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1.
Nat Commun ; 11(1): 3859, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737298

RESUMO

Non-enzymatic proteins including antibodies function as biomarkers and are used as biopharmaceuticals in several diseases. Protein-responsive soft materials capable of the controlled release of drugs and proteins have potential for use in next-generation diagnosis and therapies. Here, we describe a supramolecular/agarose hydrogel composite that can release a protein in response to a non-enzymatic protein. A non-enzymatic protein-responsive system is developed by hybridization of an enzyme-sensitive supramolecular hydrogel with a protein-triggered enzyme activation set. In situ imaging shows that the supramolecular/agarose hydrogel composite consists of orthogonal domains of supramolecular fibers and agarose, which play distinct roles in protein entrapment and mechanical stiffness, respectively. Integrating the enzyme activation set with the composite allows for controlled release of the embedded RNase in response to an antibody. Such composite hydrogels would be promising as a matrix embedded in a body, which can autonomously release biopharmaceuticals by sensing biomarker proteins.


Assuntos
Anidrase Carbônica II/química , Preparações de Ação Retardada/síntese química , Hidrogéis/química , Ribonucleases/química , Sefarose/química , Animais , Anticorpos/química , Avidina/química , Biotina/química , Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Bovinos , Ativação Enzimática , Transição de Fase , Reologia , Ribonucleases/antagonistas & inibidores , Sulfonamidas/química
2.
Science ; 369(6503): 524-530, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732418

RESUMO

RNA molecules are frequently modified with a terminal 2',3'-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2',3'-cyclic phosphates into 2',3'-OH nucleotides. We analyzed ANGEL2's substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box-binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)-mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2',3'-cyclic phosphates.


Assuntos
Nucleotidases/química , Ribonucleases/química , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Nucleotidases/genética , Estrutura Secundária de Proteína , Precursores de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Especificidade por Substrato , Proteína 1 de Ligação a X-Box/genética
3.
Science ; 369(6504): 656-663, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32586950

RESUMO

Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA-like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo-electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.


Assuntos
Endorribonucleases/química , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Ribonucleases/química , Ribonucleoproteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Holoenzimas/química , Conformação Proteica , RNA Catalítico/química , Especificidade por Substrato
4.
J Phys Chem Lett ; 11(9): 3263-3270, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32251595

RESUMO

Molecular recognition is a fundamental step in essentially any biological process. However, the kinetic processes during association and dissociation are difficult to be efficiently sampled by direct all-atom molecular dynamics simulations because of the large spatial and temporal scales. Here we propose an arbitrary resolution with two bead types (ART) coarse-grained (CG) strategy that is adept in molecular recognition. ART is a universal user-customized CG strategy that can generate a system-specific CG force field anytime and be applied to any system with an arbitrary CG resolution according to research requirements. ART CG simulations can be very efficiently performed with implicit solvation in prevalent simulation packages and provide interfaces for any enhanced sampling method. We used three applications, HLA-HIV epitope recognition, barnase-barstar association, and trimeric TRAF2 self-assembly, to validate the feasibility of the ART CG strategy, its advantages in protein recognition, and its high performance in simulations. Regular CG simulations can successfully achieve valid protein recognitions without any prior bound structure.


Assuntos
Proteínas de Bactérias/química , Epitopos de Linfócito T/química , Antígeno HLA-B27/química , Modelos Moleculares , Ribonucleases/química , Fator 2 Associado a Receptor de TNF/química , Linfócitos T CD8-Positivos , Simulação por Computador , Proteína do Núcleo p24 do HIV , HIV-1
5.
Science ; 368(6488)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32299921

RESUMO

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.


