlates with its more pronounced flexibility and stronger interaction with ANS, an indicator of hydrophobic regions. Dose-dependent ThT fluorescence kinetics and SAXS experiments reveal that Hsp60 does not change the nature of the molecular processes stochastically leading to the formation of seeds, but strongly delays them by recognition of hydrophobic sites of some peptide species crucial for triggering amyloid formation. Hsp60 reduces the initial chaotic heterogeneity of Aβ1-42 sample at high concentration regimes. The understanding of chaperone action in counteracting pathological aggregation could be a starting point for potential new therapeutic strategies against neurodegenerative diseases.

BACKGROUND: Molecular chaperones are a very special class of proteins that play essential roles in many cellular processes like folding, targeting and transport of proteins. Moreover, recent evidence indicates that chaperones can act as potentially strong suppressor agents in Alzheimer's disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aβ peptide and in vivo studies reveal that treatment with specific chaperones or their overexpression can ameliorate some distinct pathological signs characterizing AD. METHODS: Here we investigate using a biophysical approach (fluorescence, circular dichroism (CD), transmission electron (TEM) and atomic force (AFM) microscopy, size exclusion chromatography (SEC)) the effect of the human chaperonin Hsp60 on Aβ fibrillogenesis. RESULTS: We found that Hsp60 powerfully inhibits Aβ amyloid aggregation, by closing molecular pathways leading to peptide fibrillogenesis. CONCLUSIONS: We observe that Hsp60 inhibits Aβ aggregation through a more complex mechanism than a simple folding chaperone action. The action is specifically directed toward the early oligomeric species behaving as aggregation seeds for on-pathway amyloid fibrillogenesis. GENERAL SIGNIFICANCE: Understanding the specificity of the molecular interactions of Hsp60 with amyloid Aβ peptide allowed us to emphasize the important aspects to be taken into consideration when considering the recent promising therapeutic strategies for neurodegeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

International audience; Curcumin is a natural polyphenol able to bind the amyloid beta peptide, which is related to Alzheimer's disease, and modify its self-assembly pathway. This paper focuses on a multi-disciplinary study that starts from the design of curcumin-like compounds with the key chemical features required for inhibiting amyloid beta aggregation, and reports the effects of these compounds on the in vitro aggregation of amyloid beta peptides. Chemoinformatic screening was performed through the calculation of molecular descriptors that were able to highlight the drug-like profile, followed by docking studies with an amyloid beta peptide fibril. The computational design underlined two different scaffolds that were easily synthesized in good yields. In vitro experiments, ranging from fluorescence spectroscopy and confocal microscopy up to small angle X-ray scattering, provided evidence that the synthesized compounds are able to modify the aggregation pattern of amyloid beta peptides both in the secondary structures, and in terms of the overall structure dimensions. The cytotoxic potential of the synthesized compounds was finally tested in vitro with a model neuronal cell line (LAN5). The overall view of this study suggests new concepts and potential difficulties in the design of novel drugs against diverse amyloidoses, including Alzheimer's disease

In the large class of molecules that maintain protein homeostasis, called molecular chaperones, chaperonins constitute a subclass that specifically assist the correct folding of newly synthesized proteins. Among them, Hsp60 is composed of a double heptameric ring structure with a large central cavity where the unfolded protein binds via hydrophobic interactions and is supported, in this function, by the co-chaperonin Hsp10. Hsp60 is typically located in the mitochondria, but in some pathological situations, such as cancers and chronic inflammatory diseases, Hsp60 accumulates in the cytoplasm. In these cases, cytoplasmatic Hsp60 is a mixture of mitochondrial Hsp60 secreted from mitochondria upon stress, and its precursor, called naive Hsp60, never entered into the organella. The difference between the naive and mitochondrial Hsp60s resides in the absence of the mitochondrial import signal (MIS) in the mitochondrial form, but information on their different structure and stability is still lacking. We present here a study on the stability against a chemical denaturant, of the different cytoplasmic Hsp60 species. By combining Circular Dichroism and Small Angle X-ray Scattering as experimental biophysical techniques to investigate Hsp60, we find that naive and mitochondrial Hsp60 (mtHsp60) forms differ in their stability. Furthermore, specific responses from the two forms are discussed in terms of the biological environment they are working in, thus opening new questions on their biological function.

