Publications by authors named "Fernando L Palhano"

39 Publications

A water-soluble manganese(II) octanediaoate/phenanthroline complex acts as an antioxidant and attenuates alpha-synuclein toxicity.

Biochim Biophys Acta Mol Basis Dis 2022 10 28;1868(10):166475. Epub 2022 Jun 28.

Departamento de Bioquímica, Instituto de Química, Centro de Tecnologia, Cidade Universitária, Universidade Federal do Rio de Janeiro, Brazil; Rede de Micrologia RJ-FAPERJ, Brazil. Electronic address:

The overproduction of reactive oxygen species (ROS) induces oxidative stress, a well-known process associated with aging and several human pathologies, such as cancer and neurodegenerative diseases. A large number of synthetic compounds have been described as antioxidant enzyme mimics, capable of eliminating ROS and/or reducing oxidative damage. In this study, we investigated the antioxidant activity of a water-soluble 1,10-phenantroline-octanediaoate Mn-complex on cells under oxidative stress, and assessed its capacity to attenuate alpha-synuclein (aSyn) toxicity and aggregation, a process associated with increased oxidative stress. This Mn-complex exhibited a significant antioxidant potential, reducing intracelular oxidation and increasing oxidative stress resistance in S. cerevisiae cells and in vivo, in G. mellonella, increasing the activity of the intracellular antioxidant enzymes superoxide dismutase and catalase. Strikingly, the Mn-complex reduced both aSyn oligomerization and aggregation in human cell cultures and, using NMR and DFT/molecular docking we confirmed its interaction with the C-terminal region of aSyn. In conclusion, the Mn-complex appears as an excellent lead for the design of new phenanthroline derivatives as alternative compounds for preventing oxidative damages and oxidative stress - related diseases.
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http://dx.doi.org/10.1016/j.bbadis.2022.166475DOI Listing
October 2022

A systematic structural comparison of all solved small proteins deposited in PDB. The effect of disulfide bonds in protein fold.

Comput Struct Biotechnol J 2021 17;19:6255-6262. Epub 2021 Nov 17.

Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil.

Defensins are small proteins, usually ranging from 3 to 6 kDa, amphipathic, disulfide-rich, and with a small or even absent hydrophobic core. Since a hydrophobic core is generally found in globular proteins that fold in an aqueous solvent, the peculiar fold of defensins can challenge tertiary protein structure predictors. We performed a Protein Data Bank survey of small proteins (3-6 kDa) to understand the similarities of defensins with other small disulfide-rich proteins. We found no differences when we compared defensins with non-defensins regarding the proportion of apolar, polar and charged residues and their exposure to the solvent. Then we divided all small proteins (3-6 kDa) in the Protein Data Bank into two groups, one group with at least one disulfide bond (bonded, defensins included) and another group without any disulfide bond (unbonded). The group of bonded proteins contained apolar residues more exposed to the solvent than the unbonded group. The algorithm for tertiary protein structure prediction Robetta was more accurate at predicting unbonded than bonded proteins. On the other hand, the trRosetta algorithm, which uses artificial intelligence, improved the prediction of most bonded proteins, while for the unbonded group no improvement was obtained. Our work highlights one more layer of complexity for the prediction of protein tertiary structure: The ability of small disulfide-rich proteins to fold even with a poorly hydrophobic core.
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http://dx.doi.org/10.1016/j.csbj.2021.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8712280PMC
November 2021

Green Tea Polyphenol Epigallocatechin-Gallate in Amyloid Aggregation and Neurodegenerative Diseases.

Front Neurosci 2021 14;15:718188. Epub 2021 Sep 14.

Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

The accumulation of protein aggregates in human tissues is a hallmark of more than 40 diseases called amyloidoses. In seven of these disorders, the aggregation is associated with neurodegenerative processes in the central nervous system such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The aggregation occurs when certain soluble proteins lose their physiological function and become toxic amyloid species. The amyloid assembly consists of protein filament interactions, which can form fibrillar structures rich in β-sheets. Despite the frequent incidence of these diseases among the elderly, the available treatments are limited and at best palliative, and new therapeutic approaches are needed. Among the many natural compounds that have been evaluated for their ability to prevent or delay the amyloidogenic process is epigallocatechin-3-gallate (EGCG), an abundant and potent polyphenolic molecule present in green tea that has extensive biological activity. There is evidence for EGCG's ability to inhibit the aggregation of α-synuclein, amyloid-β, and huntingtin proteins, respectively associated with PD, AD, and HD. It prevents fibrillogenesis ( and ), reduces amyloid cytotoxicity, and remodels fibrils to form non-toxic amorphous species that lack seed propagation. Although it is an antioxidant, EGCG in an oxidized state can promote fibrils' remodeling through formation of Schiff bases and crosslinking the fibrils. Moreover, microparticles to drug delivery were synthesized from oxidized EGCG and loaded with a second anti-amyloidogenic molecule, obtaining a synergistic therapeutic effect. Here, we describe several pre-clinical and clinical studies involving EGCG and neurodegenerative diseases and their related mechanisms.
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http://dx.doi.org/10.3389/fnins.2021.718188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8477582PMC
September 2021

Cerebral dopamine neurotrophic factor reduces α-synuclein aggregation and propagation and alleviates behavioral alterations in vivo.

Mol Ther 2021 09 1;29(9):2821-2840. Epub 2021 May 1.

Institute of Biotechnology, HiLIFE, University of Helsinki, 00014 Helsinki, Finland; Neuroscience Center, HiLIFE, University of Helsinki, 00014 Helsinki, Finland; Faculty of Pharmacy, University of Helsinki, Helsinki, Finland. Electronic address:

A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a K = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.
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http://dx.doi.org/10.1016/j.ymthe.2021.04.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417450PMC
September 2021

Pentameric Thiophene as a Probe to Monitor EGCG's Remodeling Activity of Mature Amyloid Fibrils: Overcoming Signal Artifacts of Thioflavin T.

ACS Omega 2021 Mar 16;6(12):8700-8705. Epub 2021 Mar 16.

Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.

Thioflavin T fluorescence is a gold standard probe for the detection of amyloid fibrils. Herein, we showed that mature amyloid fibrils incubated with polyphenol epigallocatechin gallate (EGCG) present a fast reduction of the thioflavin T fluorescence, which is not related to remodeling activity. We propose the use of the pentameric thiophene fluorescence for monitoring the polyphenol remodeling activity.
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http://dx.doi.org/10.1021/acsomega.1c00680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015118PMC
March 2021

Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex.

J Biol Chem 2021 Jan-Jun;296:100586. Epub 2021 Mar 24.

Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Electronic address:

Previous work has suggested that highly positively charged protein segments coded by rare codons or poly (A) stretches induce ribosome stalling and translational arrest through electrostatic interactions with the negatively charged ribosome exit tunnel, leading to inefficient elongation. This arrest leads to the activation of the Ribosome Quality Control (RQC) pathway and results in low expression of these reporter proteins. However, the only endogenous yeast proteins known to activate the RQC are Rqc1, a protein essential for RQC function, and Sdd1, a protein with unknown function, both of which contain polybasic sequences. To explore the generality of this phenomenon, we investigated whether the RQC complex controls the expression of other proteins with polybasic sequences. We showed by ribosome profiling data analysis and western blot that proteins containing polybasic sequences similar to, or even more positively charged than those of Rqc1 and Sdd1, were not targeted by the RQC complex. We also observed that the previously reported Ltn1-dependent regulation of Rqc1 is posttranslational, independent of the RQC activity. Taken together, our results suggest that RQC should not be regarded as a general regulatory pathway for the expression of highly positively charged proteins in yeast.
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http://dx.doi.org/10.1016/j.jbc.2021.100586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102910PMC
August 2021

Green Tea Polyphenol Microparticles Based on the Oxidative Coupling of EGCG Inhibit Amyloid Aggregation/Cytotoxicity and Serve as a Platform for Drug Delivery.

ACS Biomater Sci Eng 2020 08 9;6(8):4414-4423. Epub 2020 Jul 9.

Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.

