Publications by authors named "Anderson S Pinheiro"

31 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

Phase separation of the mammalian prion protein: Physiological and pathological perspectives.

J Neurochem 2022 Feb 11. Epub 2022 Feb 11.

Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, Rio de Janeiro-RJ, Brazil.

Abnormal phase transitions have been implicated in the occurrence of proteinopathies. Disordered proteins with nucleic acidbinding ability drive the formation of reversible micron-sized condensates capable of controlling nucleic acid processing/transport. This mechanism, achieved via liquid-liquid phase separation (LLPS), underlies the formation of long-studied membraneless organelles (e.g., nucleolus) and various transient condensates formed by driver proteins. The prion protein (PrP) is not a classical nucleic acid-binding protein. However, it binds nucleic acids with high affinity, undergoes nucleocytoplasmic shuttling, contains a long intrinsically disordered region rich in glycines and evenly spaced aromatic residues, among other biochemical/biophysical properties of bona fide drivers of phase transitions. Because of this, our group and others have characterized LLPS of recombinant PrP. In vitro phase separation of PrP is modulated by nucleic acid aptamers, and depending on the aptamer conformation, the liquid droplets evolve to solid-like species. Herein, we discuss recent studies and previous evidence supporting PrP phase transitions. We focus on the central role of LLPS related to PrP physiology and pathology, with a special emphasis on the interaction of PrP with different ligands, such as proteins and nucleic acids, which can play a role in prion disease pathogenesis. Finally, we comment on therapeutic strategies directed at the non-functional phase separation that could potentially tackle prion diseases or other protein misfolding disorders.
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http://dx.doi.org/10.1111/jnc.15586DOI Listing
February 2022

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

Int J Biol Macromol 2022 Apr 22;203:466-480. Epub 2022 Jan 22.

Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; National Center of Nuclear Magnetic Resonance (CNRMN), CENABIO, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; Rio BioNMR Network, Rio de Janeiro, Brazil. Electronic address:

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to β-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.
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http://dx.doi.org/10.1016/j.ijbiomac.2022.01.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8783401PMC
April 2022

Dynamics of the SARS-CoV-2 nucleoprotein N-terminal domain triggers RNA duplex destabilization.

Biophys J 2021 07 29;120(14):2814-2827. Epub 2021 Jun 29.

Institute of Medical Biochemistry Leopoldo de Meis and National Center for Structural Biology and Bioimaging, Rio de Janeiro, Brazil. Electronic address:

The nucleocapsid (N) protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. The N protein N-terminal domain (N-NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRSs), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of the severe acute respiratory syndrome coronavirus 2 N-NTD to nonspecific (NS) and TRS dsRNAs. We probed dsRNAs' Watson-Crick basepairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, triggering melting initiation. dsRNA destabilization driven by N-NTD was more efficient for dsTRSs than dsNS. N-NTD dynamics, especially a tweezer-like motion of β2-β3 and Δ2-β5 loops, seems to play a key role in Watson-Crick basepairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD/dsRNA), matching MD simulations and raising different possibilities for N-NTD action: 1) two N-NTD arms of dimeric N would bind to two different RNA sites, either closely or spatially spaced in the viral genome, in a cooperative manner; and 2) monomeric N-NTD would be active, opening up the possibility of a regulatory dissociation event.
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http://dx.doi.org/10.1016/j.bpj.2021.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239202PMC
July 2021

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

Front Mol Biosci 2021 10;8:653148. Epub 2021 May 10.

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, United States.

The highly infectious disease COVID-19 caused by the SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on , we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
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http://dx.doi.org/10.3389/fmolb.2021.653148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141814PMC
May 2021

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest .

J Venom Anim Toxins Incl Trop Dis 2021 Mar 12;27:e20200127. Epub 2021 Mar 12.

Department of Biochemistry, Institute of Chemistry, Center of Mathematical and Natural Sciences, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil.

Abstract:

Background: Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests.