A range of debilitating human diseases is known to be associated with the formation of stable highly organized protein aggregates known as amyloid fibrils. The early prefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca2+ that lead to apoptotic or necrotic cell death. However, specific signaling pathways that underlie amyloid pathogenicity remain still unclear. This work aimed to clarify cell impairment induced by amyloid aggregated. To this end, we used a combined proteomic and one-dimensional 1H-NMR approach on NIH-3T3 cells exposed to prefibrillar aggregates from the amyloidogenic apomyoglobin mutant W7FW14F. The results indicated that cell exposure to prefibrillar aggregates induces changes of the expression level of proteins and metabolites involved in stress response. The majority of the proteins and metabolites detected are reported to be related to oxidative stress, perturbation of calcium homeostasis, apoptotic and survival pathways, and membrane damage. In conclusion, the combined proteomic and 1H-NMR metabonomic approach, described in this study, contributes to unveil novel proteins and metabolites that could take part to the general framework of the toxicity induced by amyloid aggregates. These findings offer new insights in therapeutic and diagnostic opportunities. J. Cell. Physiol. 228: 1359–1367, 2013. © 2012 Wiley Periodicals, Inc.

The inhibition of the aggregation in protein solutions is currently a subject of great interest in many research fields, from the study of protein-misfolding related diseases to pharmaceutics, biotechnology, and food science. α(s1)-Casein, one of the four types of caseins, which are the largest protein component of bovine milk, has been found to hinder the aggregation process of several proteins, including the amyloid β-peptide, involved in Alzheimer's disease. To shed light into the mechanisms by which casein exerts this chaperon-like protective action, we studied its effect on the different steps of the aggregation process of concanavalin A, by means of both static and dynamic light scattering, thioflavin T and ANS fluorescence, circular dichroism, and atomic force microscopy. Our results show that casein has a poor effect on the first step of the process leading to the formation of amyloid-like structures. On the contrary, it has a marked effect on the second step of the process, ascribable to clusters condensation and compaction, up to the formation of very large aggregates. Such an effect requires a molar ratio of casein larger than that necessary to inhibit the fibrillogenesis of the amyloid β-peptide, thus, suggesting a different mechanism of interaction of casein, depending on both conformational properties and relative size of the aggregating molecules.

Molecular chaperones are a class of proteins able to prevent non-specific aggregation of mitochondrial proteins and to promote their proper folding. Among them, human Hsp60 is currently considered as a ubiquitous molecule with multiple roles both in maintaining health conditions and as a trigger of several diseases. Of particular interest is its role in neurodegenerative disorders since it is able to inhibit the formation of amyloid fibrils.Hsp60 structure was considered, until recent years, analogue to the one of its bacterial homolog GroEL, one of the most investigated chaperones, whose crystallographic structure is a homo-tetradecamer, made up of two seven member rings. On the contrary, it has been recently demonstrated by some of us that Hsp60 shows an equilibrium of tetradecamers and heptamers in solution.We present the results of combined investigations including complementary approaches such as molecular dynamics (MD), circular dichroism (CD) and small-angle X-ray scattering (SAXS) concerning structure, self-organization, and stability of naive Hsp60 in solution in comparison with bacterial GroEL. We analyzed the structural differences between the two proteins in solution, evidencing their different oligomeric equilibria, too. Experiments at increasing concentrations of guanidinium hydrochloride provided further information on the stability of the oligomeric species, on the dissociation of tetradecamers into heptamers and monomers, until the unfolding. The unfolding free energy was obtained, proving that GroEL is more stable in respect to Hsp60.