The accumulation of cross-β-sheet amyloid fibrils is a hallmark of all human amyloid diseases. The compound epigallocatechin-3-gallate (EGCG), the main polyphenol present in green tea, has been described to have beneficial effects in several pathologies, including amyloidogenic diseases. This polyphenol blocks amyloidogenesis and disaggregates a broad range of amyloidogenic peptides comprising amyloid fibrils . The mechanism by which EGCG acts in the context of amyloid aggregation is not clear. Most of the biological effects of EGCG are attributable to its antioxidant activity. However, EGCG-oxidized products appear to be sufficient for the majority of EGCG amyloid remodeling observed against some polypeptides. If controlled, EGCG oxidation can afford homogenous microparticles (MPs) and can serve as drug delivery agents. Herein, we produced EGCG MPs by oxidative coupling and analyzed their activity during the aggregation of the protein α-synuclein (α-syn), the main protein related to Parkinson's disease. The MPs modestly remodeled mature amyloid fibrils and efficiently inhibited the amyloidogenic aggregation of α-syn. The MPs showed low cytotoxicity against both dopaminergic cells and microglial cells. The MPs reduced the cytotoxic effects of α-syn oligomers. Interestingly, the MPs were loaded with another antiamyloidogenic compound, increasing their activity against amyloid aggregation. We propose the use of EGCG MPs as a bifunctional strategy, blocking amyloid aggregation directly and carrying a molecule that can act synergistically to alleviate the symptoms caused by the amyloidogenic pathway.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00188DOI Listing
August 2020

New Cardiomyokine Reduces Myocardial Ischemia/Reperfusion Injury by PI3K-AKT Pathway Via a Putative KDEL-Receptor Binding.

J Am Heart Assoc 2021 01 29;10(1):e019685. Epub 2020 Dec 29.

Institute of Medical Biochemistry Leopoldo de Meis Rio de Janeiro Federal, University of Rio de Janeiro Brazil.

Background CDNF (cerebral dopamine neurotrophic factor) belongs to a new family of neurotrophic factors that exert systemic beneficial effects beyond the brain. Little is known about the role of CDNF in the cardiac context. Herein we investigated the effects of CDNF under endoplasmic reticulum-stress conditions using cardiomyocytes (humans and mice) and isolated rat hearts, as well as in rats subjected to ischemia/reperfusion (I/R). Methods and Results We showed that CDNF is secreted by cardiomyocytes stressed by thapsigargin and by isolated hearts subjected to I/R. Recombinant CDNF (exoCDNF) protected human and mouse cardiomyocytes against endoplasmic reticulum stress and restored the calcium transient. In isolated hearts subjected to I/R, exoCDNF avoided mitochondrial impairment and reduced the infarct area to 19% when administered before ischemia and to 25% when administered at the beginning of reperfusion, compared with an infarct area of 42% in the untreated I/R group. This protection was completely abrogated by AKT (protein kinase B) inhibitor. Heptapeptides containing the KDEL sequence, which binds to the KDEL-R (KDEL receptor), abolished exoCDNF beneficial effects, suggesting the participation of KDEL-R in this cardioprotection. CDNF administered intraperitoneally to rats decreased the infarct area in an in vivo model of I/R (from an infarct area of ≈44% in the I/R group to an infarct area of ≈27%). Moreover, a shorter version of CDNF, which lacks the last 4 residues (CDNF-ΔKTEL) and thus allows CDNF binding to KDEL-R, presented no cardioprotective activity in isolated hearts. Conclusions This is the first study to propose CDNF as a new cardiomyokine that induces cardioprotection via KDEL receptor binding and PI3K/AKT activation.
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http://dx.doi.org/10.1161/JAHA.120.019685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955482PMC
January 2021

Natural variation of the cardiac transcriptome in humans.

RNA Biol 2021 10 11;18(10):1374-1381. Epub 2020 Dec 11.

Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de janeiro, Rio de Janeiro, Brazil.

We investigated the gene-expression variation among humans by analysing previously published mRNA-seq and ribosome footprint profiling of heart left-ventricles from healthy donors. We ranked the genes according to their coefficient of variation values and found that the top 5% most variable genes had special features compared to the rest of the genome, such as lower mRNA levels and shorter half-lives coupled to increased translation efficiency. We observed that these genes are mostly involved with immune response and have a pleiotropic effect on disease phenotypes, indicating that asymptomatic conditions contribute to the gene expression diversity of healthy individuals.
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http://dx.doi.org/10.1080/15476286.2020.1857508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489943PMC
October 2021

Acetylsalicylic acid and salicylic acid present anticancer properties against melanoma by promoting nitric oxide-dependent endoplasmic reticulum stress and apoptosis.