Methods: AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive () and gram-negative ( and ) bacteria.

Results: AgCecropB was expressed in BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive bacteria growth.

Conclusions: The first cecropin B-like peptide was described in and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit growth .
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http://dx.doi.org/10.1590/1678-9199-JVATITD-2020-0127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970720PMC
March 2021

Identification of Chalcone Derivatives as Inhibitors of Arginase and Promising Antileishmanial Agents.

Front Chem 2020 14;8:624678. Epub 2021 Jan 14.

Graduate Program in Pharmaceutical Sciences, School of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

Arginase catalyzes the hydrolysis of l-arginine into l-ornithine and urea, acting as a key enzyme in the biosynthesis of polyamines. growth and survival is dependent on polyamine biosynthesis; therefore, inhibition of arginase may be a promising therapeutic strategy. Here, we evaluated a series of thirty-six chalcone derivatives as potential inhibitors of arginase (LiARG). In addition, the activity of selected inhibitors against parasites was assessed . Seven compounds exhibited LiARG inhibition above 50% at 100 μM. Among them, compounds LC41, LC39, and LC32 displayed the greatest inhibition values (72.3 ± 0.3%, 71.9 ± 11.6%, and 69.5 ± 7.9%, respectively). Molecular docking studies predicted hydrogen bonds and hydrophobic interactions between the most active chalcones (LC32, LC39, and LC41) and specific residues from LiARG's active site, such as His140, Asn153, His155, and Ala193. Compound LC32 showed the highest activity against promastigotes (IC of 74.1 ± 10.0 μM), whereas compounds LC39 and LC41 displayed the best results against intracellular amastigotes (IC of 55.2 ± 3.8 and 70.4 ± 9.6 μM, respectively). Moreover, compound LC39 showed more selectivity against parasites than host cells (macrophages), with a selectivity index (SI) of 107.1, even greater than that of the reference drug Fungizone®. Computational pharmacokinetic and toxicological evaluations showed high oral bioavailability and low toxicity for the most active compounds. The results presented here support the use of substituted chalcone skeletons as promising LiARG inhibitors and antileishmanial drug candidates.
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http://dx.doi.org/10.3389/fchem.2020.624678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841069PMC
January 2021

Liquid-liquid phase separation and fibrillation of the prion protein modulated by a high-affinity DNA aptamer.

FASEB J 2020 01 22;34(1):365-385. Epub 2019 Nov 22.

Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

Structural conversion of cellular prion protein (PrP) into scrapie PrP (PrP) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs.
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http://dx.doi.org/10.1096/fj.201901897RDOI Listing
January 2020

Retinoic Acid Binding Leads to CRABP2 Rigidification and Dimerization.

Biochemistry 2019 10 7;58(41):4183-4194. Epub 2019 Oct 7.

Department of Biochemistry, Institute of Chemistry , Federal University of Rio de Janeiro , Rio de Janeiro , RJ 21941909 , Brazil.

Cellular retinoic acid-binding protein 2 (CRABP2) delivers retinoic acid (atRA) to retinoic acid receptors (RARs), allowing for the activation of specific gene transcription. The structural similarities between free and atRA-bound CRABP2 raise the questions of how atRA binding occurs and how the atRA:CRABP2 complex is recognized by downstream binding partners. Thus, to gain insights into these questions, we conducted a detailed atRA-CRABP2 interaction study using nuclear magnetic resonance spectroscopy. The data showed that free CRABP2 displays widespread intermediate-time scale dynamics that is effectively suppressed upon atRA binding. This effect is mirrored by the fast-time scale dynamics of CRABP2. Unexpectedly, CRABP2 rigidification in response to atRA binding leads to the stabilization of a homodimerization interface, which encompasses residues located on helix α2 and the βC-βD loop as well as residues on strands βI-βA and the βH-βI loop. Critically, this rigidification also affects CRABP2's nuclear localization signal and RAR-binding motif, suggesting that the loss of conformational entropy upon atRA binding may be the key for the diverse cellular functions of CRABP2.
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http://dx.doi.org/10.1021/acs.biochem.9b00672DOI Listing
October 2019

arginase: biochemical characterization and inhibition by naturally occurring phenolic substances.