Curcumin is a natural polyphenol able to bind the amyloid beta peptide, which is related to Alzheimer's disease, and modify its self-assembly pathway. This paper focuses on a multi-disciplinary study that starts from the design of curcumin-like compounds with the key chemical features required for inhibiting amyloid beta aggregation, and reports the effects of these compounds on the in vitro aggregation of amyloid beta peptides. Chemoinformatic screening was performed through the calculation of molecular descriptors that were able to highlight the drug-like profile, followed by docking studies with an amyloid beta peptide fibril. The computational design underlined two different scaffolds that were easily synthesized in good yields. In vitro experiments, ranging from fluorescence spectroscopy and confocal microscopy up to small angle X-ray scattering, provided evidence that the synthesized compounds are able to modify the aggregation pattern of amyloid beta peptides both in the secondary structures, and in terms of the overall structure dimensions. The cytotoxic potential of the synthesized compounds was finally tested in vitro with a model neuronal cell line (LAN5). The overall view of this study suggests new concepts and potential difficulties in the design of novel drugs against diverse amyloidoses, including Alzheimer's disease.

The onset of Alzheimer disease (AD) is influenced by several risk factors comprising diabetes. Within this context, antidiabetic drugs, including metformin, are investigated for their effect on AD. We report that in the C57B6/J mice, metformin is delivered to the brain where activates AMP-activated kinase (AMPK), its molecular target. This drug affects the levels of β- secretase (BACE1) and β-amyloid precursor protein (APP), promoting processing and aggregation of β-amyloid (Aβ), mainly in the cortex region. Moreover, metformin induces mitochondrial dysfunction and cell death by affecting the level and conformation of Translocase of the Outer Membrane 40 (TOM40), voltage-dependent anion-selective channels 1 (VDAC1) and hexokinase I (HKI), proteins involved in mitochondrial transport of molecules, including Aβ. By using biophysical techniques we found that metformin is able to directly interact with Aβ influencing its aggregation kinetics and features. These findings indicate that metformin induces different adverse effects, leading to an overall increase of the risk of AD onset.

""Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W -> F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.""

The potent immunomodulatory, anti-inflammatory and procoagulant properties of protein no. 4 secreted from the rat seminal vesicle epithelium (SV-IV) have previously been found to be modulated by a supramolecular monomer-trimer equilibrium. More structural details that integrate experimental data into a predictive framework have recently been reported. Unfortunately, homology modelling and fold-recognition strategies were not successful in creating a theoretical model of the structural organization of SV-IV. It was inferred that the global structure of SV-IV is not similar to that of any protein of known three-dimensional structure. Reversing the classical approach to the sequence-structure-function paradigm, in this paper we report novel information obtained by comparing the physicochemical parameters of SV-IV with two datasets composed of intrinsically unfolded and ideally globular proteins. In addition, we analyse the SV-IV sequence by several publicly available disorder-oriented predictors. Overall, disorder predictions and a re-examination of existing experimental data strongly suggest that SV-IV needs large plasticity to efficiently interact with the different targets that characterize its multifaceted biological function, and should therefore be better classified as an intrinsically disordered protein.

Heat shock protein 60kDa is a molecular chaperone (GroEL human homolog) that assists protein folding in mitochondria (mtHsp60). It is synthesized in the cell cytoplasm as a higher molecular weight precursor form (p-mtHsp60) containing a N-terminal targeting sequence, that is cleaved after import into the mitochondrial matrix [1, 2].It has been established, and demonstrated by various techniques, Hsp60 can accumulate in the cytosol, in various pathological conditions (i.e., cancer and chronic inflammatory diseases). The cytosolical Hsp60 accumulation mechanism may occur with or without mitochondrial release concomitantly, so that in the cytosol the two types of 60 kDa chaperonin proteins, (mtHsp60 and its precursor naive form, p-mtHsp60) could coexist [3]. It has been recently observed that in a wide range of concentration, the precursor naive Hsp60 is able to assemble in both heptamers and tetradecamers [4].Key questions still unanswered pertain to the differences in structure-function features that might exist between the well-studied prokaryotic GroEL and the largely unexplored eukaryotic Hsp60 proteins. Moreover, studies on human Hsp60 structure and oligomeric state in vitro could help to validate its role in physiological or pathological cases. In order to pursue this goal, we investigated the (dis)assembly and thermal stability of mtHsp60, p-mtHsp60 and GroEL in vitro, by means of Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC). Complementar Circular Dicroism (CD) measurements were done to follow the change in the secondary structure due to unfolding.[1] Ellis RJ, 2007, Adv Exp Med Biol., 594: 1-13.[2] Jonathan D, 2000, JHC., 48(1): 45-56.[3] D. Chandra, 2007, J. Biol. Chem., 282: 31289-31301.[4] Vilasi S. et al, 2014, Plos One., 9(5): e97657.

