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Find video protocols related to scientific articles indexed in Pubmed.
Amyloid Formation by Human Carboxypeptidase D Transthyretin-like Domain Under Physiological Conditions.
J. Biol. Chem.
PUBLISHED: 10-09-2014
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Protein aggregation is linked to a growing list of diseases, but is also an intrinsic property of polypeptides, since the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including M14B subfamily carboxypeptidases (CPs). We show here that the monomeric transthyretin-like domain of human CPD (h-TTL) aggregates under close to physiological conditions into amyloid structures, the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic ?-strands in the native state. These structural elements should serve for functional/structural purposes, since they have not been purged out by evolution, but at the same time they put proteins like CPD at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.
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Fluorescent dye ProteoStat to detect and discriminate intracellular amyloid-like aggregates in Escherichia coli.
Biotechnol J
PUBLISHED: 09-08-2014
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The formation of amyloid aggregates is linked to the onset of an increasing number of human disorders. Thus, there is an increasing need for methodologies able to provide insights into protein deposition and its modulation. Many approaches exist to study amyloids in vitro, but the techniques available for the study of amyloid aggregation in cells are still limited and non-specific. In this study we developed a methodology for the detection of amyloid-like aggregates inside cells that discriminates these ordered assemblies from other intracellular aggregates. We chose bacteria as model system, since the inclusion bodies formed by amyloid proteins in the cytosol of bacteria resemble toxic amyloids both structurally and functionally. Using confocal microscopy, fluorescence spectroscopy, and flow cytometry, we show that the recently developed red fluorescent dye ProteoStat can detect the presence of intracellular amyloid-like deposits in living bacterial cells with high specificity, even when the target proteins are expressed at low levels. This methodology allows quantitation of the intracellular amyloid content, shows the potential to replace in vitro screenings in the search for therapeutic anti-amyloidogenic compounds, and might be useful for identifying conditions that prevent the aggregation of therapeutic recombinant proteins.
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The small GTPase Rab11 co-localizes with ?-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity.
Hum. Mol. Genet.
PUBLISHED: 08-04-2014
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Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.
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The Importance of a Gatekeeper Residue on the Aggregation of Transthyretin: IMPLICATIONS FOR TRANSTHYRETIN-RELATED AMYLOIDOSES.
J. Biol. Chem.
PUBLISHED: 08-01-2014
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Protein aggregation into ?-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26-57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26-57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility.
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Association between foldability and aggregation propensity in small disulfide-rich proteins.
Antioxid. Redox Signal.
PUBLISHED: 05-05-2014
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Disulfide-rich domains (DRDs) are small proteins whose native structure is stabilized by the presence of covalent disulfide bonds. These domains are versatile and can perform a wide range of functions. Many of these domains readily unfold on disulfide bond reduction, suggesting that in the absence of covalent bonding they might display significant disorder.
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N-terminal protein tails act as aggregation protective entropic bristles: the SUMO case.
Biomacromolecules
PUBLISHED: 03-06-2014
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The formation of ?-sheet enriched amyloid fibrils constitutes the hallmark of many diseases but is also an intrinsic property of polypeptide chains in general, because the formation of compact globular proteins comes at the expense of an inherent sequential aggregation propensity. In this context, identification of strategies that enable proteins to remain functional and soluble in the cell has become a central issue in chemical biology. We show here, using human SUMO proteins as a model system, that the recurrent presence of disordered tails flanking globular domains might constitute yet another of these protective strategies. These short, disordered, and highly soluble protein segments would act as intramolecular entropic bristles, reducing the overall protein intrinsic aggregation propensity and favoring thus the attainment and maintenance of functional conformations.
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The mitochondrial intermembrane space oxireductase Mia40 funnels the oxidative folding pathway of the cytochrome c oxidase assembly protein Cox19.
J. Biol. Chem.
PUBLISHED: 02-25-2014
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Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an ?-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.
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Screening for amyloid aggregation: in-silico, in-vitro and in-vivo detection.
Curr. Protein Pept. Sci.
PUBLISHED: 02-21-2014
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Protein misfolding and aggregation into amyloid structures is linked with an increasing number of nonneuropathic (either localized or systemic) and neurodegenerative human disorders. In the present review, we compile and describe methods, which have been developed to predict, detect and characterize amyloid and amyloid-like protein deposits. We focus in the state-of-the-art methodologies to study and image amyloid aggregation in-vitro, from qualitative and low-resolution techniques to methods addressed to resolve protein structures at atomic level. We also recapitulate the most relevant literature describing approaches that have been demonstrated to be able to detect and characterize protein aggregation in cells and living organisms, as well as methodologies to report cytotoxicity associated to amyloid formation. Overall, the aim of this review is to illustrate computational and experimental methods to characterize and predict in-vitro and in-vivo amyloid aggregation, providing the readers valuable information to elect the most appropriate techniques at their convenience.
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PrionScan: an online database of predicted prion domains in complete proteomes.
BMC Genomics
PUBLISHED: 02-04-2014
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Prions are a particular type of amyloids related to a large variety of important processes in cells, but also responsible for serious diseases in mammals and humans. The number of experimentally characterized prions is still low and corresponds to a handful of examples in microorganisms and mammals. Prion aggregation is mediated by specific protein domains with a remarkable compositional bias towards glutamine/asparagine and against charged residues and prolines. These compositional features have been used to predict new prion proteins in the genomes of different organisms. Despite these efforts, there are only a few available data sources containing prion predictions at a genomic scale.
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Selection against toxic aggregation-prone protein sequences in bacteria.