J Enzyme Inhib Med Chem 2019 Dec;34(1):1100-1109

b Department of Biochemistry , Institute of Chemistry, Federal University of Rio de Janeiro , Rio de Janeiro , Brazil.

Inhibition of arginase leads to a decrease in parasite growth and infectivity and thus represents an attractive therapeutic strategy. We evaluated the inhibitory potential of selected naturally occurring phenolic substances on arginase (ARGLi) and investigated their antileishmanial activity . ARGLi exhibited a of 0.28 ± 0.016 mM/min and a of 5.1 ± 1.1 mM for L-arginine. The phenylpropanoids rosmarinic acid and caffeic acid (100 µM) showed percentages of inhibition of 71.48 ± 0.85% and 56.98 ± 5.51%, respectively. Moreover, rosmarinic acid and caffeic acid displayed the greatest effects against with IC values of 57.3 ± 2.65 and 60.8 ± 11 μM for promastigotes, and 7.9 ± 1.7 and 21.9 ± 5.0 µM for intracellular amastigotes, respectively. Only caffeic acid significantly increased nitric oxide production by infected macrophages. Altogether, our results broaden the current spectrum of known arginase inhibitors and revealed promising drug candidates for the therapy of visceral leishmaniasis.
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http://dx.doi.org/10.1080/14756366.2019.1616182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534257PMC
December 2019

Oligomeric transition and dynamics of RNA binding by the HuR RRM1 domain in solution.

J Biomol NMR 2018 Dec 8;72(3-4):179-192. Epub 2018 Dec 8.

Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-909, Brazil.

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central β-strands, β1 and β3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by N relaxation that, in addition to the N- and C-termini, the β2-β3 loop undergoes fast backbone dynamics (ps-ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI-IMS-MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding β-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer-dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR.
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http://dx.doi.org/10.1007/s10858-018-0217-yDOI Listing
December 2018

H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae.

J Cell Biochem 2019 04 15;120(4):5377-5385. Epub 2018 Oct 15.

Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

NSD3s, the proline-tryptophan-tryptophan-proline (PWWP) domain-containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein-dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.
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http://dx.doi.org/10.1002/jcb.27816DOI Listing
April 2019

The PWWP domain of the human oncogene WHSC1L1/NSD3 induces a metabolic shift toward fermentation.

Oncotarget 2017 Aug 12;8(33):54068-54081. Epub 2016 Aug 12.

Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, 21941-909, Rio de Janeiro, RJ, Brazil.

WHSC1L1/NSD3, one of the most aggressive human oncogenes, has two isoforms derived from alternative splicing. Overexpression of long or short NSD3 is capable of transforming a healthy into a cancer cell. NSD3s, the short isoform, contains only a PWWP domain, a histone methyl-lysine reader involved in epigenetic regulation of gene expression. With the aim of understanding the NSD3s PWWP domain role in tumorigenesis, we used as an experimental model. We identified the yeast protein Pdp3 that contains a PWWP domain that closely resembles NSD3s PWWP. Our results indicate that the yeast protein Pdp3 and human NSD3s seem to play similar roles in energy metabolism, leading to a metabolic shift toward fermentation. The swapping domain experiments suggested that the PWWP domain of NSD3s functionally substitutes that of yeast Pdp3, whose W21 is essential for its metabolic function.
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http://dx.doi.org/10.18632/oncotarget.11253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589563PMC
August 2017

Cytotoxicity and anti-Leishmania amazonensis activity of Citrus sinensis leaf extracts.