This review will analyze growing evidence suggesting a convergence between two major areas of research: Alzheimer’s disease (AD) and chaperonopathies. While AD is a widely recognized medical, public health, and social problem, the chaperonopathies have not yet been acknowledged as a related burden of similar magnitude. However, recent evidence collectively indicates that such possibility exists in that AD, or at least some forms of it, may indeed be a chaperonopathy. The importance of considering this possibility cannot be overemphasized since it provides a novel point of view to examine AD and potentially suggests new therapeutic avenues. In this review, we focus on the mitochondrial chaperone HSP60 and discuss some of its biological, molecular, and pathological facets as they pertain to AD. We further illustrate how HSP60 may be an etiologic-pathogenic factor in AD and, as such, it could become a novel, effective therapeutic target. This possibility is discussed both in the light of negative chaperonotherapy, namely the development of means to inhibit HSP60 in the event its excessive activity is a disease-promoting event in AD, as well as positive chaperonotherapy, that is boosting its activity if, on the other hand, it is demonstrated that HSP60 insufficiency is a key feature of AD with such pathological consequences as causing mitochondrial dysfunction.

In this paper we present the control system we implemented for test the control of a suspended mass using monolithic tunable folded pendulum sensors in the band 0.01 - 100 Hz. The main goal of this experiment is to test the performances of monolithic sensors as main acceleration sensors for application in inertial damping control systems. The main advantages of these sensors with respect to the others are the sensitivity (better than 10-11 m/s2/√(Hz) in the band 0.1 ÷ 10Hz), the large band (0.01 ÷ 100Hz), the tuneability of the resonance frequency (necessary to match the sensors to the suspension mechanics) and the high sensitive integrated laser optical readout, consisting of an optical lever and an interferometer, and a very good immunity to environmental noises. Preliminary results are presented and discussed in this paper together with the planned further developments and improvements of monolithic sensors.

Molecular chaperones play essential and several roles in many cellular processes, including protein folding, targeting, transport, and are essential in fighting the consequences of protein misfolding and aggregation or enhancing disaggregation of toxic aggregates by clearance mechanisms. In particular, recent studies have attributed to some chaperones the capability to directly interact with amyloid aggregation-prone species of peptide involved in Alzheimer Disease in solution and/or suppress the associated toxicity. Among them are the principal heat-shock proteins (HSPs), the small HSPs, but also molecules the exhibit marked chaperon like activity, like milk caseins or BRICHOS domains. There are several mechanisms by which chaperones exert their protective action, many of which involve protein disordered regions. However, in many cases, the scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms, that result crucially important when considering how to screen for and characterize potential inhibitors of amyloidosis. Here we show how a human chaperonin, Hsp60, and intrinsically disorder colloids with marked chaperon-like activity, the milk caseins, can specifically influence Aβ40 fibrillogenesis. Inhibition studies are correlated with investigation on structural, self organization and stability properties of the chaperones under study performed by a battery of biophysical methods and aimed to understand what are molecular determinants responsible for their inhibitory action.