Biochim. Biophys. Acta
PUBLISHED: 01-16-2014
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Despite genetic variation has the potential to arise new protein functions, spontaneous mutations usually destabilize the native fold. Misfolded proteins tend to form cytotoxic intracellular aggregates, decreasing cell fitness and leading to degenerative disorders in humans. Therefore, it is thought that selection against protein misfolding and aggregation constrains the evolution of protein sequences. However, obtaining experimental data to validate this hypothesis has been traditionally difficult. Here we exploit bacteria as a model organism to address this question. Using variants of the Alzheimer's related A?42 peptide designed to exhibit different in vivo aggregation propensities we show here that, in cell competition experiments, the most aggregation-prone variants are always purged out from the growing population. Flow cytometry analysis of cellular metabolism and viability demonstrates that this purifying effect responds to a clear correlation between physiological burden and intrinsic aggregation propensity. Interestingly, the fitness cost of aggregation appears to be associated with aggregation rates rather than with overall protein solubility. Accordingly, we show that, by reducing in vivo aggregation rates, the model osmolyte proline is able to buffer the metabolic impact of protein aggregation. Overall, our data provide experimental support for the role of toxic protein aggregation on the cell fitness landscape and the evolution of natural protein sequences.
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Human stefin B role in cell's response to misfolded proteins and autophagy.
PLoS ONE
PUBLISHED: 01-01-2014
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Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.
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Trifluoroethanol modulates amyloid formation by the all ?-helical URN1 FF domain.
Int J Mol Sci
PUBLISHED: 07-26-2013
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Amyloid fibril formation is implicated in different human diseases. The transition between native ?-helices and nonnative intermolecular ?-sheets has been suggested to be a trigger of fibrillation in different conformational diseases. The FF domain of the URN1 splicing factor (URN1-FF) is a small all-? protein that populates a molten globule (MG) at low pH. Despite the fact that this conformation maintains most of the domain native secondary structure, it progressively converts into ?-sheet enriched and highly ordered amyloid fibrils. In this study, we investigated if 2,2,2-trifluoroethanol (TFE) induced conformational changes that affect URN1-FF amyloid formation. Despite TFE having been shown to induce or increase the aggregation of both globular and disordered proteins at moderate concentrations, we demonstrate here that in the case of URN1-FF it reinforces its intrinsic ?-helical structure, which competes the formation of aggregated assemblies. In addition, we show that TFE induces conformational diversity in URN1-FF fibrils, in such a way that the fibrils formed in the presence and absence of the cosolvent represent different polymorphs. It is suggested that the effect of TFE on both the soluble and aggregated states of URN1-FF depends on its ability to facilitate hydrogen bonding.
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Thioflavin-T excimer formation upon interaction with amyloid fibers.
Chem. Commun. (Camb.)
PUBLISHED: 05-21-2013
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The molecular mechanism of the Thioflavin-T (Th-T) binding to amyloids remains unknown. By combining experimental analysis of Th-T excitation and emission spectra with theoretical calculations we suggest that Th-T fluorescence changes upon interaction with amyloids may arise from the formation of an excimer with an oblique angle of ~120 degrees.
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Protein aggregation propensity is a crucial determinant of intracellular inclusion formation and quality control degradation.
Biochim. Biophys. Acta
PUBLISHED: 05-14-2013
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Protein aggregation is linked to many pathological conditions, including several neurodegenerative diseases. The aggregation propensities of proteins are thought to be controlled to a large extent by the physicochemical properties encoded in the primary sequence. We have previously exploited a set of amyloid ? peptide (A?42) variants exhibiting a continuous gradient of intrinsic aggregation propensities to demonstrate that this rule applies in vivo in bacteria. In the present work we have characterized the behavior of these A?42 mutants when expressed in yeast. In contrast to bacteria, the intrinsic aggregation propensity is gated by yeast, in such a way that this property correlates with the formation of intracellular inclusions only above a specific aggregation threshold. Proteins displaying solubility levels above this threshold escape the inclusion formation pathway. In addition, the most aggregation-prone variants are selectively cleared by the yeast quality control degradation machinery. Thus, both inclusion formation and proteolysis target the same aggregation-prone variants and cooperate to minimize the presence of these potentially dangerous species in the cytosol. The demonstration that sorting to these pathways in eukaryotes is strongly influenced by protein primary sequence should facilitate the development of rational approaches to predict and hopefully prevent in vivo protein deposition.
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Discovering putative prion sequences in complete proteomes using probabilistic representations of Q/N-rich domains.
BMC Genomics
PUBLISHED: 05-06-2013
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Prion proteins conform a special class among amyloids due to their ability to transmit aggregative folds. Prions are known to act as infectious agents in neurodegenerative diseases in animals, or as key elements in transcription and translation processes in yeast. It has been suggested that prions contain specific sequential domains with distinctive amino acid composition and physicochemical properties that allow them to control the switch between soluble and ?-sheet aggregated states. Those prion-forming domains are low complexity segments enriched in glutamine/asparagine and depleted in charged residues and prolines. Different predictive methods have been developed to discover novel prions by either assessing the compositional bias of these stretches or estimating the propensity of protein sequences to form amyloid aggregates. However, the available algorithms hitherto lack a thorough statistical calibration against large sequence databases, which makes them unable to accurately predict prions without retrieving a large number of false positives.
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The N-terminal helix controls the transition between the soluble and amyloid states of an FF domain.
PLoS ONE
PUBLISHED: 02-01-2013
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Protein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native ?-helical structures and their self-assembly into nonnative intermolecular ?-sheets has been proposed to trigger amyloid fibril formation in Alzheimers and Parkinsons diseases.
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Cross-?-sheet supersecondary structure in amyloid folds: techniques for detection and characterization.
Methods Mol. Biol.