Assuntos
Códon , Elongação Traducional da Cadeia Peptídica , Estabilidade de RNA , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Microscopia Crioeletrônica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/química , Ribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
6.
FASEB J ; 34(2): 3051-3068, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908032

RESUMO

Bacterial toxin-antitoxin (TA) system has gained attention for its essential roles in cellular maintenance and survival under harsh environmental conditions such as nutrient deficiency and antibiotic treatment. There are at least 14 TA systems in Salmonella enterica serovar Typhimurium LT2, a pathogenic bacterium, and none of the structures of these TA systems have been determined. We determined the crystal structure of the VapBC TA complex from S. Typhimurium LT2 in proteolyzed and DNA-bound forms at 2.0 Å and 2.8 Å resolution, respectively. The VapC toxin possesses a pilT N-terminal domain (PIN-domain) that shows ribonuclease activity, and the VapB antitoxin has an AbrB-type DNA binding domain. In addition, the structure revealed details of interaction mode between VapBC and the cognate promoter DNA, including the inhibition of VapC by VapB and linear conformation of bound DNA in the VapBC complex. The complexation of VapBC with the linear DNA is not consistent with known structures of VapBC homologs in complex with bent DNA. We also identified VapC from S. Typhimurium LT2 as a putative Ca2+ -dependent ribonuclease, which differs from previous data showing that VapC homologs have Mg2+ or Mn2+ -dependent ribonuclease activities. The present studies could provide structural understanding of the physiology of VapBC systems and foundation for the development of new antibiotic drugs against Salmonella infection.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Ribonucleases/química , Salmonella typhimurium/enzimologia , Cristalografia por Raios X , Domínios Proteicos , Estrutura Quaternária de Proteína
7.
Biochemistry ; 59(6): 755-765, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31909602

RESUMO

Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologues, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show the conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogues. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5'-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B2 subsite ligand preferences, our results show a lack of typical B2 subsite interactions normally observed in homologous ligand-bound RNases. A comparison of the dynamical properties of RNase 6 in its apo-, substrate-, and product-bound states highlight the unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ribonucleases/química , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação/fisiologia , Humanos , Ligantes , Estrutura Secundária de Proteína
8.
Proc Natl Acad Sci U S A ; 117(5): 2406-2411, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964809

RESUMO

As the area of small molecules interacting with RNA advances, general routes to provide bioactive compounds are needed as ligands can bind RNA avidly to sites that will not affect function. Small-molecule targeted RNA degradation will thus provide a general route to affect RNA biology. A non-oligonucleotide-containing compound was designed from sequence to target the precursor to oncogenic microRNA-21 (pre-miR-21) for enzymatic destruction with selectivity that can exceed that for protein-targeted medicines. The compound specifically binds the target and contains a heterocycle that recruits and activates a ribonuclease to pre-miR-21 to substoichiometrically effect its cleavage and subsequently impede metastasis of breast cancer to lung in a mouse model. Transcriptomic and proteomic analyses demonstrate that the compound is potent and selective, specifically modulating oncogenic pathways. Thus, small molecules can be designed from sequence to have all of the functional repertoire of oligonucleotides, including inducing enzymatic degradation, and to selectively and potently modulate RNA function in vivo.


Assuntos
Neoplasias da Mama/tratamento farmacológico , MicroRNAs/metabolismo , Ribonucleases/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Humanos , Camundongos , MicroRNAs/química , Estrutura Molecular , Metástase Neoplásica , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ribonucleases/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
9.
Biol Pharm Bull ; 42(12): 2054-2061, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31787719

RESUMO

RNase He1 is a guanylic acid-specific ribonuclease of the RNase T1 family from Hericium erinaceus (Japanese name: Yamabushitake). Its RNA degrading activity is strongly inhibited by Zn2+, similar to other T1 family RNases. However, RNase He1 shows little inhibition of human tumor cell proliferation, unlike RNase Po1, another T1 family RNase from Pleurotus ostreatus (Japanese name: Hiratake). Here, we determined the three-dimensional X-ray crystal structure of RNase He1 in complex with Zn, which revealed that Zn binding most likely prevents substrate entry into the active site due to steric hindrance. This could explain why RNase He1 and other T1 family RNases are inhibited by Zn. The X-ray crystal structures revealed that RNase He1 and RNase Po1 are almost identical in their catalytic sites and in the cysteine residues involved in disulfide bonds that increase their stability. However, our comparison of the electrostatic potentials of their molecular surfaces revealed that RNase He1 is negative whereas RNase Po1 is positive; thus, RNase He1 may not be able to electrostatically bind to the plasma membrane, potentially explaining why it does not exhibit antitumor activity. Hence, we suggest that the cationic characteristics of RNase Po1 are critical to the anti-tumor properties of the protein.