Beta-amyloid (1-40) is one of the two most abundant species of amyloid-beta peptides present as fibrils in the extracellular senile plaques in the brain of Alzheimer's patients. Recently, the molecular aggregates constituting the early stage of fibril formation, i.e., oligomers and protofibrils, have been investigated as the main responsible for amyloid-beta cytotoxic effect. The molecular mechanism leading to neurodegeneration is still under debate, and it is common opinion that it may reside in the interaction between amyloid species and the neural membrane. In this investigation Atomic Force Microscopy and spectroscopy have been used to understand how structural (and mechanical) properties of POPC/POPS lipid bilayers, simulating the phospholipid composition and negative net charge of neuritic cell membranes, are influenced by the interaction with A beta(1-40), in different stages of the peptide aggregation. Substantial differences in the damage caused to the lipid bilayers have been observed, confirming the toxic effect exerted especially by A beta(1-40) prefibrillar oligomers. (c) 2013 Elsevier B.V. All rights reserved.

Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH.

The paper describes the application of a monolithic folded pendulum (FP) as a tiltmeter for geophysical applications, developed at the University of Salerno. Both the theoretical model and the experimental results of a tunable mechanical monolithic FP tiltmeter prototype are presented and discussed. Some of the most important characteristics, like the possibility of tuning its resonance frequency to values as low as 70mHz and its measured resolution of ≈ 0.1 nrad at 100mHz, are detailed. Among the scientific results, earth tilt tides have been already observed with a monolithic FP tiltmeter prototype.

Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aβ-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid β-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis.

This paper describes a new implementation of monolithic horizontal sensor, developed at the University of Salerno, based on the Folded Pendulum architecture, configurable both as seismometer and as accelerometer. The large low-frequency band (10-6 ÷ 10Hz), the high sensitivity ( in the band 0.1 ÷ 10 Hz) and the high quality factor in air (Q > 1500) are largely better than all the previous Folded Pendulum implementations. Moreover its monolithic implementation of the whole mechanics, coupled with a full tunability of its resonance frequency (70 mHz ÷ 1.2 Hz) obtained with a specially designed calibration procedure and with an integrated laser optical readout, guarantees both compactness, robustness and immunity to environmental noises. This makes this sensor suitable for a large number of scientific applications, also in high vacuum and cryogeny. Applications of this sensor are already started in the field of geophysics, including the study of seismic and newtonian noise for characterization of suitable sites for future underground interferometric detectors of gravitational waves.

The description of the fibrillogenesis pathway and the identification of "on-pathway" or "off-pathway" intermediates are key issues in amyloid research as they are concerned with the mechanism for onset of certain diseases and with therapeutic treatments. Recent results on the fibril formation process revealed an unexpected complexity both in the number and in the types of species involved, but the early aggregation events are still largely unknown, mainly because of their experimental inaccessibility. To provide information on the early stage events of self-assembly of an amyloidogenic protein, during the so-called lag phase, stopped-flow time-resolved small angle x-ray scattering (SAXS) experiments were performed. Using a global fitting analysis, the structural and aggregation properties of the apomyoglobin W7FW14F mutant, which is monomeric and partly folded at acidic pH but forms amyloid fibrils after neutralization, were derived from the first few milliseconds onward. SAXS data indicated that the first aggregates appear in less than 20 ms after the pH jump to neutrality and further revealed the simultaneous presence of diverse species. In particular, worm-like unstructured monomers, very large assemblies, and elongated particles were detected, and their structural features and relative concentrations were derived as a function of time on the basis of our model. The final results show that, during the lag phase, early assembling occurs due to the presence of transient monomeric species very prone to association and through successive competing aggregation and rearrangement processes leading to coexisting on-pathway and off-pathway transient species.