PUBLISHED: 02-01-2013
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The formation of protein aggregates is linked to the onset of several human disorders of increasing prevalence, ranging from dementia to diabetes. In most of these diseases, the toxic effect is exerted by the self-assembly of initially soluble proteins into insoluble amyloid-like fibrils. Independently of the protein origin, all these macromolecular assemblies share a common supersecondary structure: the cross-?-sheet conformation, in which a core of ?-strands is aligned perpendicularly to the fibril axis forming extended regular ?-sheets. Due to this ubiquity, the presence of cross-?-sheet conformational signatures is usually exploited to detect, characterize, and screen for amyloid fibrils in protein samples. Here we describe in detail some of the most commonly used methods to analyze such supersecondary structure.
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Oxidative folding in the mitochondrial intermembrane space in human health and disease.
Int J Mol Sci
PUBLISHED: 01-21-2013
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Oxidative folding in the mitochondrial intermembrane space (IMS) is a key cellular event associated with the folding and import of a large and still undetermined number of proteins. This process is catalyzed by an oxidoreductase, Mia40 that is able to recognize substrates with apparently little or no homology. Following substrate oxidation, Mia40 is reduced and must be reoxidized by Erv1/Alr1 that consequently transfers the electrons to the mitochondrial respiratory chain. Although our understanding of the physiological relevance of this process is still limited, an increasing number of pathologies are being associated with the impairment of this pathway; especially because oxidative folding is fundamental for several of the proteins involved in defense against oxidative stress. Here we review these aspects and discuss recent findings suggesting that oxidative folding in the IMS is modulated by the redox state of the cell.
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Inhibition of human transthyretin aggregation by non-steroidal anti-inflammatory compounds: a structural and thermodynamic analysis.
Int J Mol Sci
PUBLISHED: 01-08-2013
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Transthyretin (TTR) is a homotetrameric protein that circulates in plasma and cerebral spinal fluid (CSF) whose aggregation into amyloid fibrils has been associated with at least two different amyloid diseases: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). In SSA aggregates are composed of WT-TTR, while in FAP more than 100 already-described variants have been found in deposits. Until now, TTR-related diseases have been untreatable, although a new drug called Tafamidis has been approved only in Europe to specifically treat V30M patients. Thus, new strategies are still necessary to treat FAP caused by other variants of TTR. TTR has two channels in the dimer interface that bind to the hormone thyroxin and that have been used to accommodate anti-amyloidogenic compounds. These compounds stabilize the tetramers, rendering TTR less amyloidogenic. Here, we investigated the effects of three non-steroidal anti-inflammatory compounds-sulindac (SUL), indomethacin (IND) and lumiracoxib (LUM)-as tetramer stabilizers and aggregation inhibitors. WT-TTR and the very aggressive TTR variant L55P were used as models. These compounds were able to stabilize TTR against high hydrostatic pressure (HHP), increasing the ?Gf by several kcal. They were also effective in inhibiting WT-TTR and L55P acid- or HHP-induced aggregation; in particular, LUM and IND were very effective, inhibiting almost 100% of the aggregation of both proteins under certain conditions. The species formed when aggregation was performed in the presence of these compounds were much less toxic to cells in culture. The crystal structures of WT-TTR bound to the three compounds were solved at high resolution, allowing the identification of the relevant protein:drug interactions. We discuss here the ligand-binding features of LUM, IND and SUL to TTR, emphasizing the critical interactions that render the protein more stable and less amyloidogenic.
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Structure-Based Analysis of A19D, a Variant of Transthyretin Involved in Familial Amyloid Cardiomyopathy.
PLoS ONE
PUBLISHED: 01-01-2013
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Transthyretin (TTR) is a tetrameric beta-sheet-rich protein. Its deposits have been implicated in four different amyloid diseases. Although aggregation of the wild-type sequence is responsible for the senile form of the disease, more than one hundred variants have been described thus far, most of which confer a more amyloidogenic character to TTR, mainly because they compromise the stability of the protein in relation to monomer formation, which upon misfolding is intrinsically aggregation-prone. We report the case of a Brazilian patient suffering from a severe cardiomyopathy who carries a rare mutation in exon 2 of the TTR gene that results in an Ala to Asp substitution at position 19 (A19D). The putative pathogenic mechanisms of this variant were analyzed in silico. We constructed a structural model for the A19D tetramer from which its thermodynamic stability was compared to that displayed by the V30M (more amyloidogenic than WT-TTR) and T119M (non-amyloidogenic) variants. The FoldX force field predicted that A19D and V30M are 10.88 and 8.07 kCal/mol less stable than the WT-TTR, while T119M is 5.15 kCal/mol more stable, which is consistent with the aggregation propensities exhibited by these variants. We analyzed the step in which the tetramer-dimer-monomer-unfolded monomer equilibrium might contribute the most to the increased or decreased amyloidogenicity in each variant. Our results suggest that the concentration of four non-native negative charges occur inside thyroxine-binding channels, and the loss of contacts at both the tetrameric and dimeric interfaces would account for an overall decreased stability of the tetramer and the consequent enhanced amyloidogenicity of the A19D variant. As far as we know, this is the first description of a non-V30M mutation in Brazil.
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Temperature dependence of the aggregation kinetics of Sup35 and Ure2p yeast prions.
Biomacromolecules
PUBLISHED: 12-29-2011
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Fungal prions are protein-based genetic elements. Sup35 and Ure2p constitute the best-characterized prion proteins in the yeast Saccharomyces cerevisiae. No high-resolution molecular models of the amyloid conformations adopted by the prion domains of these proteins are available yet. A quantitative description of the kinetics and thermodynamics of their self-assembly processes might provide clues on the nature of the structural changes originating their heritable and transmissible phenotypes. Here we study the temperature dependence of Sup35 and Ure2p amyloid fibril nucleation and elongation reactions at physiological pH. Both processes follow the Arrhenius law, allowing calculation of their associated thermodynamic activation parameters. Although the Gibbs energies (?G*) for the nucleation and elongation of both prions are similar, the enthalpic and entropic contributions to these two processes are dramatically different. In addition, the structural properties of the two types of prion fibrils exhibit different dependence on the polymerization temperature. Overall, we show here that the amyloidogenic pathways of Sup35 and Ure2p prions diverge significantly.