Pharm Biol 2017 Dec;55(1):1780-1786

a Graduate Program in Pharmaceutical Sciences, School of Pharmacy , Federal University of Rio de Janeiro , RJ , Brazil.

Context: Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease characterized by lesional polymorphism and the commitment of skin surface. Previous reports demonstrated that the Citrus genus possess antimicrobial activity.

Objective: This study evaluated the anti-L. amazonensis activity of Citrus sinensis (L.) Osbeck (Rutaceae) extracts.

Materials And Methods: Citrus sinensis dried leaves were subjected to maceration with hexane (CH), ethyl acetate (CEA), dichloromethane/ethanol (CD/Et - 1:1) or ethanol/water (CEt/W - 7:3). Leishmania amazonensis promastigotes were treated with C. sinensis extracts (1-525 μg/mL) for 120 h at 27 °C. Ultrastructure alterations of treated parasites were evaluated by transmission electron microscopy. Cytotoxicity of the extracts was assessed on RAW 264.7 and J774.G8 macrophages after 48-h treatment at 37 °C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10-80 μg/mL).

Results: CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC) of 25.91 ± 4.87, 54.23 ± 3.78 and 62.74 ± 5.04 μg/mL, respectively. Parasites treated with CD/Et (131.2 μg/mL) presented severe alterations including mitochondrial swelling, lipid body formation and intense cytoplasmic vacuolization. CH and CD/Et demonstrated cytotoxic effects similar to that of amphotericin B in the anti-amastigote assays (SI of 2.16, 1.98 and 1.35, respectively). Triterpene amyrins were the main substances in CH and CD/Et extracts. In addition, 80 μg/mL of CD/Et reduced the number of intracellular amastigotes and the percentage of infected macrophages in 63% and 36%, respectively.

Conclusion: The results presented here highlight C. sinensis as a promising source of antileishmanial agents.
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http://dx.doi.org/10.1080/13880209.2017.1325380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130762PMC
December 2017

PWWP domains and their modes of sensing DNA and histone methylated lysines.

Biophys Rev 2016 Mar 14;8(1):63-74. Epub 2016 Jan 14.

Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-909, Brazil.

Chromatin plays an important role in gene transcription control, cell cycle progression, recombination, DNA replication and repair. The fundamental unit of chromatin, the nucleosome, is formed by a DNA duplex wrapped around an octamer of histones. Histones are susceptible to various post-translational modifications, covalent alterations that change the chromatin status. Lysine methylation is one of the major post-translational modifications involved in the regulation of chromatin function. The PWWP domain is a member of the Royal superfamily that functions as a chromatin methylation reader by recognizing both DNA and histone methylated lysines. The PWWP domain three-dimensional structure is based on an N-terminal hydrophobic β-barrel responsible for histone methyl-lysine binding, and a C-terminal α-helical domain. In this review, we set out to discuss the most recent literature on PWWP domains, focusing on their structural features and the mechanisms by which they specifically recognize DNA and histone methylated lysines at the level of the nucleosome.
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http://dx.doi.org/10.1007/s12551-015-0190-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425739PMC
March 2016

Refolding, purification, and preliminary structural characterization of the DNA-binding domain of the quorum sensing receptor RhlR from Pseudomonas aeruginosa.

Protein Expr Purif 2016 May 11;121:31-40. Epub 2016 Jan 11.

Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil. Electronic address:

RhlR is a 241-residue quorum sensing receptor that controls the expression of a myriad of virulence genes in Pseudomonas aeruginosa. Here, the DNA sequence encoding the carboxi-terminal DNA-binding domain of RhlR was cloned into the pET-RP1B plasmid and expressed as an N-terminal fusion protein to the expression/purification Thio6His6 tag. The fusion construct expressed insolubly in Escherichia coli BL21 (DE3) cells. The recombinant protein was extracted from the bacterial inclusion bodies and refolded in the presence of the charged amino acids l-arginine and l-glutamate. The refolded protein was purified by a combination of Ni(+2)-affinity and size exclusion chromatography, allowing the production of 2 mg of highly purified protein (>95% purity) per 5 mg of wet cells derived from 1 L culture. (1)H 1D NMR analysis revealed that the recombinant protein is folded. Moreover, a fluorescence anisotropy DNA-binding assay showed that the refolded protein is functional, as it recognizes the rhlAB promoter. This is the first time that a domain of the quorum sensing regulator RhlR was produced in sufficient amounts for structural studies, enabling the investigation of the molecular basis for RhlR specific interaction with DNA promoters.
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http://dx.doi.org/10.1016/j.pep.2016.01.006DOI Listing
May 2016