This paper describes an optimized version of the mechanical version of the monolithic tunable folded pendulum, developed at the University of Salerno, configurable both as seismometer and, in a force-feedback configuration, as accelerometer. Typical application of the sensors are in the field of geophysics, including the study of seismic and newtonian noise for characterization of suitable sites for underground interferometer for gravitational waves detection. The sensor, shaped with precision machining and electric-discharge-machining, like the previous version, is a very compact instrument, very sensitive in the low-frequency seismic noise band, with a very good immunity to environmental noises. Important characteristics are the tunability of the resonance frequency and the integrated laser optical readout, consisting of an optical lever and an interferometer. The theoretical sensitivity curves, largely improved due to a new design of the pendulum arms and of the electronics, are in a very good agreement with the measurements. The very large measurement band (10 -6 ± 10Hz) is couple to a very good sensitivity (10 -12 m/√ Hz in the band 0.1 ± 10 Hz ), as seismometer. Prototypes of monolithic seismometers are already operational in selected sites around the world both to acquire seismic data for scientific analysis of seismic noise and to collect all the useful information to understand their performances in the very low frequency band (f < 1mHz). The results of the monolithic sensor as accelerometer (force feed-back configuration) are also presented and discussed. Particular relevance has their sensitivity that is better than 10 -11 m/s 2 /√Hz in the band 0.1 ± 10Hz. Finally, hypotheses are made on further developments and improvements of monolithic sensors.

Glycosaminoglycans (GAGs) are frequently associated with amyloid deposits in most amyloid diseases, and there is evidence to support their active role in amyloid fibril formation. The purpose of this study was to obtain structural insight into GAG-protein interactions and to better elucidate the molecular mechanism underlying the effect of GAGs on the amyloid aggregation process and on the related cytotoxicity. To this aim, using Fourier transform infrared and circular diochroism spectroscopy, electron microscopy and thioflavin fluorescence dye we examined the effect of heparin and other GAGs on the fibrillogenesis and cytotoxicity of aggregates formed by the amyloidogenic W7FW14 apomyoglobin mutant. Although this protein is unrelated to human disease, it is a suitable model for in vitro studies because it forms amyloid-like fibrils under physiological conditions of pH and temperature. Heparin strongly stimulated aggregation into amyloid fibrils, thereby abolishing the lag-phase normally detected following the kinetics of the process, and increasing the yield of fibrils. Moreover, the protein aggregates were harmless when assayed for cytotoxicity in vitro. Neutral or positive compounds did not affect the aggregation rate, and the early aggregates were highly cytotoxic. The surprising result that heparin induced amyloid fibril formation in wild-type apomyoglobin and in the partially folded intermediate state of the mutant, i.e., proteins that normally do not show any tendency to aggregate, suggested that the interaction of heparin with apomyoglobin is highly specific because of the presence, in protein turn regions, of consensus sequences consisting of alternating basic and non-basic residues that are capable of binding heparin molecules. Our data suggest that GAGs play a dual role in amyloidosis, namely, they promote beneficial fibril formation, but they also function as pathological chaperones by inducing amyloid aggregation.

MatCAKE (www.cake.unisa.it) is a toolbox for integrating 2D NMR spectra by the CAKE (Monte CArlo peaK volume Estimation)1 algorithm within the Matlab environment (www.mathworks.com). Quantitative information from multidimensional NMR experiments can be obtained by peak volume integration. The standard procedure (selection of a region around the chosen peak and addition of all values) is often biased by poor peak definition because of peak overlap. CAKE is a simple algorithm designed for volume integration of (partially) overlapping peaks. Assuming the axial symmetry of two-dimensional NMR peaks, as it occurs in NOESY and TOCSY when Lorentz-Gauss transformation of the signals is carried out, CAKE estimates the peak volume by multiplying a volume fraction by a factor R. It represents a proportionality ratio between the total and the fractional volume, which is identified as a slice in an exposed region of the overlapping peaks. The volume fraction is obtained via Monte Carlo Hit-or-Miss technique, which proved to be the most efficient because of the small region and the limited number of points within the selected area. Due to the large number of software packages available for processing nuclear magnetic resonance data, MatCAKE is designed just for implementing the new CAKE algorithm. In MatCAKe, in fact, only already processed bidimensional spectra are imported and, at the moment, the only volume integration (by CAKE and by the most simple standard procedure) are allowed. MatCAKE is a free software at disposal for the scientific community and can be obtained on line at the web address cake.unisa.it.