Assuntos
Basidiomycota/enzimologia , Proteínas Fúngicas/química , Ribonucleases/química , Zinco/química , Cristalografia por Raios X , Conformação Proteica , RNA Fúngico/química
10.
Int J Mol Sci ; 20(21)2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683668

RESUMO

Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40-50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5'-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease.


Assuntos
Cisteína/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa/metabolismo , Ribonucleases/metabolismo , Animais , Bovinos , Dissulfetos/metabolismo , Ácido Ditionitrobenzoico/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução , Estresse Oxidativo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Ribonucleases/química , Compostos de Sulfidrila/metabolismo , Espectrometria de Massas em Tandem
11.
Chem Commun (Camb) ; 55(84): 12623-12626, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31580354

RESUMO

We described a ribonuclease-dependent cleavable beacon primer, an energy-transfer-tagged oligonucleotide inserted with a ribonucleotide, which can be cleaved by ribonuclease to generate enhanced fluorescence signals and initiate DNA amplification for single nucleotide mutation detection with ultrahigh sensitivity and selectivity.


Assuntos
Clivagem do DNA , Primers do DNA , DNA/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Ribonucleases/química , Ribonucleotídeos/química , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Cinética , Sondas de Oligonucleotídeos/química , Mutação Puntual , Imagem Individual de Molécula/métodos , Temperatura
12.
Environ Microbiol ; 21(12): 4822-4835, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31631506

RESUMO

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Flavonoides/química , Rhizobiaceae/enzimologia , Ribonucleases/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Inibidores Enzimáticos/metabolismo , Flavonoides/metabolismo , Doenças das Plantas/microbiologia , Rhizobiaceae/química , Rhizobiaceae/genética , Ribonucleases/química , Ribonucleases/genética , Ribonucleases/metabolismo
13.
J Chem Theory Comput ; 15(11): 5829-5844, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31593627

RESUMO

A powerful computational strategy to determine the equilibrium association constant of two macromolecules with explicit-solvent molecular dynamics (MD) simulations is the "geometric route", which considers the reversible physical separation of the bound complex in solution. Nonetheless, multiple challenges remain to render this type of methodology reliable and computationally efficient in practice. In particular, in one, formulation of the geometric route relies on the potential of mean force (PMF) for physically separating the two binding partners restrained along a straight axis, which must be selected prior to the calculation. However, practical applications indicate that the calculation of the separation PMF along the predefined rectilinear pathway may be suboptimal and slowly convergent. Recognizing that a rectilinear straight separation pathway is generally not representative of how the protein complex physically separates in solution, we put forth a novel theoretical framework for binding free-energy calculations, leaning on the optimal curvilinear minimum free-energy path (MFEP) determined from the string method. The proposed formalism is validated by comparing the results obtained using both rectilinear and curvilinear pathways for a prototypical host-guest complex formed by cucurbit[7]uril (CB[7]) binding benzene, and for the barnase-barstar protein complex. On the basis of multi-microsecond MD calculations, we find that the calculations following the traditional rectilinear pathway and the string-based curvilinear pathway agree quantitatively, but convergence is faster with the latter.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzeno/química , Benzeno/metabolismo , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Ligação Proteica , Proteínas/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Termodinâmica
14.
PLoS One ; 14(9): e0223008, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31568482

RESUMO

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.


Assuntos
Desoxirribonuclease I/química , Fragmentos de Peptídeos/análise , Ribonuclease Pancreático/química , Ribonucleases/química , DNA/química , Desoxirribonuclease I/efeitos dos fármacos , Desoxirribonuclease I/efeitos da radiação , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Hidrólise , Micro-Ondas , Peso Molecular , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , RNA/química , Ribonuclease Pancreático/efeitos dos fármacos , Ribonuclease Pancreático/efeitos da radiação , Ribonucleases/efeitos dos fármacos , Ribonucleases/efeitos da radiação , Soluções
15.
Int J Mol Sci ; 20(18)2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31540052