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The effect of amyloidogenic peptides on bacterial aging correlates with their intrinsic aggregation propensity.
J. Mol. Biol.
PUBLISHED: 09-23-2011
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The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimers-disease-related amyloid ? peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness.
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Contribution of disulfide bonds to stability, folding, and amyloid fibril formation: the PI3-SH3 domain case.
Antioxid. Redox Signal.
PUBLISHED: 09-15-2011
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The failure of proteins to fold or to remain folded very often leads to their deposition into amyloid fibrils and is the origin of a variety of human diseases. Accordingly, mutations that destabilize the native conformation are associated with pathological phenotypes in several protein models. Protein backbone cyclization by disulfide bond crosslinking strongly reduces the entropy of the unfolded state and, usually, increases protein stability. The effect of protein cyclization on the thermodynamic and kinetics of folding has been extensively studied, but little is know on its effect on aggregation reactions.
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Biological role of bacterial inclusion bodies: a model for amyloid aggregation.
FEBS J.
PUBLISHED: 05-31-2011
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Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cells cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of inclusion bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make inclusion bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background.
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The aggregation properties of Escherichia coli proteins associated with their cellular abundance.
Biotechnol J
PUBLISHED: 01-21-2011
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Proteins are key players in most cellular processes. Therefore, their abundances are thought to be tightly regulated at the gene-expression level. Recent studies indicate, however, that steady-state cellular-protein concentrations correlate better across species than the levels of the corresponding mRNAs; this supports the existence of selective forces to maintain precise cellular-protein concentrations and homeostasis, even if gene-expression levels diverge. One of these forces might be the avoidance of protein aggregation because, in the cell, the folding of proteins into functional conformations might be in competition with anomalous aggregation into non-functional and usually toxic structures in a concentration-dependent manner. The data in the present work provide support for this hypothesis because, in E. coli, the experimental solubility of proteins correlates better with the cellular abundance than with the gene-expression levels. We found that the divergence between protein and mRNAs levels is low for high-abundance proteins. This suggests that because abundant proteins are at higher risk of aggregation, cellular concentrations need to be stringently regulated by gene expression.
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Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes.
Biotechnol J
PUBLISHED: 01-21-2011
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In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of ?-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation.
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Linking amyloid protein aggregation and yeast survival.
Mol Biosyst
PUBLISHED: 01-14-2011
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Protein aggregation and amyloid formation lie behind an increasing number of human diseases. Here we describe the application of an "aggregation reporter", in which the test protein is fused to dihydrofolate reductase, as a general method to assess the intracellular solubility of amyloid proteins in eukaryotic background. Because the aggregation state of the target protein is linked directly to yeast cells survival in the presence of methotrexate, protein solubility can be monitored in vivo without the requirement of a functional assay for the protein of interest. In addition, the approach allows the in vivo visualization of the cellular location and aggregated state of the target protein. To demonstrate the applicability of the assay in the screening of genes or compounds that modulate amyloid protein aggregation in living cells, we have used as models the Alzheimers amyloid ? peptide, polyglutamine expansions of huntingtin, ?-synuclein and non-aggregating variants thereof. Moreover, the anti-aggregational properties of small molecules and the effects of the yeast protein quality control machinery have also been evaluated using this method.
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Bacterial inclusion bodies of Alzheimers disease ?-amyloid peptides can be employed to study native-like aggregation intermediate states.
Chembiochem
PUBLISHED: 01-10-2011
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The structures of oligomeric intermediate states in the aggregation process of Alzheimers disease ?-amyloid peptides have been the subject of debate for many years. Bacterial inclusion bodies contain large amounts of small heat shock proteins (sHSPs), which are highly homologous to those found in the plaques of the brains of Alzheimers disease patients. sHSPs break down amyloid fibril structure in vitro and induce oligomeric assemblies. Prokaryotic protein overexpression thus mimics the conditions encountered in the cell under stress and allows the structures of A? aggregation intermediate states to be investigated under native-like conditions, which is not otherwise technically possible. We show that IB40/IB42 fulfil all the requirements to be classified as amyloids: they seed fibril growth, are Congo red positive and show characteristic ?-sheet-rich CD spectra. However, IB40 and IB42 are much less stable than fibrils formed in vitro and contain significant amounts of non-?-sheet regions, as seen from FTIR studies. Quantitative analyses of solution-state NMR H/D exchange rates show that the hydrophobic cores involving residues V18-F19-F20 adopt ?-sheet conformations, whereas the C termini adopt ?-helical coiled-coil structures. In the past, an ?-helical intermediate-state structure has been postulated, but could not be verified experimentally. In agreement with the current literature, in which A? oligomers are described as the most toxic state of the peptides, we find that IB42 contains SDS-resistant oligomers that are more neurotoxic than A?42 fibrils. E. coli inclusion bodies formed by the Alzheimers disease ?-amyloid peptides A?40 and A?42 thus behave structurally like amyloid aggregation intermediate states and open the possibility of studying amyloids in a native-like, cellular environment.
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Deciphering the role of the thermodynamic and kinetic stabilities of SH3 domains on their aggregation inside bacteria.