This paper describes a new mechanical architecture of the monolithic tunable folded pendulum, developed at the University of Salerno, configurable both as seismometer and, in a force-feedback configuration, as accelerometer. Typical applications are the remote monitoring of seismic and newtonian noises for geophysical applications. This sensor, shaped with precision machining and electric-discharge-machining, like the previous versions, is a very compact instrument, very sensitive in the low-frequency seismic noise band, with a very good immunity to environmental noises. Important characteristics are the large band (10-6 - 10mHz), the tunability of the resonance frequency and the laser optical readout, that integrates an optical lever and a laser interferometer. The theoretical sensitivity curves, largely improved due to a new design of the pendulum arms and of the electronics, are in a very good agreement with the measurements. In particular, a very good sensitivity (10-12 m/Hz1/2) has been obtained in the band 0.1 - 10 Hz). Prototypes of monolithic seismometers are already operational in selected sites around the world both to remotely acquire data for scientific analysis of seismic noise and to collect all the useful information to understand their performances in the very low frequency band. The results of the monolithic sensor as accelerometer (force feed-back configuration) are also presented and discussed.

In this paper we present and discuss the scientific data recorded along one month of data taking of two mechanical monolithic horizontal sensor prototypes located in a blind-ended (side) tunnel 2000 ft deep in the Homestake mine (South Dakota, USA), chosen to host the Deep Underground Science and Engineering Laboratory (DUSEL). The main goal of this test is to provide preliminary data to characterize the Homestake site in the frequency band 10^(-4) ÷ 30Hz and to estimate the level of Newtonian noise, information necessary to understand the feasibility of underground gravitational-wave interferometers sensitive at 1Hz and below. The recorded scientific data and all the technical information obtained with this test are very useful also to understand the performances of the monolithic sensors in the very low frequency band (10^(-6) ÷ 10^(-3) Hz) and to organize an experiment for the low frequency seismic characterization of the Homestake site with monolithic sensors positioned at different levels and orientations.

This paper describes a new mechanical version of the monolithic tunable folded pendulum, developed at the University of Salerno, configurable both as seismometer and, in a force-feedback configuration, as accelerometer. Typical applications of the sensors are in the field of geophysics, including the study of seismic and newtonian noise for characterization of suitable sites for underground interferometers for gravitational waves detection. The sensor, shaped with precision machining and electric-discharge machining, is a very compact instrument, very sensitive in the low-frequency seismic noise band, with a very good immunity to environmental noises. Important characteristics are the tunable resonance frequency and the integrated laser optical readout, consisting of an optical lever and/or an interferometer. The theoretical sensitivity curves, largely improved due to a new design of the pendulum arms and of the electronics, are in a very good agreement with the measurements. The results of the monolithic sensor as accelerometer (force feed-back configuration) are also presented and discussed.

Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in 'toxic' and 'immunogenic' classes, which elicit an innate immune response and an HLA-mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31-43 (P31-43) and the 56-68 (P56-68) A-gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane-mimetic medium. We show that P31-43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56-68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD.

The potent immunomodulatory, anti-inflammatory and procoagulant properties of the protein no. 4 secreted from the rat seminal vesicle epithelium (SV-IV) have been previously found to be modulated by a supramolecular monomer-trimer equilibrium. More structural details that integrate experimental data into a predictive framework have recently been reported. Unfortunately, homology modelling and fold-recognition strategies were not successful in creating a theoretical model of the structural organization of SV-IV. It was inferred that the global structure of SV-IV is not similar to any protein of known three-dimensional structure. Reversing the classical approach to the sequence-structure-function paradigm, in this paper we report on novel information obtained by comparing physicochemical parameters of SV-IV with two datasets made of intrinsically unfolded and ideally globular proteins. In addition, we have analysed the SV-IV sequence by several publicly available disorder-oriented predictors. Overall, disorder predictions and a re-examination of existing experimental data strongly suggest that SV-IV needs large plasticity to efficiently interact with the different targets that characterize its multifaceted biological function and should be therefore better classified as an intrinsically disordered protein.

