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Annalisa Pastore

Publications and source records attributed to Annalisa Pastore.

At least 19 recordsLinked to original sources
PubMed2006

The interplay between PolyQ and protein context delays aggregation by forming a reservoir of protofibrils.

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway.

Carrier Proteins↗
PubMed2006

Acidic residues of yeast frataxin have an essential role in Fe-S cluster assembly.

Friedreich ataxia is caused by decreased levels of frataxin, a mitochondrial acidic protein that is assumed to act as chaperone in the assembly of Fe-S clusters on the scaffold Isu protein. Frataxin has the in vitro capacity to form iron-loaded multimers, which also suggests an iron storage function. It has been reported that alanine substitution of residues in an acidic ridge of yeast frataxin (Yfh1) elicits loss of iron binding in vitro but has no effect on Fe-S cluster synthesis in vivo. Here, we show that a marked change in the electrostatic properties of a specific region of Yfh1 surface - by substituting two or four acidic residues by lysine or alanine, respectively - impairs Fe-S cluster assembly, weakens the interaction between Yfh1 and Isu1, and increases oxidative damage. Therefore, the acidic ridge is essential for the Yfh1 function and is likely to be involved in iron-mediated protein-protein interactions.

Adrenodoxin↗
PubMed2006

Structure validation of the Josephin domain of ataxin-3: conclusive evidence for an open conformation.

The availability of new and fast tools in structure determination has led to a more than exponential growth of the number of structures solved per year. It is therefore increasingly essential to assess the accuracy of the new structures by reliable approaches able to assist validation. Here, we discuss a specific example in which the use of different complementary techniques, which include Bayesian methods and small angle scattering, resulted essential for validating the two currently available structures of the Josephin domain of ataxin-3, a protein involved in the ubiquitin/proteasome pathway and responsible for neurodegenerative spinocerebellar ataxia of type 3. Taken together, our results demonstrate that only one of the two structures is compatible with the experimental information. Based on the high precision of our refined structure, we show that Josephin contains an open cleft which could be directly implicated in the interaction with polyubiquitin chains and other partners.

Ataxin-3↗
PubMed2006

Conformational stability of human frataxin and effect of Friedreich's ataxia-related mutations on protein folding.

The neurodegenerative disorder FRDA (Friedreich's ataxia) results from a deficiency in frataxin, a putative iron chaperone, and is due to the presence of a high number of GAA repeats in the coding regions of both alleles of the frataxin gene, which impair protein expression. However, some FRDA patients are heterozygous for this triplet expansion and contain a deleterious point mutation on the other allele. In the present study, we investigated whether two particular FRDA-associated frataxin mutants, I154F and W155R, result in unfolded protein as a consequence of a severe structural modification. A detailed comparison of the conformational properties of the wild-type and mutant proteins combining biophysical and biochemical methodologies was undertaken. We show that the FRDA mutants retain the native fold under physiological conditions, but are differentially destabilized as reflected both by their reduced thermodynamic stability and a higher tendency towards proteolytic digestion. The I154F mutant has the strongest effect on fold stability as expected from the fact that the mutated residue contributes to the hydrophobic core formation. Functionally, the iron-binding properties of the mutant frataxins are found to be partly impaired. The apparently paradoxical situation of having clinically aggressive frataxin variants which are folded and are only significantly less stable than the wild-type form in a given adverse physiological stress condition is discussed and contextualized in terms of a mechanism determining the pathology of FRDA heterozygous.

Enzyme Stability↗
PubMed2006

The structure of the N-terminal domain of the fragile X mental retardation protein: a platform for protein-protein interaction.

FMRP, whose lack of expression causes the X-linked fragile X syndrome, is a modular RNA binding protein thought to be involved in posttranslational regulation. We have solved the structure in solution of the N-terminal domain of FMRP (NDF), a functionally important region involved in multiple interactions. The structure consists of a composite fold comprising two repeats of a Tudor motif followed by a short alpha helix. The interactions between the three structural elements are essential for the stability of the NDF fold. Although structurally similar, the two repeats have different dynamic and functional properties. The second, more flexible repeat is responsible for interacting both with methylated lysine and with 82-FIP, one of the FMRP nuclear partners. NDF contains a 3D nucleolar localization signal, since destabilization of its fold leads to altered nucleolar localization of FMRP. We suggest that the NDF composite fold determines an allosteric mechanism that regulates the FMRP functions.