Proteomics
PUBLISHED: 11-19-2010
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The formation of insoluble deposits by globular proteins underlies the onset of many human diseases. Recent studies suggest a relationship between the thermodynamic stability of proteins and their in vivo aggregation. However, it has been argued that, in the cell, the occurrence of irreversible aggregation might shift the system from equilibrium, in such a way that it could be the rate of unfolding and associated kinetic stability instead of the conformational stability that controls protein deposition. This is an important but difficult to decipher question, because kinetic and thermodynamic stabilities appear usually correlated. Here we address this issue by comparing the in vitro folding kinetics and stability features of a set of non-natural SH3 domains with their aggregation properties when expressed in bacteria. In addition, we compare the in vitro stability of the isolated domains with their effective stability in conditions that mimic the cytosolic environment. Overall, the data argue in favor of a thermodynamic rather than a kinetic control of the intracellular aggregation propensities of small globular proteins in which folding and unfolding velocities largely exceed aggregation rates. These results have implications regarding the evolution of proteins.
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Protease inhibitors as models for the study of oxidative folding.
Antioxid. Redox Signal.
PUBLISHED: 10-12-2010
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The correct balance between proteases and their natural protein inhibitors is of great importance in living systems. Protease inhibitors usually comprise small folds that are crosslinked by a high number of disulfide bonds, making them perfect models for the study of oxidative folding. To date, the oxidative folding of numerous protease inhibitors has been analyzed, revealing a great diversity of folding pathways that differ mainly in the heterogeneity and native disulfide-bond content of their intermediates. The two extremes of this diversity are represented by bovine pancreatic trypsin inhibitor and hirudin, which fold, respectively, via few native intermediates and heterogeneous scrambled isomers. Other proteins, such as leech carboxypeptidase inhibitor, share characteristics of both models displaying mixed folding pathways. The study of the oxidative folding of two-domain inhibitors, such as secretory leukocyte protease inhibitor, tick carboxypeptidase inhibitor, and Ascaris carboxypeptidase inhibitor, has provided some clues about how two-domain protease inhibitors may fold, that is, either by folding each domain autonomously or with one domain assisting in the folding of the other. Finally, the recent determination of the structures of the major intermediates of protease inhibitors has shed light on the molecular mechanisms guiding the oxidative folding of small disulfide-rich proteins.
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Modulation of Abeta42 fibrillogenesis by glycosaminoglycan structure.
FASEB J.
PUBLISHED: 06-28-2010
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The role of amyloid ? (A?) peptide in the onset and progression of Alzheimers disease is linked to the presence of soluble A? species. Sulfated glycosaminoglycans (GAGs) promote A? fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment A?(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable A?(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-Å separating A?(42) monomers within protofibrillar ?-sheets. Incubation of A?(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, A?(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased ?3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between A?(42) fibrils induced by PVS and A?(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble A?(42) fibrils trapping soluble toxic forms.
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Amyloid-like protein inclusions in tobacco transgenic plants.
PLoS ONE
PUBLISHED: 05-28-2010
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The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered ?-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.
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The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils.
J. Mol. Biol.
PUBLISHED: 04-22-2010
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The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar ?-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, ?-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.
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Protein aggregation profile of the bacterial cytosol.
PLoS ONE
PUBLISHED: 01-30-2010
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Protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity.
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Protein folding and aggregation in bacteria.
Cell. Mol. Life Sci.
PUBLISHED: 01-18-2010
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Proteins might experience many conformational changes and interactions during their lifetimes, from their synthesis at ribosomes to their controlled degradation. Because, in most cases, only folded proteins are functional, protein folding in bacteria is tightly controlled genetically, transcriptionally, and at the protein sequence level. In addition, important cellular machinery assists the folding of polypeptides to avoid misfolding and ensure the attainment of functional structures. When these redundant protective strategies are overcome, misfolded polypeptides are recruited into insoluble inclusion bodies. The protein embedded in these intracellular deposits might display different conformations including functional and beta-sheet-rich structures. The latter assemblies are similar to the amyloid fibrils characteristic of several human neurodegenerative diseases. Interestingly, bacteria exploit the same structural principles for functional properties such as adhesion or cytotoxicity. Overall, this review illustrates how prokaryotic organisms might provide the bedrock on which to understand the complexity of protein folding and aggregation in the cell.
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Deciphering the structural basis that guides the oxidative folding of leech-derived tryptase inhibitor.
J. Biol. Chem.
PUBLISHED: 10-09-2009
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Protein folding mechanisms have remained elusive mainly because of the transient nature of intermediates. Leech-derived tryptase inhibitor (LDTI) is a Kazal-type serine proteinase inhibitor that is emerging as an attractive model for folding studies. It comprises 46 amino acid residues with three disulfide bonds, with one located inside a small triple-stranded antiparallel beta-sheet and with two involved in a cystine-stabilized alpha-helix, a motif that is widely distributed in bioactive peptides. Here, we analyzed the oxidative folding and reductive unfolding of LDTI by chromatographic and disulfide analyses of acid-trapped intermediates. It folds and unfolds, respectively, via sequential oxidation and reduction of the cysteine residues that give rise to a few 1- and 2-disulfide intermediates. Species containing two native disulfide bonds predominate during LDTI folding (IIa and IIc) and unfolding (IIa and IIb). Stop/go folding experiments demonstrate that only intermediate IIa is productive and oxidizes directly into the native form. The NMR structures of acid-trapped and further isolated IIa, IIb, and IIc reveal global folds similar to that of the native protein, including a native-like canonical inhibitory loop. Enzyme kinetics shows that both IIa and IIc are inhibitory-active, which may substantially reduce proteolysis of LDTI during its folding process. The results reported show that the kinetics of the folding reaction is modulated by the specific structural properties of the intermediates and together provide insights into the interdependence of conformational folding and the assembly of native disulfides during oxidative folding.