Natural Products: Insights into Leishmaniasis Inflammatory Response.

Mediators Inflamm 2015 11;2015:835910. Epub 2015 Oct 11.

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

Leishmaniasis is a vector-borne disease that affects several populations worldwide, against which there are no vaccines available and the chemotherapy is highly toxic. Depending on the species causing the infection, the disease is characterized by commitment of tissues, including the skin, mucous membranes, and internal organs. Despite the relevance of host inflammatory mediators on parasite burden control, Leishmania and host immune cells interaction may generate an exacerbated proinflammatory response that plays an important role in the development of leishmaniasis clinical manifestations. Plant-derived natural products have been recognized as bioactive agents with several properties, including anti-protozoal and anti-inflammatory activities. The present review focuses on the antileishmanial activity of plant-derived natural products that are able to modulate the inflammatory response in vitro and in vivo. The capability of crude extracts and some isolated substances in promoting an anti-inflammatory response during Leishmania infection may be used as part of an effective strategy to fight the disease.
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http://dx.doi.org/10.1155/2015/835910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619978PMC
August 2016

Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL).

J Biol Chem 2015 Nov 17;290(46):27660-79. Epub 2015 Sep 17.

the Department of Biochemistry, Weill Cornell Medical College, New York, New York 10065

Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.
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http://dx.doi.org/10.1074/jbc.M115.686584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646016PMC
November 2015

A structural perspective on the mechanisms of quorum sensing activation in bacteria.

An Acad Bras Cienc 2015 Oct-Dec;87(4):2189-203. Epub 2015 Aug 4.

Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.

Bacteria are able to synchronize the population behavior in order to regulate gene expression through a cell-to-cell communication mechanism called quorum sensing. This phenomenon involves the production, detection and the response to extracellular signaling molecules named autoinducers, which directly or indirectly regulate gene expression in a cell density-dependent manner. Quorum sensing may control a wide range of biological processes in bacteria, such as bioluminescence, virulence factor production, biofilm formation and antibiotic resistance. The autoinducers are recognized by specific receptors that can either be membrane-bound histidine kinase receptors, which work by activating cognate cytoplasmic response regulators, or cytoplasmic receptors acting as transcription factors. In this review, we focused on the cytosolic quorum sensing regulators whose three-dimensional structures helped elucidate their mechanisms of action. Structural studies of quorum sensing receptors may enable the rational design of inhibitor molecules. Ultimately, this approach may represent an effective alternative to treat infections where classical antimicrobial therapy fails to overcome the microorganism virulence.
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http://dx.doi.org/10.1590/0001-3765201520140482DOI Listing
April 2016

(1)H, (15)N and (13)C resonance assignments of the RRM1 domain of the key post-transcriptional regulator HuR.

Biomol NMR Assign 2015 Oct 9;9(2):281-4. Epub 2014 Dec 9.

Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, 21941-909, Brazil.