Allosteric Regulation↗
PubMed2006

YfhJ, a molecular adaptor in iron-sulfur cluster formation or a frataxin-like protein?

The yfhJ gene is part of the isc operon, which encodes the machinery devoted to assemble iron-sulfur clusters in prokaryotes. Its transcript is a small acidic protein that binds the desulfurase IscS, which is essential in iron-specific metabolic pathways. To understand its cellular role, we have characterized the structure of YfhJ in solution and its interactions with potential cellular partners. It contains a modified winged helix motif, usually present in DNA binding proteins, and is able to bind iron cations. The IscS interaction surface is the same as that involved in iron binding. This observation and the pattern of conservation through species strongly suggest that YfhJ is a molecular adaptor that is able to modulate the function of IscS in iron-sulfur cluster formation. The remarkable similarity between the in vitro behavior of YfhJ and that of the protein frataxin also suggests new hypotheses regarding the functional role of both proteins.

Amino Acid Sequence↗
PubMed2006

New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae.

SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.

Binding Sites↗
PubMed2005

Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein.

A family of neurodegenerative diseases is associated with anomalous expansion of a polyglutamine tract in the coding region of the corresponding proteins. The current working hypothesis is that polyglutamine diseases are caused by misfolding and aggregation of the proteins with a process dictated by the polyglutamine tracts, although increasing evidence suggests an involvement of the protein context in modulating these properties. Here, we show that the AXH domain of ataxin-1, the protein involved in spinocerebellar ataxia type-1, is the region responsible for the transcriptional repression activity of ataxin-1 and participates in protein aggregation. In vitro, the isolated domain undergoes a conformational transition towards a beta-enriched structure associated with aggregation and amyloid fibre formation spontaneously and without need for destabilizing conditions. Using a transfected cell line, we demonstrate that, while determined by polyglutamine expansion, ataxin-1 aggregation is noticeably reduced by deletion of AXH or by replacement with the homologous sequence from the transcription factor HBP1, which has no known tendency to aggregate. These results provide the first direct evidence of an involvement of a region other than the polyglutamine tract in polyglutamine pathologies.

Amyloid↗
PubMed2005

The solution structure of the Josephin domain of ataxin-3: structural determinants for molecular recognition.

The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity. By using the Josephin structure and the availability of NMR chemical shift assignments, we have mapped the enzyme active site by using the typical cysteine protease inhibitors, transepoxysuccinyl-L-eucylamido-4-guanidino-butane (E-64) and [L-3-trans-(propylcarbamyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline (CA-074). We also demonstrate that the specific interaction of Josephin with the ubiquitin-like domain of the ubiquitin- and proteasome-binding factor HHR23B involves complementary exposed hydrophobic surfaces. The structural similarity with other deubiquitinating enzymes suggests a model for the proteolytic enzymatic activity of ataxin-3.

Ataxin-3↗
PubMed2005

Fragile X mental retardation protein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-binding motif.

Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. This region does not contain a motif known to specifically recognize RNA and thus constitutes a new RNA-binding motif. We further demonstrated that FMRP recognition involves the 5' stem loop of BC1 and that this is the region that exhibits complementarity to FMRP target mRNAs, raising the possibility that FMRP plays a direct role in BC1/mRNA annealing.

Base Sequence↗
PubMed2005

Mechanical properties of the domains of titin in a Go-like model.

