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1.
Nucleic Acids Res ; 49(11): 6529-6548, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34057470

RESUMO

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.


Assuntos
Adenosina Desaminase/química , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Movimento Celular , Cristalografia por Raios X , Ferredoxinas/química , Inosina/metabolismo , Camundongos , Modelos Moleculares , Mutação , Neurônios/fisiologia , Domínios Proteicos , RNA de Transferência/química , RNA de Transferência/metabolismo
2.
Stroke ; 53(10): 3235-3237, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36039755

RESUMO

Stroke burden is substantially increasing but current therapeutic drugs are still far from ideal. Here we highlight the vast potential of staphylokinase as an efficient, fibrin-selective, inexpensive, and evolvable thrombolytic agent. The emphasis is escalated by new recent findings. Staphylokinase nonimmunogenic variant was proven noninferior to alteplase in a clinical trial, with decreased risk of intracranial hemorrhage and the advantage of single bolus administration. Furthermore, our detailed kinetic analysis revealed a new staphylokinase limiting bottleneck whose elimination might provide up to 1000-fold higher activity than the clinically approved alteplase. This knowledge of limitations unlocks new possibilities for improvements that are now achievable by the community of protein engineers who have the required expertise and are ready to transform staphylokinase into a powerful molecule. Collectively, the noninferiority and safety of nonimmunogenic staphylokinase together with the newly identified effectivity limitation make staphylokinase a perfect candidate for further exploration, modification, and advancement to make it the next-generation widely accessible thrombolytic drug effectively treating stroke all around the world, including middle- and low-income countries.


Assuntos
Fibrinolíticos , Acidente Vascular Cerebral , Fibrina , Fibrinolíticos/uso terapêutico , Humanos , Cinética , Metaloendopeptidases/metabolismo , Metaloendopeptidases/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/uso terapêutico
3.
Nature ; 505(7485): 648-53, 2014 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-24463511

RESUMO

H2A.Z is an essential histone variant implicated in the regulation of key nuclear events. However, the metazoan chaperones responsible for H2A.Z deposition and its removal from chromatin remain unknown. Here we report the identification and characterization of the human protein ANP32E as a specific H2A.Z chaperone. We show that ANP32E is a member of the presumed H2A.Z histone-exchange complex p400/TIP60. ANP32E interacts with a short region of the docking domain of H2A.Z through a new motif termed H2A.Z interacting domain (ZID). The 1.48 Å resolution crystal structure of the complex formed between the ANP32E-ZID and the H2A.Z/H2B dimer and biochemical data support an underlying molecular mechanism for H2A.Z/H2B eviction from the nucleosome and its stabilization by ANP32E through a specific extension of the H2A.Z carboxy-terminal α-helix. Finally, analysis of H2A.Z localization in ANP32E(-/-) cells by chromatin immunoprecipitation followed by sequencing shows genome-wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators.


Assuntos
Cromatina/química , Cromatina/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/genética , Imunoprecipitação da Cromatina , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Genoma Humano/genética , Histonas/química , Histonas/isolamento & purificação , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Dados de Sequência Molecular , Proteínas Nucleares/química , Nucleossomos/química , Nucleossomos/metabolismo , Fosfoproteínas/química , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
4.
Biochemistry ; 58(48): 4777-4789, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31682411

RESUMO

We developed a one-step direct assay for the determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety with sulfur, resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiencies ranging from not accepted for HDAC1 to kinetic constants similar to that of the parent oxo substrate for HDAC8. Class II HDACs can hydrolyze thioacylated substrates with approximately 5-10-fold reduced kcat values, which resembles the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficiently with an ∼5-fold reduced specificity constant. On the basis of the unique spectroscopic properties of thioamide bonds (strong absorption in spectral range of 260-280 nm and efficient fluorescence quenching), HDAC-mediated cleavage of thioamides could be followed by ultraviolet-visible and fluorescence spectroscopy in a continuous manner. The HDAC activity assay is compatible with microtiter plate-based screening formats up to 1536-well plates with Z' factors of >0.75 and signal-to-noise ratios of >50. Using thioacylated lysine residues in p53-derived peptides, we optimized substrates for HDAC8 with a catalytic efficiency of >250000 M-1 s-1, which are more than 100-fold more effective than most of the known substrates. We determined inhibition constants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values from the literature. On the other hand, we could introduce N-methylated, N-acylated lysine residues as inhibitors for HDACs with an IC50 value of 1 µM for an N-methylated, N-myristoylated peptide derivative and human HDAC11.