Human antigen R (HuR) is a ubiquitous protein that recognizes adenylate and uridylate-rich elements in mRNA, thereby interfering with the fate of protein translation. This protein plays a central role in the outcome of the inflammatory response as it may stabilize or silence mRNAs of key components of the immune system. HuR is able to interact with other RNA-binding proteins, reflecting a complex network that dictates mRNAs post-transcriptional control. HuR is composed of three functional domains, known as RNA-recognition motifs (RRM1, RRM2 and RRM3). It is known that RRM1 is the most important domain for mRNA-binding affinity. In this study, we completed the NMR chemical shift assignment of the RRM1 domain of HuR, as a first step to further establishing the structure, dynamics and function relationship for this protein.
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http://dx.doi.org/10.1007/s12104-014-9592-9DOI Listing
October 2015

Pitfalls associated with the use of Thioflavin-T to monitor anti-fibrillogenic activity.

Bioorg Med Chem Lett 2014 Jul 29;24(14):3194-8. Epub 2014 Apr 29.

Department of Physical Chemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil. Electronic address:

Thioflavin-T (ThT) is a cationic benzothiazole dye that displays enhanced fluorescence upon binding to amyloid fibrils. This property makes ThT the current reagent of choice for the quantification of amyloid fibrils. Herein, we investigate the main pitfalls associated with the use of ThT-based assays to monitor the fibrillation of α-synuclein (α-syn), a protein linked to Parkinson's disease and other α-synucleinopathies. We demonstrated for the first time that ThT interacts with α-syn disordered monomer and accelerates the protein fibrillation in vitro. As a consequence, misleading conclusions may arise from the use of ThT-based real-time assays in the evaluation of anti-fibrillogenic compounds. Interestingly, NMR experiments indicated that C-terminal domain of α-syn is the main region perturbed by ThT interaction, similarly to that found for the pesticide paraquat, a well-documented accelerator of α-syn fibrillation. Moreover, we demonstrated that certain potent inhibitors of α-syn fibrillation, such as oxidized catecholamines and polyphenols, undergo spontaneous oxidation in aqueous solution, generating compounds that strongly quench ThT fluorescence. In light of these findings, we alert for possible artifacts associated to the measure of the anti-fibrillogenic activity based only on ThT fluorescence approach.
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http://dx.doi.org/10.1016/j.bmcl.2014.04.072DOI Listing
July 2014

UV-induced selective oxidation of Met5 to Met-sulfoxide leads to the formation of neurotoxic fibril-incompetent α-synuclein oligomers.

Amyloid 2014 Sep 30;21(3):163-74. Epub 2014 Apr 30.

Department of Physical Chemistry and.

Oxidative stress and the formation of cytotoxic aggregates of the presynaptic protein α-synuclein (AS) are two important events associated with the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative diseases. In this context, extensive efforts have been made to elucidate the molecular basis of the cytotoxic synergy between oxidative stress and AS aggregation. In this study, we demonstrate that the exposure of AS to oxidative stress induced by UV radiation (ASUV) blocks the protein fibrillation, leading to the formation of highly toxic fibril-incompetent oligomers. In addition, ASUV exhibited stronger anti-fibrillogenic properties than H2O2-treated AS, inhibiting the fibrillation of unmodified AS at notably low concentrations. Mass spectrometry indicated that Met5 oxidation to Met-sulfoxide was the only modification promoted by UV exposure, which is reinforced by NMR data indicating that Met5 is the only residue whose amide resonance completely disappeared from the (1)H-(15)N HSQC spectrum after UV exposure. This result is supported by previous data that indicate that C-terminal Met residues (Met116 and Met127) and N-terminal Met1 are less susceptible to oxidation than Met5 because of the residual structure of the disordered AS monomer. Overall, our findings suggest that specific oxidation of Met5 might be sufficient to promote the formation of highly neurotoxic oligomers of AS.
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http://dx.doi.org/10.3109/13506129.2014.912208DOI Listing
September 2014

From structure to catalysis: recent developments in the biotechnological applications of lipases.

Biomed Res Int 2014 24;2014:684506. Epub 2014 Mar 24.

Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos, 21941-909 Rio de Janeiro, RJ, Brazil.

Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to "order" a "customized" enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts.
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http://dx.doi.org/10.1155/2014/684506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982246PMC
December 2014

Hydration and conformational equilibrium in yeast thioredoxin 1: implication for H(+) exchange.

