Amanda Penco - Academia.edu (original) (raw)

Papers by Amanda Penco

Research paper thumbnail of The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli

PLoS ONE, 2012

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) s... more Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291D). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291D strongly reduced the growth rate in the early stages (2-4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291D on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular b-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.

Research paper thumbnail of The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli

PLoS ONE, 2012

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) s... more Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291D). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291D strongly reduced the growth rate in the early stages (2-4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291D on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular b-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.

Research paper thumbnail of Wild Type Beta-2 Microglobulin and DE Loop Mutants Display a Common Fibrillar Architecture

PLOS ONE, 2015

Beta-2 microglobulin (β2m) is the protein responsible for a pathologic condition known as dialysi... more Beta-2 microglobulin (β2m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining β2m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity. However, it remains unclear whether the aggregates formed by wild type (wt) β2m and by the DE loop variants are of the same kind, or whether the mutations open new aggregation pathways. In order to address this question, fibrillar samples of wt and mutated β2m variants have been analysed by means of atomic force microscopy and infrared spectroscopy. The data here reported indicate that the DE loop mutants form aggregates with morphology and structural organisation very similar to the wt protein. Therefore, the main effect of β2m DE loop mutations is proposed to stem from the different stabilities of the native fold. Considerations on the structural role of the DE loop in the free monomeric β2m and as part of the Major Histocompatibility Complex are also presented.

Research paper thumbnail of Structural and morphological characterization of aggregated species of α-synuclein induced by docosahexaenoic acid

The Journal of biological chemistry, Jan 24, 2011

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding o... more 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 b-hematin (bH). 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 bH. 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.

Research paper thumbnail of TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells

PloS one, 2014

Accumulation of ubiquitin-positive, tau-and a-synuclein-negative intracellular inclusions of TDP-... more Accumulation of ubiquitin-positive, tau-and a-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting fulllength and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

Research paper thumbnail of Aligning Amyloid-Like Fibrils on Nanopatterned Graphite

BioNanoScience, 2012

ABSTRACT Amyloid-like fibrils have been obtained upon incubation of a diluted aqueous solution of... more ABSTRACT Amyloid-like fibrils have been obtained upon incubation of a diluted aqueous solution of cytochrome c at neutral pH in the presence of gold nanoparticles. The interaction of the cytochrome c suspension, containing proteins in both the monomeric and fibrillar state, with the freshly cleaved highly ordered pyrolytic graphite (HOPG) surface results in a double process. First, it leads to a nanopatterning of the HOPG surface and, second, it promotes the self-aligning of cytochrome c protofilaments on the nanopatterned HOPG surface.

Research paper thumbnail of The H50Q Mutation Induces a 10-fold Decrease in the Solubility of α-Synuclein

Journal of Biological Chemistry, 2014

The basis of the pathogenicity of the H50Q variant ␣-synuclein is unknown. Results: The critical ... more The basis of the pathogenicity of the H50Q variant ␣-synuclein is unknown. Results: The critical concentration of ␣-synuclein is decreased by 10-fold by the H50Q mutation, and its aggregation is modulated by the wild-type isoform. Conclusion: Key effects of the H50Q mutation on the aggregation of ␣-synuclein can be quantified. Significance: Our data provide insights into the mechanism of Lewy body formation in vivo. The abbreviations used are: ␣-Syn, ␣-Synuclein; ThT, thioflavin T; EM, electron microscopy; AFM, atomic force microscopy; BEST, band-selective excitation short transient.