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Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion.
Microb. Cell Fact.
PUBLISHED: 07-20-2009
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The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied.
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Amyloidogenic regions and interaction surfaces overlap in globular proteins related to conformational diseases.
PLoS Comput. Biol.
PUBLISHED: 03-04-2009
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Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.
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Amyloids in bacterial inclusion bodies.
Trends Biochem. Sci.
PUBLISHED: 02-06-2009
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Protein misfolding and aggregation into amyloid structures are associated with dozens of human diseases. Recent studies have provided compelling evidence for the existence of highly ordered, amyloid-like conformations in the insoluble inclusion bodies produced during heterologous protein expression in bacteria. Thus, amyloid aggregation seems to be an omnipresent process in both eukaryotic and prokaryotic organisms. Amyloid formation inside cell factories raises important safety concerns with regard to the toxicity and infectivity of recombinant proteins. Yet such findings also suggest that prokaryotic cells could be useful systems for studying how and why proteins aggregate in vivo, and they could also provide a biologically relevant background for screening therapeutic approaches to pathologic protein deposition.
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Protein complementation assays: approaches for the in vivo analysis of protein interactions.
FEBS Lett.
PUBLISHED: 02-03-2009
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The in vivo identification and characterization of protein-protein interactions (PPIs) are essential to understand cellular events in living organisms. In this review, we focus on protein complementation assays (PCAs) that have been developed to detect in vivo protein interactions as well as their modulation or spatial and temporal changes. The uses of PCAs are increasing, spanning different areas such as the study of biochemical networks, screening for protein inhibitors and determination of drug effects. Emphasis is given to approaches that rely on signals of spectroscopic nature (i.e. fluorescence or luminescence), the ones that are more directly related to bioimaging.
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Detecting and interfering protein interactions: towards the control of biochemical pathways.
Curr. Med. Chem.
PUBLISHED: 01-20-2009
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Proteins almost never act in an isolated manner; they interact with other proteins in order to perform essential roles in many important cellular processes. Apart from their ability to form stable multiprotein complexes, proteins associate transiently with their targets to modify, regulate by steric effects, or translocate them to different cellular compartments. Therefore, the identification of molecules able to modulate such protein contacts is of significant interest for drug discovery and chemical biology, since it provides a means to exert control over cellular events. Nevertheless, finding antagonists of protein interactions displaying both target affinity and selectivity in the complex context of the cell proteome is a challenging task, because of the generally large, noncontiguous, interfaces involved in protein interactions. In this review we focus on recent advances in the detection, analysis and specific interference of protein interactions. These studies provide the basis for a promising avenue in medicinal chemistry towards the selective regulation of biochemical pathways.
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Designing out disulfide bonds of leech carboxypeptidase inhibitor: implications for its folding, stability and function.
J. Mol. Biol.
PUBLISHED: 01-13-2009
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Leech carboxypeptidase inhibitor (LCI) is a 67-residue, tight-binding metallocarboxypeptidase inhibitor composed of a compact domain with a five-stranded beta-sheet and a short alpha-helix that are strongly stabilized by four disulfide bonds. In this study, we investigated the contribution of each particular disulfide to the folding, stability and function of LCI by constructing a series of single and multiple mutants lacking one to four disulfide bonds. The results allow a better understanding of how individual disulfide bonds shape and restrict the conformational space that LCI must explore before attaining its native conformation. The work also dissected the role played by intramolecular rearrangements of disulfides during LCI folding, providing a new kinetic scheme in which the 2S ensemble suffers a non-specific oxidation into the 3S ensemble. These 3-disulfide-bonded species reshuffle to preferentially form III-A and III-B, two major native-like folding intermediates that need structural rearrangements through the formation of scrambled isomers to finally render native LCI. The designed multiple mutants of LCI are unable to fold correctly, displaying a highly unstructured conformation and a very low inhibitory capability, which indicates the importance of disulfide bonds in LCI for both correct folding and achievement of a functional structure. In contrast, the elimination of a single disulfide bond in LCI only results in a significant reduction of conformational stability, but the mutations have a rather moderate impact on carboxypeptidase inhibition, allowing the possibility to target the intrinsic stability and specific activity of LCI independently. In this way, the findings reported provide a basis for the design of novel variants of the molecule with improved therapeutic properties.
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Design, selection, and characterization of thioflavin-based intercalation compounds with metal chelating properties for application in Alzheimers disease.
J. Am. Chem. Soc.
PUBLISHED: 01-13-2009
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Metal chelation is considered a rational therapeutic approach for interdicting Alzheimers amyloid pathogenesis. At present, enhancing the targeting and efficacy of metal-ion chelating agents through ligand design is a main strategy in the development of the next generation of metal chelators. Inspired by the traditional dye Thioflavin-T, we have designed new multifunctional molecules that contain both amyloid binding and metal chelating properties. In silico techniques have enabled us to identify commercial compounds that enclose the designed molecular framework (M1), include potential antioxidant properties, facilitate the formation of iodine-labeled derivatives, and can be permeable through the blood-brain barrier. Iodination reactions of the selected compounds, 2-(2-hydroxyphenyl)benzoxazole (HBX), 2-(2-hydroxyphenyl)benzothiazole (HBT), and 2-(2-aminophenyl)-1H-benzimidazole (BM), have led to the corresponding iodinated derivatives HBXI, HBTI, and BMI, which have been characterized by X-ray diffraction. The chelating properties of the latter compounds toward Cu(II) and Zn(II) have been examined in the solid phase and in solution. The acidity constants of HBXI, HBTI, and BMI and the formation constants of the corresponding ML and ML2 complexes [M = Cu(II), Zn(II)] have been determined by UV-vis pH titrations. The calculated values for the overall formation constants for the ML2 complexes indicate the suitability of the HBXI, HBTI, and BMI ligands for sequestering Cu(II) and Zn(II) metal ions present in freshly prepared solutions of beta-amyloid (Abeta) peptide. This was confirmed by Abeta aggregation studies showing that these compounds are able to arrest the metal-promoted increase in amyloid fibril buildup. The fluorescence features of HBX, HBT, BM, and the corresponding iodinated derivatives, together with fluorescence microscopy studies on two types of pregrown fibrils, have shown that HBX and HBT compounds could behave as potential markers for the presence of amyloid fibrils, whereas HBXI and HBTI may be especially suitable for radioisotopic detection of Abeta deposits. Taken together, the results reported in this work show the potential of new multifunctional thioflavin-based chelating agents as Alzheimers disease therapeutics.