Biochemistry 2014 May 29;53(18):2890-902. Epub 2014 Apr 29.

Institute of Medical Biochemistry, National Center of Nuclear Magnetic Resonance Jiri Jonas, Federal University of Rio de Janeiro-Institute of Structural Biology and Bioimaging , Rio de Janeiro, Brazil.

One of the ancestral features of thioredoxins is the presence of a water cavity. Here, we report that a largely hydrated, conserved, buried aspartic acid in the water cavity modulates the dynamics of the interacting loops of yeast thioredoxin 1 (yTrx1). It is well-established that the aspartic acid, Asp24 for yTrx1, works as a proton acceptor in the reduction of the target protein. We propose a complementary role for Asp24 of coupling hydration and conformational motion of the water cavity and interacting loops. The intimate contact between the water cavity and the interacting loops means that motion at the water cavity will affect the interacting loops and vice versa. The D24N mutation alters the conformational equilibrium for both the oxidized and reduced states, quenching the conformational motion in the water cavity. By measuring the hydration and molecular dynamics simulation of wild-type yTrx1 and the D24N mutant, we showed that Asn24 is more exposed to water than Asp24 and the water cavity is smaller in the mutant, closing the inner part of the water cavity. We discuss how the conformational equilibrium contributes to the mechanism of catalysis and H(+) exchange.
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http://dx.doi.org/10.1021/bi401542vDOI Listing
May 2014

Structural and functional analysis of the NLRP4 pyrin domain.

Biochemistry 2012 Sep 6;51(37):7330-41. Epub 2012 Sep 6.

Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2-α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD-PYD interaction specificity.
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http://dx.doi.org/10.1021/bi3007059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445046PMC
September 2012

The NLRP12 pyrin domain: structure, dynamics, and functional insights.

J Mol Biol 2011 Nov 28;413(4):790-803. Epub 2011 Sep 28.

Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02903, USA.

The initial line of defense against infection is sustained by the innate immune system. Together, membrane-bound Toll-like receptors and cytosolic nucleotide-binding domain and leucine-rich repeat-containing receptors (NLR) play key roles in the innate immune response by detecting bacterial and viral invaders as well as endogenous stress signals. NLRs are multi-domain proteins with varying N-terminal effector domains that are responsible for regulating downstream signaling events. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP12 (NLRP12 PYD) determined using NMR spectroscopy. NLRP12 is a non-inflammasome NLR that has been implicated in the regulation of Toll-like receptor-dependent nuclear factor-κB activation. NLRP12 PYD adopts a typical six-helical bundle death domain fold. By direct comparison with other PYD structures, we identified hydrophobic residues that are essential for the stable fold of the NLRP PYD family. In addition, we report the first in vitro confirmed non-homotypic PYD interaction between NLRP12 PYD and the pro-apoptotic protein Fas-associated factor 1 (FAF-1), which links the innate immune system to apoptotic signaling. Interestingly, all residues that participate in this protein:protein interaction are confined to the α2-α3 surface, a region of NLRP12 PYD that differs most between currently reported NLRP PYD structures. Finally, we experimentally highlight a significant role for tryptophan 45 in the interaction between NLRP12 PYD and the FAF-1 UBA domain.
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http://dx.doi.org/10.1016/j.jmb.2011.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3202057PMC
November 2011

Structural signature of the MYPT1-PP1 interaction.

J Am Chem Soc 2011 Jan 10;133(1):73-80. Epub 2010 Dec 10.

Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02903, USA.