Research paper thumbnail of Photochromic and photomechanical responses of an amorphous diarylethene-based polymer: A spectroscopic ellipsometry investigation of ultrathin films

This work deals with very thin (14-40 nm) films of a polyester containing diarylethene units in t... more This work deals with very thin (14-40 nm) films of a polyester containing diarylethene units in the main chain spin cast on a silicon wafer. By irradiation with UV light the colourless form turns blue due to the appearance of a strong absorption band centred at about 600 nm. The coloured state is thermally stable and the backward conversion can be triggered with visible light. Comparison of broadband (245-1700 nm) spectroscopic ellipsometry data with simulations based on an isotropic, Kramers-Kronig consistent, multiple-resonance model allowed us to determine the complex index of refractionñ of the film in its blue and colourless forms. The refractive index of the blue phase neatly exceeds that of the transparent form for wavelengths in the NIR. In particular, out of resonance, at l $ 1700 nm, DRe(ñ) $ 0.05. Parallel to the DRe(ñ) increase, the analysis of ellipsometry data suggests a decrease of the film thickness (about 1.5%) during the transition from the open (transparent) to the closed (coloured) form. † Electronic supplementary information (ESI) available: Denition of the MSE function; UV-vis absorption spectra of p-DTE in chloroform solution; AFM images of lms; the extinction coefficient of the polymer lm as a function of energy; details about the comparison between experimental and simulated spectra of representative samples with different thicknesses. See

Research paper thumbnail of Epigallocatechin-3-gallate and tetracycline differently affect ataxin-3 fibrillogenesis and reduce toxicity in spinocerebellar ataxia type 3 model

Human molecular genetics, Jan 15, 2014

The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative diseas... more The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid b-rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in b-sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared with the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP.

Research paper thumbnail of Yeast Cytochrome c Monolayer on Flat and Nanostructured Gold Films Studied by UV–Vis Spectroscopic Ellipsometry

... by UV–Vis Spectroscopic Ellipsometry Chiara Toccafondi · Mirko Prato · Emanuele Barborini · S... more ... by UV–Vis Spectroscopic Ellipsometry Chiara Toccafondi · Mirko Prato · Emanuele Barborini · Simone Vinati · Giulia Maidecchi · Amanda Penco · Ornella Cavalleri · Francesco Bisio ·Maurizio Canepa ... 10. Barborini, E., Piseri, P., & Milani, P. (1999). ...

Research paper thumbnail of Nanopatterning by protein unfolding

Soft Matter, 2008

ABSTRACT Molecular layers patterned on the nanoscale, with long-range order properties extending ... more ABSTRACT Molecular layers patterned on the nanoscale, with long-range order properties extending over the microscopic scale, have been obtained upon adsorption of commonly available proteins onto the hydrophobic and long-range ordered surface of pyrolytic graphite (HOPG). Proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. This behaviour is rather general since it is observed for different proteins irrespective of their specific structural properties.

Research paper thumbnail of Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

Proceedings of the National Academy of Sciences, 2012

Chaperones are the primary regulators of the proteostasis network and are known to facilitate pro... more Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.

Research paper thumbnail of Rapid oligomer formation of human muscle acylphosphatase induced by heparan sulfate

Nature Structural & Molecular Biology, 2012

Many human diseases are caused by the conversion of proteins from their native state into amyloid... more Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail.

Research paper thumbnail of Salt Anions Promote the Conversion of HypF-N into Amyloid-Like Oligomers and Modulate the Structure of the Oligomers and the Monomeric Precursor State

Journal of Molecular Biology, 2012

An understanding of the solution factors contributing to the rate of aggregation of a protein int... more An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichiacoli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO(4), NaI, Na(2)SO(4)) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO(4)(2-)≥ ClO(4)(-)>I(-)>Cl(-)). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes.

Research paper thumbnail of Structure, Folding Dynamics, and Amyloidogenesis of D76N  2-Microglobulin: ROLES OF SHEAR FLOW, HYDROPHOBIC SURFACES, AND  -CRYSTALLIN

Journal of Biological Chemistry, 2013

Research paper thumbnail of Different ataxin-3 amyloid aggregates induce intracellular Ca2+ deregulation by different mechanisms in cerebellar granule cells

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2013

This work aims at elucidating the relation between morphological and physicochemical properties o... more This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca 2+ levels and the abnormal Ca 2+ signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca 2+ responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca 2+ response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.