Sci Rep 2020 11 12;10(1):19617. Epub 2020 Nov 12.

Laboratório de Oncobiologia Molecular, Departamento de Biotecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil.

Melanoma is the most aggressive and fatal type of skin cancer due to being highly proliferative. Acetylsalicylic acid (ASA; Aspirin) and salicylic acid (SA) are ancient drugs with multiple applications in medicine. Here, we showed that ASA and SA present anticancer effects against a murine model of implanted melanoma. These effects were also validated in 3D- and 2D-cultured melanoma B16F10 cells, where the drugs promoted pro-apoptotic effects. In both in vivo and in vitro models, SA and ASA triggered endoplasmic reticulum (ER) stress, which culminates with the upregulation of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). These effects are initiated by ASA/SA-triggered Akt/mTOR/AMPK-dependent activation of nitric oxide synthase 3 (eNOS), which increases nitric oxide and reactive oxygen species production inducing ER stress response. In the end, we propose that ASA and SA instigate anticancer effects by a novel mechanism, the activation of ER stress.
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http://dx.doi.org/10.1038/s41598-020-76824-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665072PMC
November 2020

Influence of nascent polypeptide positive charges on translation dynamics.

Biochem J 2020 08;477(15):2921-2934

Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil.

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.
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http://dx.doi.org/10.1042/BCJ20200303DOI Listing
August 2020

Viruses with different genome types adopt a similar strategy to pack nucleic acids based on positively charged protein domains.

Sci Rep 2020 03 25;10(1):5470. Epub 2020 Mar 25.

Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Rio de Janeiro, 21941-902, Brazil.

Capsid proteins often present a positively charged arginine-rich sequence at their terminal regions, which has a fundamental role in genome packaging and particle stability for some icosahedral viruses. These sequences show little to no conservation and are structurally dynamic such that they cannot be easily detected by common sequence or structure comparisons. As a result, the occurrence and distribution of positively charged domains across the viral universe are unknown. Based on the net charge calculation of discrete protein segments, we identified proteins containing amino acid stretches with a notably high net charge (Q > + 17), which are enriched in icosahedral viruses with a distinctive bias towards arginine over lysine. We used viral particle structural data to calculate the total electrostatic charge derived from the most positively charged protein segment of capsid proteins and correlated these values with genome charges arising from the phosphates of each nucleotide. We obtained a positive correlation (r = 0.91, p-value <0001) for a group of 17 viral families, corresponding to 40% of all families with icosahedral structures described to date. These data indicated that unrelated viruses with diverse genome types adopt a common underlying mechanism for capsid assembly based on R-arms.
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http://dx.doi.org/10.1038/s41598-020-62328-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096446PMC
March 2020

From reporters to endogenous genes: the impact of the first five codons on translation efficiency in .

RNA Biol 2019 12 5;16(12):1806-1816. Epub 2019 Sep 5.

Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5' UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3 to the 5 codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as , suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.
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http://dx.doi.org/10.1080/15476286.2019.1661213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844562PMC
December 2019

Inflammatory profiling of patients with familial amyloid polyneuropathy.

BMC Neurol 2019 Jun 28;19(1):146. Epub 2019 Jun 28.

Instituto de Bioquímica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Background: Familial amyloid polyneuropathy (FAP) or ATTRv (amyloid TTR variant) amyloidosis is a fatal hereditary disease characterized by the deposition of amyloid fibrils composed of transthyretin (TTR). The current diagnosis of ATTRv relies on genetic identification of TTR mutations and on Congo Red-positive amyloid deposits, which are absent in most ATTRv patients that are asymptomatic or early symptomatic, supporting the need for novel biomarkers to identify patients in earlier disease phases allowing disease control.

Methods: In an effort to search for new markers for ATTRv, our group searched for nine inflammation markers in ATTRv serum from a cohort of 28 Brazilian ATTRv patients.