Muscle relaxation is triggered by the dephosphorylation of Ser19 in the myosin regulatory light chain. This reaction is catalyzed by the holoenzyme myosin phosphatase (MP), which includes the catalytic subunit protein phosphatase 1 (PP1) and the regulatory targeting subunit (MYPT). MYPT1 (myosin phosphatase targeting subunit 1) is responsible for both targeting the holoenzyme to subcellular compartments in the muscle and directing PP1 specificity toward myosin. To understand the molecular events leading to the MYPT1-PP1 holoenzyme formation, we used NMR spectroscopy to determine the structural and dynamic characteristics of unbound MYPT1. This allowed the conformations of MYPT1 in the free, unbound state to be directly compared to the PP1-bound state. Our results show that MYPT1(1-98) behaves like a two-domain protein in solution. The first 40 residues of MYPT1(1-98), the disordered region, are intrinsically disordered and highly dynamic, whereas residues 41-98, the folded ankyrin-repeat region, are well-structured and rigid. Furthermore, the integrated use of NMR and biophysical data enabled us to calculate an ensemble model for MYPT1(1-98). The most prominent structural feature of the MYPT1(1-98) ensemble is a 25% populated transient α-helix in the disordered region of MYPT1(1-98). This α-helix becomes fully populated when bound to PP1 and, as we show, likely plays a central role in the formation of the MYPT1-PP1 holoenzyme complex. Finally, this combined analysis shows that the structural and dynamic behaviors exhibited by MYPT1 for PP1 are distinct from those of any other previously analyzed PP1 regulatory protein. Collectively, these data enable us to present a new model of the molecular events that drive MYPT1-PP1 holoenzyme formation and demonstrate that there are structural differences in unbound PP1 regulators that have not been previously observed. Thus, this work adds significant insights to the currently limited data for molecular structures and dynamics of PP1 regulators.
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http://dx.doi.org/10.1021/ja107810rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3016445PMC
January 2011

Backbone and side chain 1H, 15N and 13C assignments of the KSR1 CA1 domain.

Biomol NMR Assign 2011 Apr 25;5(1):39-41. Epub 2010 Aug 25.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02903, USA.

The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [(13)C, (15)N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain.
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http://dx.doi.org/10.1007/s12104-010-9262-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083359PMC
April 2011

Three-dimensional structure of the NLRP7 pyrin domain: insight into pyrin-pyrin-mediated effector domain signaling in innate immunity.

J Biol Chem 2010 Aug 11;285(35):27402-27410. Epub 2010 Jun 11.

Departments of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912, Austria. Electronic address:

The innate immune system provides an initial line of defense against infection. Nucleotide-binding domain- and leucine-rich repeat-containing protein (NLR or (NOD-like)) receptors play a critical role in the innate immune response by surveying the cytoplasm for traces of intracellular invaders and endogenous stress signals. NLRs themselves are multi-domain proteins. Their N-terminal effector domains (typically a pyrin or caspase activation and recruitment domain) are responsible for driving downstream signaling and initiating the formation of inflammasomes, multi-component complexes necessary for cytokine activation. However, the currently available structures of NLR effector domains have not yet revealed the mechanism of their differential modes of interaction. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP7 (NLRP7 PYD) obtained by NMR spectroscopy. The NLRP7 PYD adopts a six-alpha-helix bundle death domain fold. A comparison of conformational and dynamics features of the NLRP7 PYD with other PYDs showed distinct differences for helix alpha3 and loop alpha2-alpha3, which, in NLRP7, is stabilized by a strong hydrophobic cluster. Moreover, the NLRP7 and NLRP1 PYDs have different electrostatic surfaces. This is significant, because death domain signaling is driven by electrostatic contacts and stabilized by hydrophobic interactions. Thus, these results provide new insights into NLRP signaling and provide a first molecular understanding of inflammasome formation.
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http://dx.doi.org/10.1074/jbc.M110.113191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930738PMC
August 2010

1H, 13C and 15N resonance assignments for the reduced forms of thioredoxin 1 and 2 from S. cerevisiae.

J Biomol NMR 2006 ;36 Suppl 1:35

Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil.

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http://dx.doi.org/10.1007/s10858-006-0025-7DOI Listing
August 2007
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