RESUMO

Candida albicans is a polymorphic fungus responsible for mucosal and skin infections. Candida cells establish themselves into biofilm communities resistant to most currently available antifungal agents. An increase of severe infections ensuing in fungal septic shock in elderly or immunosuppressed patients, along with the emergence of drug-resistant strains, urge the need for the development of alternative antifungal agents. In the search for novel antifungal drugs our laboratory demonstrated that two human ribonucleases from the vertebrate-specific RNaseA superfamily, hRNase3 and hRNase7, display a high anticandidal activity. In a previous work, we proved that the N-terminal region of the RNases was sufficient to reproduce most of the parental protein bactericidal activity. Next, we explored their potency against a fungal pathogen. Here, we have tested the N-terminal derived peptides that correspond to the eight human canonical RNases (RN1-8) against planktonic cells and biofilms of C. albicans. RN3 and RN7 peptides displayed the most potent inhibitory effect with a mechanism of action characterized by cell-wall binding, membrane permeabilization and biofilm eradication activities. Both peptides are able to eradicate planktonic and sessile cells, and to alter their gene expression, reinforcing its role as a lead candidate to develop novel antifungal and antibiofilm therapies.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Ribonucleases/química , Antifúngicos/química , Biofilmes/efeitos dos fármacos , Candida albicans/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Proteína Catiônica de Eosinófilo/química , Proteína Catiônica de Eosinófilo/metabolismo , Proteína Catiônica de Eosinófilo/farmacologia , Humanos , Peptídeos/metabolismo , Ribonucleases/metabolismo , Ribonucleases/farmacologia
16.
Nucleic Acids Res ; 47(19): 10400-10413, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501867

RESUMO

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress.


Assuntos
Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Estabilidade Proteica , Ribonucleases/química , Sequência de Aminoácidos/genética , Toxinas Bacterianas/genética , Códon/química , Códon/genética , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Resposta ao Choque Térmico/genética , Estabilidade de RNA/genética , RNA Mensageiro , RNA Ribossômico 16S/genética , Ribonucleases/genética , Ribossomos/química , Ribossomos/genética
17.
Food Funct ; 10(10): 6342-6350, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31441483

RESUMO

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.


Assuntos
Agaricales/química , Diferenciação Celular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ribonucleases/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ribonucleases/química , Ribonucleases/isolamento & purificação
18.
J Phys Chem Lett ; 10(18): 5463-5467, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31442055

RESUMO

Nuclear spin hyperpolarized water is utilized to obtain protein spectra not only in the folded state but also during the refolding process. Polarization transfer to Ribonuclease Sa through proton exchange and the nuclear Overhauser effect (NOE) results in NMR signal enhancements of amide protons by up to 24-fold. These enhancements enable the measurement of fast two-dimensional NMR spectra on the same time scale as the folding. Resolved amide proton signals corresponding to the folded protein are observed both under folded and refolding conditions, whereby the refolding protein shows smaller transferred signals. Residue-specific evaluation of contributions to the polarization transfer indicates that signals attributed to a relayed intramolecular NOE are not observable in the refolding experiment. These differences are explained by the absence of long-range contacts and faster molecular motions in the unfolded protein. Applications of this method include accessing residue-specific information on structure and dynamics during multistate protein folding.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Dobramento de Proteína , Proteínas/química , Amidas/química , Sequência de Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Conformação Proteica , Prótons , Ribonucleases/química , Água
19.
Mol Cell ; 75(5): 933-943.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31326272

RESUMO

Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cAn) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cAn second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppAn), and products (cAn), to decipher mechanistic aspects of cAn formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppAn intermediates and subsequent cAn formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cAn signaling pathway.


Assuntos
Nucleotídeos de Adenina/química , Proteínas Arqueais/química , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , Ribonucleases/química , Sistemas do Segundo Mensageiro , Thermococcus/química , Nucleotídeos de Adenina/metabolismo , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , Oligorribonucleotídeos/metabolismo , Ribonucleases/metabolismo , Thermococcus/metabolismo , Thermococcus/ultraestrutura
20.
Mol Cell ; 75(5): 944-956.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31326273

RESUMO

Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cAn) from ATP and subsequent cAn-mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity.


Assuntos
Nucleotídeos de Adenina/química , Proteínas Arqueais/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , Ribonucleases/química , Thermococcus/química , Sítios de Ligação , Domínios Proteicos
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