Results: We found that the levels of six markers were increased (TNF-α, IL-1β, IL-8, IL-33, IFN-β and IL-10), one had decreased levels (IL-12) and two of them were unchanged (IL-6 and cortisol). Interestingly, asymptomatic patients already presented high levels of IL-33, IL-1β and IL-10, suggesting that inflammation may take place before fibril deposition.

Conclusions: Our findings shed light on a new, previously unidentified aspect of ATTRv, which might help define new criteria for disease management, as well as provide additional understanding of ATTRv aggressiveness.
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http://dx.doi.org/10.1186/s12883-019-1369-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599258PMC
June 2019

Codon stabilization coefficient as a metric to gain insights into mRNA stability and codon bias and their relationships with translation.

Nucleic Acids Res 2019 03;47(5):2216-2228

Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil.

The codon stabilization coefficient (CSC) is derived from the correlation between each codon frequency in transcripts and mRNA half-life experimental data. In this work, we used this metric as a reference to compare previously published Saccharomyces cerevisiae mRNA half-life datasets and investigate how codon composition related to protein levels. We generated CSCs derived from nine studies. Four datasets produced similar CSCs, which also correlated with other independent parameters that reflected codon optimality, such as the tRNA abundance and ribosome residence time. By calculating the average CSC for each gene, we found that most mRNAs tended to have more non-optimal codons. Conversely, a high proportion of optimal codons was found for genes coding highly abundant proteins, including proteins that were only transiently overexpressed in response to stress conditions. We also used CSCs to identify and locate mRNA regions enriched in non-optimal codons. We found that these stretches were usually located close to the initiation codon and were sufficient to slow ribosome movement. However, in contrast to observations from reporter systems, we found no position-dependent effect on the mRNA half-life. These analyses underscore the value of CSCs in studies of mRNA stability and codon bias and their relationships with protein expression.
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http://dx.doi.org/10.1093/nar/gkz033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412131PMC
March 2019

Selective and Sensitive Pull Down of Amyloid Fibrils Produced in Vitro and in Vivo by the Use of Pentameric-Thiophene-Coupled Resins.

ACS Chem Neurosci 2018 11 31;9(11):2807-2814. Epub 2018 May 31.

Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural , Universidade Federal do Rio de Janeiro , Rio de Janeiro , RJ 21941-590 , Brazil.

Protein aggregation is a hallmark of several degenerative diseases, including Alzheimer's disease, Parkinson's disease and familial amyloidosis (Finnish type) (FAF). A method to isolate and detect amyloids is desired for the diagnosis of amyloid diseases. Here, we report the synthesis of pentameric thiophene amyloid ligand (p-FTAA) linked to agarose resin for selective purification of amyloid aggregates produced in vitro and in vivo. Using amyloid fibrils produced in vitro from α-synuclein, gelsolin, and Aβ and gelsolin amyloid aggregates extracted from tissue homogenates of a mouse model of FAF, we observed that p-FTAA resin was able to pull down amyloid aggregates. The functionalized resin was also able to pull down oligomers produced in vitro from the A30P variant of α-synuclein. The methodology described here can be useful for the diagnosis of amyloidogenic disease and also can be used to purify amyloid fibrils from biological samples, rendering the fibrils available for more accurate structural and biochemical characterization.
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http://dx.doi.org/10.1021/acschemneuro.8b00222DOI Listing
November 2018

Protein charge distribution in proteomes and its impact on translation.

PLoS Comput Biol 2017 05 22;13(5):e1005549. Epub 2017 May 22.

Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

As proteins are synthesized, the nascent polypeptide must pass through a negatively charged exit tunnel. During this stage, positively charged stretches can interact with the ribosome walls and slow the translation. Therefore, charged polypeptides may be important factors that affect protein expression. To determine the frequency and distribution of positively and negatively charged stretches in different proteomes, the net charge was calculated for every 30 consecutive amino acid residues, which corresponds to the length of the ribosome exit tunnel. The following annotated and reviewed proteins in the UniProt database (Swiss-Prot) were analyzed: 551,705 proteins from different organisms and a total of 180 million protein segments. We observed that there were more negative than positive stretches and that super-charged positive sequences (i.e., net charges ≥ 14) were underrepresented in the proteomes. Overall, the proteins were more positively charged at their N-termini and C-termini, and this feature was present in most organisms and subcellular localizations. To investigate whether the N-terminal charges affect the elongation rates, previously published ribosomal profiling data obtained from S. cerevisiae, without translation-interfering drugs, were analyzed. We observed a nonlinear effect of the charge on the ribosome occupancy in which values ≥ +5 and ≤ -6 showed increased and reduced ribosome densities, respectively. These groups also showed different distributions across 80S monosomes and polysomes. Basic polypeptides are more common within short proteins that are translated by monosomes, whereas negative stretches are more abundant in polysome-translated proteins. These findings suggest that the nascent peptide charge impacts translation and can be one of the factors that regulate translation efficiency and protein expression.
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http://dx.doi.org/10.1371/journal.pcbi.1005549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460897PMC
May 2017

An ortho-Iminoquinone Compound Reacts with Lysine Inhibiting Aggregation while Remodeling Mature Amyloid Fibrils.

ACS Chem Neurosci 2017 08 4;8(8):1704-1712. Epub 2017 May 4.

Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro , Rio de Janeiro 21941-590, Brazil.

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aβ as model amyloidogenic proteins, we observed that IQ was able to react with lysine residues and reduce amyloid aggregation. We also observed that IQ reacted with free amines within the amyloid fibrils preventing their dissociation and seeding capacity.
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http://dx.doi.org/10.1021/acschemneuro.7b00017DOI Listing
August 2017

Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca-binding affinity in a C-domain mutant of cardiac troponin C.

Sci Rep 2017 04 6;7(1):691. Epub 2017 Apr 6.

Department of Biomedical Sciences, Florida State University College of Medicine, 1115 West Call Street, Tallahassee, FL, 32306-4300, USA.

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.
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http://dx.doi.org/10.1038/s41598-017-00777-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429600PMC
April 2017

Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation.

Sci Rep 2017 03 24;7:44709. Epub 2017 Mar 24.

Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain.

More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.
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http://dx.doi.org/10.1038/srep44709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364509PMC
March 2017

Increased ribosome density associated to positively charged residues is evident in ribosome profiling experiments performed in the absence of translation inhibitors.

RNA Biol 2016 06 11;13(6):561-8. Epub 2016 Apr 11.

a Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro , Rio de Janeiro , RJ , Brazil.

It has been proposed that polybasic peptides cause slower movement of ribosomes through an electrostatic interaction with the highly negative ribosome exit tunnel. Ribosome profiling data-the sequencing of short ribosome-bound fragments of mRNA-is a powerful tool for the analysis of mRNA translation. Using the yeast Saccharomyces cerevisiae as a model, we showed that reduced translation efficiency associated with polybasic protein sequences could be inferred from ribosome profiling. However, an increase in ribosome density at polybasic sequences was evident only when the commonly used translational inhibitors cycloheximide and anisomycin were omitted during mRNA isolation. Since ribosome profiling performed without inhibitors agrees with experimental evidence obtained by other methods, we conclude that cycloheximide and anisomycin must be avoided in ribosome profiling experiments.
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http://dx.doi.org/10.1080/15476286.2016.1172755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962802PMC
June 2016

Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/M)P from Fungi.

PLoS One 2015 1;10(9):e0136761. Epub 2015 Sep 1.

Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Pyridoxinamine 5'-phosphate oxidases (P(N/M)P oxidases) that bind flavin mononucleotide (FMN) and oxidize pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate (PLP) are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/M)P oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/M)P oxidases, we found that the protein domain (PF01243) that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/M)P oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136761PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556617PMC
May 2016

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation.

J Biol Chem 2015 Sep 21;290(36):22174-83. Epub 2015 Jul 21.

From the Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902 and

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation.
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http://dx.doi.org/10.1074/jbc.M115.640094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571968PMC
September 2015

The Solution Structure and Dynamics of Full-length Human Cerebral Dopamine Neurotrophic Factor and Its Neuroprotective Role against α-Synuclein Oligomers.