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Modeling amyloids in bacteria.
Microb. Cell Fact.
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An increasing number of proteins are being shown to assemble into amyloid structures, self-seeding fibrillar aggregates that may lead to pathological states or play essential biological functions in organisms. Bacterial cell factories have raised as privileged model systems to understand the mechanisms behind amyloid assembly and the cellular fitness cost associated to the formation of these aggregates. In the near future, these bacterial systems will allow implementing high-throughput screening approaches to identify effective modulators of amyloid aggregation.
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Multiple ?-sheet molecular dynamics of amyloid formation from two ABl-SH3 domain peptides.
Biopolymers
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Molecular dynamics simulations in explicit water were carried out for two stacks, each composed of six 10-strand antiparallel ?-sheets for two peptides corresponding to the diverging turn of two homologous Abl-SH3 domains. The first system, referred to as 10×6×MK contained the DLSFMKGE sequence from the Drosophila, while the second one, referred to as 10×6×KK, contained the human DLSFKKGE sequence. It was found that the 10×6×MK ?-sheet stack is stable, but the 10×6×KK ?-sheet stack is not. The stability of the 10×6×MK ?-sheet stack results from the hydrophobic interactions of the methionine and phenylalanine residues and the leucine residues of the neighboring sheets. The Met, Phe, and Leu hydrophobic units make a hydrophobic core for the stack of ?-sheets. During the MD run, the Met, Phe, and Leu residues of the neighboring ?-sheets acted as a conformational switch moving the ?-sheets so that the Phe residue interacted with the Met residue from the neighboring ?-sheet. Replacement of Met by Lys destroys the hydrophobic core, which is the stability factor of the ?-sheet stack. For the 10×6×KK system, individual ?-sheets were preserved during simulations, but the interactions between the ?-sheets were lost. The calculations of a six ?-sheet stack confirm the conclusion drawn from our earlier studies of single ?-sheet systems that the ?-sheets must form stacks to be stabilized. These results suggest that the two conserved basic residues at the diverging turn of SH3 domains could act as gatekeepers to avoid aggregation.
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Protein aggregation profile of the human kinome.
Front Physiol
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Protein aggregation into amyloid fibrils is associated with the onset of an increasing number of human disorders, including Alzheimers disease, diabetes, and some types of cancer. The ability to form toxic amyloids appears to be a property of most polypeptides. Accordingly, it has been proposed that reducing aggregation and its effect in cell fitness is a driving force in the evolution of proteins sequences. This control of protein solubility should be especially important for regulatory hubs in biological networks, like protein kinases. These enzymes are implicated in practically all processes in normal and abnormal cell physiology, and phosphorylation is one of the most frequent protein modifications used to control protein activity. Here, we use the AGGRESCAN algorithm to study the aggregation propensity of kinase sequences. We compared them with the rest of globular proteins to decipher whether they display differential aggregation properties. In addition, we compared the human kinase complement with the kinomes of other organisms to see if we can identify any evolutionary trend in the aggregational properties of this protein superfamily. Our analysis indicates that kinase domains display significant aggregation propensity, a property that decreases with increasing organism complexity.
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Zinc induced folding is essential for TIM15 activity as an mtHsp70 chaperone.
Biochim. Biophys. Acta
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TIM15/Zim17 in yeast and its mammalian ortholog Hep are Zn(2+) finger (Cys4) proteins that assist mtHsp70 in protein import into the mitochondrial matrix.
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Discovery of novel inhibitors of amyloid ?-peptide 1-42 aggregation.
J. Med. Chem.
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Alzheimers disease, characterized by deposits of amyloid ?-peptide (A?), is the most common neurodegenerative disease, but it still lacks a specific treatment. We have discovered five chemically unrelated inhibitors of the in vitro aggregation of the A?17-40 peptide by screening two commercial chemical libraries. Four of them (1-4) exhibit relatively low MCCs toward HeLa cells (17-184 ?M). The usefulness of compounds 1-4 to inhibit the in vivo aggregation of A?1-42 has been demonstrated using two fungi models, Saccharomyces cerevisiae and Podospora anserina, previously transformed to express A?1-42. Estimated IC(50)s are around 1-2 ?M. Interestingly, addition of any of the four compounds to sonicated preformed P. anserina aggregates completely inhibited the appearance of SDS-resistant oligomers. This combination of HTP in vitro screening with validation in fungi models provides an efficient way to identify novel inhibitory compounds of A?1-42 aggregation for subsequent testing in animal models.
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Evolutionary selection for protein aggregation.
Biochem. Soc. Trans.