Research paper thumbnail of The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli

PLoS ONE, 2012

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) s... more Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291D). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291D strongly reduced the growth rate in the early stages (2-4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291D on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular b-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.

Research paper thumbnail of The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli

PLoS ONE, 2012

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) s... more Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291D). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291D strongly reduced the growth rate in the early stages (2-4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291D on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular b-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.

Research paper thumbnail of Wild Type Beta-2 Microglobulin and DE Loop Mutants Display a Common Fibrillar Architecture

PLOS ONE, 2015

Beta-2 microglobulin (β2m) is the protein responsible for a pathologic condition known as dialysi... more Beta-2 microglobulin (β2m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining β2m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity. However, it remains unclear whether the aggregates formed by wild type (wt) β2m and by the DE loop variants are of the same kind, or whether the mutations open new aggregation pathways. In order to address this question, fibrillar samples of wt and mutated β2m variants have been analysed by means of atomic force microscopy and infrared spectroscopy. The data here reported indicate that the DE loop mutants form aggregates with morphology and structural organisation very similar to the wt protein. Therefore, the main effect of β2m DE loop mutations is proposed to stem from the different stabilities of the native fold. Considerations on the structural role of the DE loop in the free monomeric β2m and as part of the Major Histocompatibility Complex are also presented.

Research paper thumbnail of Structural and morphological characterization of aggregated species of α-synuclein induced by docosahexaenoic acid

The Journal of biological chemistry, Jan 24, 2011

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding o... more 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 b-hematin (bH). 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 bH. 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.

Research paper thumbnail of TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells

PloS one, 2014

Accumulation of ubiquitin-positive, tau-and a-synuclein-negative intracellular inclusions of TDP-... more Accumulation of ubiquitin-positive, tau-and a-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting fulllength and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

Research paper thumbnail of Aligning Amyloid-Like Fibrils on Nanopatterned Graphite

BioNanoScience, 2012

ABSTRACT Amyloid-like fibrils have been obtained upon incubation of a diluted aqueous solution of... more ABSTRACT Amyloid-like fibrils have been obtained upon incubation of a diluted aqueous solution of cytochrome c at neutral pH in the presence of gold nanoparticles. The interaction of the cytochrome c suspension, containing proteins in both the monomeric and fibrillar state, with the freshly cleaved highly ordered pyrolytic graphite (HOPG) surface results in a double process. First, it leads to a nanopatterning of the HOPG surface and, second, it promotes the self-aligning of cytochrome c protofilaments on the nanopatterned HOPG surface.

Research paper thumbnail of The H50Q Mutation Induces a 10-fold Decrease in the Solubility of α-Synuclein

Journal of Biological Chemistry, 2014

The basis of the pathogenicity of the H50Q variant ␣-synuclein is unknown. Results: The critical ... more The basis of the pathogenicity of the H50Q variant ␣-synuclein is unknown. Results: The critical concentration of ␣-synuclein is decreased by 10-fold by the H50Q mutation, and its aggregation is modulated by the wild-type isoform. Conclusion: Key effects of the H50Q mutation on the aggregation of ␣-synuclein can be quantified. Significance: Our data provide insights into the mechanism of Lewy body formation in vivo. The abbreviations used are: ␣-Syn, ␣-Synuclein; ThT, thioflavin T; EM, electron microscopy; AFM, atomic force microscopy; BEST, band-selective excitation short transient.