Assuntos
Histona Desacetilases/química , Histona Desacetilases/metabolismo , Biocatálise , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Humanos , Cinética , Lisina/química , Lisina/metabolismo , Tioamidas/química , Tioamidas/metabolismo
5.
Molecules ; 23(3)2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29498707

RESUMO

A promising means in the search of new small molecules for the treatment of schistosomiasis (amongst other parasitic ailments) is by targeting the parasitic epigenome. In the present study, a docking based virtual screening procedure using the crystal structure of histone deacetylase 8 from Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 values ranging from 4.4-20.3 µM against smHDAC8. These newly identified inhibitors were further tested against human histone deacetylases (hsHDAC1, 6 and 8), and were found also to be exerting interesting activity against them. In silico prediction of the docking pose of the compounds was confirmed by the resolved crystal structure of one of the identified hits. This confirmed these compounds were able to chelate the catalytic zinc ion in a bidentate fashion, whilst showing an inverted binding mode of the hydroxamate group when compared to the reported smHDAC8/hydroxamates crystal structures. Therefore, they can be considered as new potential scaffold for the development of new smHDAC8 inhibitors by further investigation of their structure-activity relationship.


Assuntos
Anti-Helmínticos/síntese química , Quelantes/síntese química , Proteínas de Helminto/antagonistas & inibidores , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/química , Ácidos Hidroxâmicos/síntese química , Pirrolidinas/síntese química , Schistosoma mansoni/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Quelantes/farmacologia , Cristalografia por Raios X , Expressão Gênica , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Pirrolidinas/farmacologia , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Relação Estrutura-Atividade , Zinco/química , Zinco/metabolismo
6.
Arch Pharm (Weinheim) ; 350(8)2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28639720

RESUMO

Schistosoma mansoni histone deacetylase 8 (SmHDAC8) has been recently identified as a new potential target for the treatment of schistosomiasis. A series of newly designed and synthesized alkoxyamide-based and hydrazide-based HDAC inhibitors were tested for inhibitory activity against SmHDAC8 and human HDACs 1, 6, and 8. The front runner compounds showed submicromolar activity against SmHDAC8 and modest preference for SmHDAC8 over its human orthologue hHDAC8. Docking studies provided insights into the putative binding mode in SmHDAC8 and allowed rationalization of the observed selectivity profile.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Ftálicos/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Esquistossomicidas/farmacologia , Animais , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Histona Desacetilases , Humanos , Simulação de Acoplamento Molecular , Ácidos Ftálicos/síntese química , Ácidos Ftálicos/química , Schistosoma mansoni/enzimologia , Esquistossomicidas/síntese química , Esquistossomicidas/química , Especificidade da Espécie
7.
PLoS Pathog ; 9(9): e1003645, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086136

RESUMO

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/ß HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.


Assuntos
Epigênese Genética , Proteínas de Helminto/química , Histona Desacetilases/química , Dobramento de Proteína , Schistosoma mansoni/enzimologia , Animais , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Inibidores de Histona Desacetilases/química , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Estrutura Secundária de Proteína , Schistosoma mansoni/genética
8.
J Virol ; 87(15): 8465-80, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23720732