J Biol Chem 2015 Aug 6;290(33):20527-40. Epub 2015 Jul 6.

From the Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21.941-902, Brazil,

Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.
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http://dx.doi.org/10.1074/jbc.M115.662254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536457PMC
August 2015

Immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils.

PLoS One 2014 21;9(8):e105433. Epub 2014 Aug 21.

Departments of Chemistry and Molecular and Experimental Medicine and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America; Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Amyloid fibrils are associated with many maladies, including Alzheimer's disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ(1-40) amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ(1-40) amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ(1-40) amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105433PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140755PMC
May 2015

Conformational changes in human Hsp70 induced by high hydrostatic pressure produce oligomers with ATPase activity but without chaperone activity.

Biochemistry 2014 May 29;53(18):2884-9. Epub 2014 Apr 29.

Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro , Rio de Janeiro 21941-590, Brazil.

We investigated the folding of the 70 kDa human cytosolic inducible protein (Hsp70) in vitro using high hydrostatic pressure as a denaturing agent. We followed the structural changes in Hsp70 induced by high hydrostatic pressure using tryptophan fluorescence, molecular dynamics, circular dichroism, high-performance liquid chromatography gel filtration, dynamic light scattering, ATPase activity, and chaperone activity. Although monomeric, Hsp70 is very sensitive to hydrostatic pressure; after pressure had been removed, the protein did not return to its native sate but instead formed oligomeric species that lost chaperone activity but retained ATPase activity.
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http://dx.doi.org/10.1021/bi500004qDOI Listing
May 2014

Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes.

Protein Sci 2013 Nov 30;22(11):1531-41. Epub 2013 Sep 30.

Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, 92037; Department of Molecular and Experimental Medicine and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, 92037.

The accumulation of cross-β-sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as Aβ disaggregation in vitro. Here, we show that Aβ fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of Aβ amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate Aβ amyloid fibrils in vitro at 37°C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused.
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http://dx.doi.org/10.1002/pro.2339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831668PMC
November 2013

Toward the molecular mechanism(s) by which EGCG treatment remodels mature amyloid fibrils.

J Am Chem Soc 2013 May 7;135(20):7503-10. Epub 2013 May 7.

Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Protein misfolding and/or aggregation has been implicated as the cause of several human diseases, such as Alzheimer's and Parkinson's diseases and familial amyloid polyneuropathy. These maladies are referred to as amyloid diseases, named after the cross-β-sheet amyloid fibril aggregates or deposits common to these disorders. Epigallocatechin-3-gallate (EGCG), the principal polyphenol present in green tea, has been shown to be effective at preventing aggregation and is able to remodel amyloid fibrils comprising different amyloidogenic proteins, although the mechanistic underpinnings are unclear. Herein, we work toward an understanding of the molecular mechanism(s) by which EGCG remodels mature amyloid fibrils made up of Aβ(1-40), IAPP(8-24), or Sup35NM(7-16). We show that EGCG amyloid remodeling activity in vitro is dependent on auto-oxidation of the EGCG. Oxidized and unoxidized EGCG binds to amyloid fibrils, preventing the binding of thioflavin T. This engagement of the hydrophobic binding sites in Aβ(1-40), IAPP(8-24), or Sup35NM(Ac7-16) Y→F amyloid fibrils seems to be sufficient to explain the majority of the amyloid remodeling observed by EGCG treatment, although how EGCG oxidation drives remodeling remains unclear. Oxidized EGCG molecules react with free amines within the amyloid fibril through the formation of Schiff bases, cross-linking the fibrils, which may prevent dissociation and toxicity, but these aberrant post-translational modifications do not appear to be the major driving force for amyloid remodeling by EGCG treatment. These insights into the molecular mechanism of action of EGCG provide boundary conditions for exploring amyloid remodeling in more detail.
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http://dx.doi.org/10.1021/ja3115696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674815PMC
May 2013

Amyloid fibrils trigger the release of neutrophil extracellular traps (NETs), causing fibril fragmentation by NET-associated elastase.

J Biol Chem 2012 Oct 23;287(44):37206-18. Epub 2012 Aug 23.

Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil.

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.
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http://dx.doi.org/10.1074/jbc.M112.369942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3481320PMC
October 2012
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