Trehalose, a disaccharide present in many nonmammalian species, protects cells against various environmental stresses. Trehalose has recently been shown to decrease aggregate formation and toxicity in cell models and to alleviate amyloid-induced diseases. The aim of our study was to use two amyloid-forming proteins, i.e., W7FW14F apomyoglobin and insulin, as model systems to elucidate the molecular mechanism by which trehalose affects the amyloid aggregation process and to investigate further its therapeutic potential. Protein aggregation was examined by far-UV circular dichroism, UV absorption, thioflavin T fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, atomic force microscopy, and Fourier transform infrared spectroscopy. Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. We found that trehalose does not inhibit protein aggregation but acts at different stages of the fibrillization process depending on the protein model used. In fact, trehalose dose-dependently inhibited fibril formation in the W7FW14F apomyoglobin model and increased the lag phase in the insulin model. In both cases, trehalose caused accumulation of toxic oligomeric species. The results suggest that trehalose may favor or inhibit the formation of "on-pathway" or "off-pathway" oligomeric intermediates depending on the nature of the aggregating protein.

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The double W/F replacement renders apomyoglobin highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions. In this work we analyze the early stage of W7FW14F apomyoglobin aggregation following the time dependence of the process by far-UV CD, Fourier-transform infrared (FTIR) spectroscopy, and heme-binding properties. The results show that the aggregation of W7FW14F apomyoglobin starts from a native-like globin state able to bind the prosthetic group with spectroscopic properties similar to those observed for wild-type apoprotein. Nevertheless, it rapidly aggregates, forming amyloid fibrils. However, when the prosthetic group is added before the beginning of aggregation, amyloid fibrillization is inhibited, although the aggregation process is not prevented. Moreover, the apomyoglobin aggregates formed in these conditions are not cytotoxic differently from what is observed for all amyloidogenic proteins. These results open new insights into the relationship between the structure adopted by the protein into the aggregates and their ability to trigger the impairment of cell viability.

In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert α-helical to β sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.

A significant number of fatal diseases are classified as protein deposition disorders, in which a normally soluble protein is deposited in an insoluble amyloid form. It has been reported that tetracycline exhibits anti-amyloidogenic activity by inhibiting aggregate formation and disaggregating preformed fibrils. In this work, we examined the effect induced by the presence of tetracycline on the fibrillogenesis and cytotoxicity of the amyloid-forming apomyoglobin mutant W7FW14F. Like other amyloid-forming proteins, early prefibrillar aggregates formed by this protein are highly cytotoxic, whereas insoluble mature fibrils are not. The effect induced by tetracycline on the fibrillation process has been examined by atomic force microscopy, light scattering, DPH staining, and thioflavin T fluorescence. The cytotoxicity of the amyloid aggregates was estimated by measuring cell viability using MTT assay. The results show that tetracycline acts as anti-aggregating agent, which inhibits the fibril elongation process but not the early aggregation steps leading to the formation of soluble oligomeric aggregates. Thus, this inhibition keeps the W7FW14F mutant in a prefibrillar, highly cytotoxic state. In this respect, a careful usage of tetracycline as fibril inhibitor is indicated.

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naive, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naive cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naive Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.

A range of debilitating human diseases is associated with the formation of stable, highly organised, protein aggregates, the amyloid fibrils. Substantial evidence suggests that Prefibrillar Oligomeric Aggregates (PFAs), preceding mature fibrils formation, are the crucial species in the onset of the neuronal degeneration even if with mechanisms to be further cleared. In this work, we show how 1H-NMR cell spectral analysis methods can prove to be very effective tools to clear the PFAs amyloid cytotoxicity mechanisms. Following the same method shown by Vilasi, we apply a new 1H-NMR analysis algorithm to identify the metabolites significantly varied in cells incubated with toxic oligomers from the amyloidogenic W7FW14F mutant of apomyoglobin. Our main aim is to confirm the results obtained by Vilasi et al., normalising a set of different data spectra with the new PRICONA algorithm here described, thus contributing to strengthen the general framework of metabolites and proteins involved in cellular amyloid toxicity.