RESUMO

Baculovirus VP1054 protein is a structural component of both of the virion types budded virus (BV) and occlusion-derived virus (ODV), but its exact role in virion morphogenesis is poorly defined. In this paper, we reveal sequence and functional similarity between the baculovirus protein VP1054 and the cellular purine-rich element binding protein PUR-alpha (PURα). The data strongly suggest that gene transfer has occurred from a host to an ancestral baculovirus. Deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp1054 gene completely prevented viral cell-to-cell spread. Electron microscopy data showed that assembly of progeny nucleocapsids is dramatically reduced in the absence of VP1054. More precisely, VP1054 is required for proper viral DNA encapsidation, as deduced from the formation of numerous electron-lucent capsid-like tubules. Complementary searching identified the presence of genetic elements composed of repeated GGN trinucleotide motifs in baculovirus genomes, the target sequence for PURα proteins. Interestingly, these GGN-rich sequences are disproportionally distributed in baculoviral genomes and mostly occurred in proximity to the gene for the major occlusion body protein polyhedrin. We further demonstrate that the VP1054 protein specifically recognizes these GGN-rich islands, which at the same time encode crucial proline-rich domains in p78/83, an essential gene adjacent to the polyhedrin gene in the AcMNPV genome. While some viruses, like human immunodeficiency virus type 1 (HIV-1) and human JC virus (JCV), utilize host PURα protein, baculoviruses encode the PURα-like protein VP1054, which is crucial for viral progeny production.


Assuntos
Baculoviridae/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Baculoviridae/crescimento & desenvolvimento , Baculoviridae/ultraestrutura , Proteínas do Capsídeo , Proteínas de Ligação a DNA/genética , Deleção de Genes , Microscopia Eletrônica de Transmissão , Especificidade por Substrato , Proteínas Estruturais Virais/genética , Vírion/ultraestrutura , Internalização do Vírus , Liberação de Vírus
9.
J Chem Inf Model ; 54(10): 3005-19, 2014 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-25243797

RESUMO

Schistosomiasis, caused by S. mansoni, is a tropical disease that affects over 200 million people worldwide. A novel approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during their life cycle. We recently identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here we present results from a virtual screening campaign on smHDAC8. Besides hydroxamates, several sulfonamide-thiazole derivatives were identified by a target-based virtual screening using a homology model of smHDAC8. In vitro testing of 75 compounds identified 8 hydroxamates as potent and lead-like inhibitors of the parasitic HDAC8. Solving of the crystal structure of smHDAC8 with two of the virtual screening hits confirmed the predicted binding mode.


Assuntos
Proteínas de Helminto/antagonistas & inibidores , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Schistosoma mansoni/química , Sulfonamidas/química , Tiazóis/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Proteínas de Helminto/química , Ensaios de Triagem em Larga Escala , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Schistosoma mansoni/enzimologia , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Interface Usuário-Computador
10.
Bioorg Chem ; 57: 116-120, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25462986

RESUMO

A series of hydroxamates (4a-4l) were prepared from p-aminobenzoic acid to inhibit HDAC8. The idea is to substitute rigid aromatic ring in place of less rigid piperazine ring of hydroxamates reported earlier by our group. It is expected to increase potency retaining the selectivity. Result obtained suggested that the modifications carried out retained the selectivity towards HDAC8 isoform and increasing the potency in very few cases. Increase in potency is also associated with variation in cap aryl region. Two compounds (4f &4l) were found to inhibit HDAC8 at concentrations (IC50) less than 20µM.


Assuntos
Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Cristalografia por Raios X , Desenho de Fármacos , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade
11.
Phytopathology ; 104(3): 306-12, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24093923

RESUMO

The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).


Assuntos
Beta vulgaris/parasitologia , DNA de Helmintos/isolamento & purificação , Doenças das Plantas/parasitologia , Extração em Fase Sólida/métodos , Tylenchoidea/isolamento & purificação , Animais , DNA de Helmintos/genética , Raízes de Plantas/parasitologia , Caules de Planta/parasitologia , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Extração em Fase Sólida/economia , Fatores de Tempo , Tylenchoidea/genética
12.
J Virol ; 86(3): 1728-38, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22090126

RESUMO

The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and in their egress from the virogenic stroma toward the nuclear periphery by a mechanism, which also includes F-actin filaments. We performed functional mapping of VP80 demonstrating that its highly conserved C-terminal region plays a crucial role in virion morphogenesis. Protein database mining identified a putative basic helix-loop-helix (bHLH) domain, a DNA-binding module typical for eukaryotic transcription factors, in the essential C-terminal region of VP80. Using a molecular modeling approach, we predicted the three-dimensional structure of this domain, revealing some unique properties. Biochemical assays proved that VP80 can form homodimers, a critical prerequisite of DNA-binding bHLH proteins. The ability of VP80 to bind DNA was subsequently confirmed by an electrophoretic mobility shift assay. We further show that AcMNPV DNA replication occurs in the absence of VP80. Immunolabeling of VP80 in baculovirus-infected cells rather points toward its involvement in nucleocapsid maturation. The competence of VP80 to interact with both F-actin and DNA provides novel insight into baculovirus morphogenesis.