Research paper thumbnail of Photochromic and photomechanical responses of an amorphous diarylethene-based polymer: A spectroscopic ellipsometry investigation of ultrathin films

This work deals with very thin (14-40 nm) films of a polyester containing diarylethene units in t... more This work deals with very thin (14-40 nm) films of a polyester containing diarylethene units in the main chain spin cast on a silicon wafer. By irradiation with UV light the colourless form turns blue due to the appearance of a strong absorption band centred at about 600 nm. The coloured state is thermally stable and the backward conversion can be triggered with visible light. Comparison of broadband (245-1700 nm) spectroscopic ellipsometry data with simulations based on an isotropic, Kramers-Kronig consistent, multiple-resonance model allowed us to determine the complex index of refractionñ of the film in its blue and colourless forms. The refractive index of the blue phase neatly exceeds that of the transparent form for wavelengths in the NIR. In particular, out of resonance, at l $ 1700 nm, DRe(ñ) $ 0.05. Parallel to the DRe(ñ) increase, the analysis of ellipsometry data suggests a decrease of the film thickness (about 1.5%) during the transition from the open (transparent) to the closed (coloured) form. † Electronic supplementary information (ESI) available: Denition of the MSE function; UV-vis absorption spectra of p-DTE in chloroform solution; AFM images of lms; the extinction coefficient of the polymer lm as a function of energy; details about the comparison between experimental and simulated spectra of representative samples with different thicknesses. See

Research paper thumbnail of Epigallocatechin-3-gallate and tetracycline differently affect ataxin-3 fibrillogenesis and reduce toxicity in spinocerebellar ataxia type 3 model

Human molecular genetics, Jan 15, 2014

The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative diseas... more The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid b-rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in b-sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared with the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP.

Research paper thumbnail of Yeast Cytochrome c Monolayer on Flat and Nanostructured Gold Films Studied by UV–Vis Spectroscopic Ellipsometry

... by UV–Vis Spectroscopic Ellipsometry Chiara Toccafondi · Mirko Prato · Emanuele Barborini · S... more ... by UV–Vis Spectroscopic Ellipsometry Chiara Toccafondi · Mirko Prato · Emanuele Barborini · Simone Vinati · Giulia Maidecchi · Amanda Penco · Ornella Cavalleri · Francesco Bisio ·Maurizio Canepa ... 10. Barborini, E., Piseri, P., & Milani, P. (1999). ...

Research paper thumbnail of Nanopatterning by protein unfolding

Soft Matter, 2008

ABSTRACT Molecular layers patterned on the nanoscale, with long-range order properties extending ... more ABSTRACT Molecular layers patterned on the nanoscale, with long-range order properties extending over the microscopic scale, have been obtained upon adsorption of commonly available proteins onto the hydrophobic and long-range ordered surface of pyrolytic graphite (HOPG). Proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. This behaviour is rather general since it is observed for different proteins irrespective of their specific structural properties.

Research paper thumbnail of Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

Proceedings of the National Academy of Sciences, 2012

Chaperones are the primary regulators of the proteostasis network and are known to facilitate pro... more Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.

Research paper thumbnail of Rapid oligomer formation of human muscle acylphosphatase induced by heparan sulfate

Nature Structural & Molecular Biology, 2012

Many human diseases are caused by the conversion of proteins from their native state into amyloid... more Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail.

Research paper thumbnail of Salt Anions Promote the Conversion of HypF-N into Amyloid-Like Oligomers and Modulate the Structure of the Oligomers and the Monomeric Precursor State

Journal of Molecular Biology, 2012

An understanding of the solution factors contributing to the rate of aggregation of a protein int... more An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichiacoli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO(4), NaI, Na(2)SO(4)) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO(4)(2-)≥ ClO(4)(-)>I(-)>Cl(-)). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes.

Research paper thumbnail of Structure, Folding Dynamics, and Amyloidogenesis of D76N  2-Microglobulin: ROLES OF SHEAR FLOW, HYDROPHOBIC SURFACES, AND  -CRYSTALLIN

Journal of Biological Chemistry, 2013

Research paper thumbnail of Different ataxin-3 amyloid aggregates induce intracellular Ca2+ deregulation by different mechanisms in cerebellar granule cells

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2013

This work aims at elucidating the relation between morphological and physicochemical properties o... more This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca 2+ levels and the abnormal Ca 2+ signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca 2+ responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca 2+ response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.