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Protein aggregation is being found to be associated with an increasing number of human diseases. Aggregation can lead to a loss of function (lack of active protein) or to a toxic gain of function (cytotoxicity associated with protein aggregates). Although potentially harmful, protein sequences predisposed to aggregation seem to be ubiquitous in all kingdoms of life, which suggests an evolutionary advantage to having such segments in polypeptide sequences. In fact, aggregation-prone segments are essential for protein folding and for mediating certain protein-protein interactions. Moreover, cells use protein aggregates for a wide range of functions. Against this background, life has adapted to tolerate the presence of potentially dangerous aggregation-prone sequences by constraining and counteracting the aggregation process. In the present review, we summarize the current knowledge of the advantages associated with aggregation-prone stretches in proteomes and the strategies that cellular systems have developed to control the aggregation process.
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Thioflavin-S staining coupled to flow cytometry. A screening tool to detect in vivo protein aggregation.
Mol Biosyst
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Amyloid deposits are associated with an increasing number of human disorders, including Alzheimers and Parkinsons diseases. Recent studies provide compelling evidence for the existence of amyloid-like conformations in the insoluble bacterial inclusion bodies (IBs) produced during the recombinant expression of amyloidogenic proteins. This makes prokaryotic cells a physiologically relevant system to study the mechanisms of in vivo amyloid deposition. We show here that the application of flow cytometry to detect Thioflavin-S (Th-S) fluorescence provides a fast, robust, quantitative, non-invasive method to screen for the presence of in vivo intracellular amyloid-like aggregates in bacteria, with potential application in the analysis of the impact of genetic mutations or chemical compounds on the aggregation of disease-associated polypeptides.
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Current use of equations for estimating glomerular filtration rate in Spanish laboratories.
Nefrologia
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In 2006 the Spanish Society of Clinical Biochemistry and Molecular Pathology (SEQC) and the Spanish Society of Nephrology (S.E.N.) developed a consensus document in order to facilitate the diagnosis and monitoring of chronic kidney disease with the incorporation of equations for estimating glomerular filtration rate (eGFR) into laboratory reports. The current national prevalence of eGFR reporting and the degree of adherence to these recommendations among clinical laboratories is unknown.
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Yeast prions form infectious amyloid inclusion bodies in bacteria.
Microb. Cell Fact.
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Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively.
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Protein aggregation: mechanisms and functional consequences.
Int. J. Biochem. Cell Biol.
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Understanding the mechanisms underlying protein misfolding and aggregation has become a central issue in biology and medicine. Compelling evidence show that the formation of amyloid aggregates has a negative impact in cell function and is behind the most prevalent human degenerative disorders, including Alzheimers Parkinsons and Huntingtons diseases or type 2 diabetes. Surprisingly, the same type of macromolecular assembly is used for specialized functions by different organisms, from bacteria to human. Here we address the conformational properties of these aggregates, their formation pathways, their role in human diseases, their functional properties and how bioinformatics tools might be of help to study these protein assemblies.
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Protein oxidative folding in the intermembrane mitochondrial space: more than protein trafficking.
Curr. Protein Pept. Sci.
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The process of oxidative folding in the intermembrane mitochondrial space (IMS) is an exciting field of research because folding is simultaneously coupled to protein translocation and functional regulation. Contrary to the endoplasmatic reticulum ER where several chaperones of the disulfide isomerase family exist, oxidative folding in the IMS is exclusively catalyzed by the oxoreductase Mia40 that recognizes a group of proteins with characteristic cysteine motifs organized in twin CX(3)C, twin CX(9)C or CX(2)C motifs. In this review, we discuss the structural and biochemical studies leading to our current understanding of the Mia40 pathway as well as the open questions on the field. In fact, despite significant advances, several key points on the Mia40 pathway remain to clarify namely on the molecular mechanism trough which substrate oxidative folding is catalyzed. This issue is receiving increasing attention since failures in the import, sorting and folding of mitochondrial proteins is related to an increasing number of debilitating human disorders.
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Native structure protects SUMO proteins from aggregation into amyloid fibrils.
Biomacromolecules
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SUMO proteins belong to the Ubiquitin-like protein family, all sharing a common fold and a similar mechanism of conjugation to target polypeptides. SUMO is ubiquitous in all eukaryotes and participates in many crucial pathways. Native SUMO proteins are highly soluble, a property that is exploited in biotechnology. Moreover, SUMO regulates the solubility of aggregation-prone proteins in neurodegenerative disorders. Despite these properties, we show here that human SUMO1, SUMO2, and SUMO3 proteins are at risk of aggregation into amyloid structures if their native conformation is perturbed. Aggregation is mediated by specific regions, which overlap with SUMO functional interfaces, illustrating a competition between function and aggregation. Aggregation of SUMOs might have important physiological implications because disruption of the SUMO pathway is lethal in different organisms. It appears that functional constraints make it difficult to avoid the competition between productive folding and deleterious aggregation in globular proteins, even for essential polypeptides.
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Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors.
Microb. Cell Fact.
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The amyloid-? peptide (A?42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimers disease (AD). The mechanism by which A?42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders.
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Effect of the surface charge of artificial model membranes on the aggregation of amyloid ?-peptide.
Biochimie
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The neurotoxicity effect of the ?-amyloid (A?) peptide, the primary constituent of senile plaques in Alzheimers disease, occurs through interactions with neuronal membranes. Here, we attempt to clarify the mechanisms and consequences of the interaction of A? with lipid membranes. We have used liposomes as a model of biological membrane, and have devoted particular attention to the bilayer charge effect. Our results show that insertion and surface association of peptide with membrane, increased in a membrane charge-dependent manner, lead to a reduction of A? soluble species, lag time elongation and an increase in the inter-molecular ?-sheet ratio of amyloid fibrils. In addition, our findings suggest that the fine balance between peptide insertion and surface association modulates A? aggregation, influencing the amyloid fibrils concentration as well as their morphology.
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