Assuntos
Baculoviridae/química , Proteínas do Capsídeo/metabolismo , DNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas do Capsídeo/química , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos
13.
Materials (Basel) ; 16(13)2023 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-37445093

RESUMO

Designing a composite, possibly strengthened by a dispersion of (fine) oxides, is a favorable way to improve the mechanical characteristics of Cu while maintaining its advantageous electric conductivity. The aim of this study was to perform mechanical alloying of a Cu powder with a powder of Al2O3 oxide, seal the powder mixture into evacuated Cu tubular containers, i.e., cans, and apply gradual direct consolidation via rotary swaging at elevated temperatures, as well as at room temperature (final passes) to find the most convenient way to produce the designed Al2O3 particle-strengthened Cu composite. The composites swaged with the total swaging degree of 1.83 to consolidated rods with a diameter of 10 mm were subjected to measurements of electroconductivity, investigations of mechanical behavior via compression testing, and detailed microstructure observations. The results revealed that the applied swaging degree was sufficient to fully consolidate the canned powders, even at moderate and ambient temperatures. In other words, the final structures, featuring ultra-fine grains, did not exhibit voids or remnants of unconsolidated powder particles. The swaged composites featured favorable plasticity regardless of the selected processing route. The flow stress curves exhibited the establishment of steady states with increasing strain, regardless of the applied strain rate. The electroconductivity of the composite swaged at elevated temperatures, featuring homogeneous distribution of strengthening oxide particles and the average grain size of 1.8 µm2, reaching 80% IACS (International Annealed Copper Standard).

14.
Acta Crystallogr D Struct Biol ; 79(Pt 11): 956-970, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37860958

RESUMO

Haloalkane dehalogenases (HLDs) are a family of α/ß-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Šresolution. The structures show a canonical αßα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.


Assuntos
Mycobacterium marinum , Mycobacterium marinum/metabolismo , Ácido Aspártico , Estereoisomerismo , Hidrolases/química , Especificidade por Substrato
15.
Methods Mol Biol ; 2652: 293-318, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37093484

RESUMO

Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.


Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Deutério/química , Medição da Troca de Deutério/métodos , Conformação Proteica , Espectrometria de Massas/métodos , Hidrogênio/química
16.
STAR Protoc ; 4(1): 102058, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36853683

RESUMO

Here, we describe a combined in cellulo and in vivo approach to identify compounds with higher potential for efficient inhibition of Trypanosoma cruzi. Phase I of in cellulo assays is designed to exclude inactive or toxic compounds, while phase II is designed for accurate IC50, CC50, and selective index (SI) determination. Compounds showing high SI are tested using in vivo infection models in parallel with benznidazole to assess their efficacy relative to a reference drug used for Chagas disease treatment. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).1.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Doença de Chagas/tratamento farmacológico
17.
ACS Catal ; 13(19): 12506-12518, 2023 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-37822856

RESUMO

Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein-water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.

18.
Protein Sci ; 32(10): e4751, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37574754

RESUMO

Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.


Assuntos
Hidrolases , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X , Especificidade por Substrato , Hidrolases/química
19.
Nat Commun ; 14(1): 7864, 2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38030625

RESUMO

NanoLuc, a superior ß-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.


Assuntos
Medições Luminescentes , Luciferases/metabolismo , Domínio Catalítico
20.
Mol Neurodegener ; 18(1): 38, 2023 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-37280636

RESUMO

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.


Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Humanos , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Apolipoproteína E3/genética , Mutação/genética , Apolipoproteínas E/genética
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