Comparison of properties of three domains of titin, I1, I27, and I28, in a simple geometry-based model shows that despite a high structural homology between their native states different domains show similar but distinguishable mechanical properties. Folding properties of the separate domains are predicted to be diversified which reflects sensitivity of the kinetics to the details of native structures. The Go-like model corresponding to the experimentally resolved native structure of the I1 domain is found to provide the biggest thermodynamic and mechanical stability compared to the other domains studied here. We analyze elastic, thermodynamic, and kinetic properties of several structures corresponding to the I28 domain as obtained through homology-based modeling. We discuss the ability of the models of the I28 domain to reproduce experimental results qualitatively. A strengthening of contacts that involve hydrophobic amino acids does not affect theoretical comparisons of the domains. Tandem linkages of up to five identical or different domains unravel in a serial fashion at low temperatures. We study the nature of the intermediate state that arises in the early stages of the serial unraveling and find it to qualitatively agree with the results of Marszalek et al.

Journal Article↗
PubMed2005

The AXH domain adopts alternative folds the solution structure of HBP1 AXH.

AXH is a protein module identified in two unrelated families that comprise the transcriptional repressor HBP1 and ataxin-1 (ATX1), the protein responsible for spinocerebellar ataxia type-1 (SCA1). SCA1 is a neurodegenerative disorder associated with protein misfolding and formation of toxic intranuclear aggregates. We have solved the structure in solution of monomeric AXH from HBP1. The domain adopts a nonclassical permutation of an OB fold and binds nucleic acids, a function previously unidentified for this region of HBP1. Comparison of HBP1 AXH with the crystal structure of dimeric ATX1 AXH indicates that, despite the significant sequence homology, the two proteins have different topologies, suggesting that AXH has chameleon properties. We further demonstrate that HBP1 AXH remains monomeric, whereas the ATX1 dimer spontaneously aggregates and forms fibers. Our results describe an entirely novel, to our knowledge, example of a chameleon fold and suggest a link between these properties and the SCA1 pathogenesis.

Amino Acid Sequence↗
PubMed2004

WHEATSHEAF: an algorithm to average protein structure ensembles.

A new algorithm is described that forms a single structure representative of ensembles of structures from files in the format used by the Protein Data Bank. A first attempt is made by averaging in the space spanned by bond lengths, inter-bond rotations and symmetry-multiplied dihedral rotations. This normally produces well formed regular secondary-structure elements, but the intervening less well ordered regions are often distorted because of the invalidity of averaging large rotations about divergent axes. For this reason, the algorithm includes a second stage that pulls the interatomic distances towards more fully representative values. Results produced by this method have proved better as judged by conventional quality checks than any input structure in nearly all cases tested so far, especially for the backbone, and much better than those produced by commonly used alternative methods.

Algorithms↗
PubMed2004

Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3.

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate.

Ataxin-3↗
PubMed2004

The C terminus of fragile X mental retardation protein interacts with the multi-domain Ran-binding protein in the microtubule-organising centre.

Absence of the fragile X mental retardation protein (FMRP) causes fragile X syndrome, the most common form of hereditary mental retardation. FMRP is a mainly cytoplasmic protein thought to be involved in repression of translation, through a complex network of protein-protein and protein-RNA interactions. Most of the currently known protein partners of FMRP recognise the conserved N terminus of the protein. No interaction has yet been mapped to the highly charged, poorly conserved C terminus, so far thought to be involved in RNA recognition through an RGG motif. In the present study, we show that a two-hybrid bait containing residues 419-632 of human FMRP fishes out a protein that spans the sequence of the Ran-binding protein in the microtubule-organising centre (RanBPM/RanBP9). Specific interaction of RanBPM with FMRP was confirmed by in vivo and in vitro assays. In brain tissue sections, RanBPM is highly expressed in the neurons of cerebral cortex and the cerebellar purkinje cells, in a pattern similar to that described for FMRP. Sequence analysis shows that RanBPM is a multi-domain protein. The interaction with FMRP was mapped in a newly identified CRA motif present in the RanBPM C terminus. Our results suggest that the functional role of RanBPM binding is modulation of the RNA-binding properties of FMRP.

Amino Acid Motifs↗
PubMed2004

Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli.

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O(2). However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.

Bacterial Proteins↗