Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 19.499
Filtrar
1.
Biochemistry (Mosc) ; 84(11): 1221-1232, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760913

RESUMO

The review covers three independent blocks of research. The first one is discovery, isolation, and investigation of snake venom RNases and their use in studying RNA macrostructure. It has been established that snake venom RNases are not specific to the primary RNA structure but rather to the RNA helical conformation (double, single, or hybrid helix). Snake venom RNases hydrolyze RNA to short oligomers with the 5'-terminal phosphate. Analysis of the kinetics and products of tRNA hydrolysis exemplifies the use of snake venom RNases for deciphering RNA macrostructure. The second block is devoted to the principle formulated by the author for analyzing the primary structure of nucleic acids and describes the method of direct RNA sequencing that has been developed with author's participation. The third block describes the results of genotyping and etiologic control of epidemic influenza A viruses circulating in the Soviet Union in 1968 to 1992. The method for comparative analysis of genome sequences of viral isolates has made it possible to detect and characterize epidemic influenza virus strains that had emerged in the circulation as a result of reactivation of inactivated vaccines.


Assuntos
RNA/química , Humanos , Hidrólise , Vírus da Influenza A/genética , Cinética , Conformação de Ácido Nucleico , RNA/metabolismo , RNA/ultraestrutura , RNA Viral/química , RNA Viral/metabolismo , Ribonucleases/metabolismo , Venenos de Serpentes/metabolismo
2.
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
3.
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 Ambiente
4.
Nat Commun ; 10(1): 4302, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541109

RESUMO

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.


Assuntos
Nucleotídeos de Adenina/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endorribonucleases/química , Oligorribonucleotídeos/química , Estabilidade de RNA , Proteínas de Ligação a RNA/química , Sulfolobus/química , Sítio Alostérico , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cristalografia por Raios X , Proteínas de Membrana/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Ribonucleases/metabolismo , Sistemas do Segundo Mensageiro , Sulfolobus/genética
5.
Nat Protoc ; 14(10): 2986-3012, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548639

RESUMO

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Ácidos Nucleicos/análise , Primers do DNA , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Humanos , Leptotrichia/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Engenharia de Proteínas/métodos , RNA Guia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/isolamento & purificação , Ribonucleases/metabolismo , Fluxo de Trabalho , Zika virus/genética , Infecção por Zika virus/sangue , Infecção por Zika virus/urina
6.
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
7.
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
8.
Nucleic Acids Res ; 47(18): 9524-9541, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392315

RESUMO

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4-Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1-decaysome and Ccr4-Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome.


Assuntos
Exorribonucleases/genética , RNA Polimerase II/genética , Estabilidade de RNA/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Impressão Genômica , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética
9.
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
10.
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
11.
Chem Commun (Camb) ; 55(62): 9112-9115, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31298670

RESUMO

We designed novel 4'-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in ≈4.9-fold more efficient knockdown than unmodified siRNA.


Assuntos
Guanidina/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Guanidina/análogos & derivados , Modelos Moleculares , RNA Interferente Pequeno/genética , Ribonucleases/sangue , Ribonucleases/metabolismo , Termodinâmica
12.
Adv Exp Med Biol ; 1157: 85-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31342438

RESUMO

RNA degradation is considered a critical posttranscriptional regulatory checkpoint, maintaining the correct functioning of organisms. When a specific RNA transcript is no longer required in the cell, it is signaled for degradation through a number of highly regulated steps. Ribonucleases (or simply RNases) are key enzymes involved in the control of RNA stability. These enzymes can perform the RNA degradation alone or cooperate with other proteins in RNA degradation complexes. Important findings over the last years have shed light into eukaryotic RNA degradation by members of the RNase II/RNB family of enzymes. DIS3 enzyme belongs to this family and represents one of the catalytic subunits of the multiprotein complex exosome. This RNase has a diverse range of functions, mainly within nuclear RNA metabolism. Humans encode two other DIS3-like enzymes: DIS3L (DIS3L1) and DIS3L2. DIS3L1 also acts in association with the exosome but is strictly cytoplasmic. In contrast, DIS3L2 acts independently of the exosome and shows a distinctive preference for uridylated RNAs. These enzymes have been shown to be involved in important cellular processes, such as mitotic control, and associated with human disorders like cancer. This review shows how the impairment of function of each of these enzymes is implicated in human disease.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo , Neoplasias , RNA , Ribonucleases , Endorribonucleases , Exorribonucleases , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos , Humanos , Neoplasias/fisiopatologia , RNA/metabolismo , Estabilidade de RNA , Ribonucleases/metabolismo
13.
Nat Commun ; 10(1): 3173, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320642

RESUMO

CCR4-NOT is a conserved multiprotein complex which regulates eukaryotic gene expression principally via shortening of poly(A) tails of messenger RNA or deadenylation. Here, we reconstitute a complete, recombinant human CCR4-NOT complex. Our reconstitution strategy permits strict compositional control to test mechanistic hypotheses with purified component variants. CCR4-NOT is more active and selective for poly(A) than the isolated exonucleases, CCR4a and CAF1, which have distinct deadenylation profiles in vitro. The exonucleases require at least two out of three conserved non-enzymatic modules (CAF40, NOT10:NOT11 or NOT) for full activity in CCR4-NOT. CAF40 and the NOT10:NOT11 module both bind RNA directly and stimulate deadenylation in a partially redundant manner. Linear motifs from different RNA-binding factors that recruit CCR4-NOT to specific mRNAs via protein-protein interactions with CAF40 can inhibit bulk deadenylation. We reveal an additional layer of regulatory complexity to the human deadenylation machinery, which may prime it either for general or target-specific degradation.


Assuntos
Exorribonucleases/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Receptores CCR4/genética , Humanos , Complexos Multiproteicos/síntese química , Complexos Multiproteicos/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Poli A/metabolismo , RNA Mensageiro/genética , Receptores CCR4/metabolismo , Proteínas Recombinantes/genética , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo
14.
Int J Mol Sci ; 20(13)2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31261992

RESUMO

Focal cerebral ischemia can cause blood-brain barrier (BBB) breakdown, which is implicated in neuroinflammation and progression of brain damage. Monocyte chemotactic protein 1-induced protein 1 (MCPIP1) is a newly identified zinc-finger protein that negatively regulates inflammatory signaling pathways. We aimed to evaluate the impact of genetic MCPIP1 deletion on BBB breakdown and expression of BBB-related matrix metalloproteinases (MMPs) and tight junction proteins after cerebral ischemia/reperfusion (I/R) using MCPIP1-deficient (MCPIP1-/-) mice. Transient middle cerebral artery occlusion was induced in the MCPIP1-/- mice and their wild-type littermates for 2 h followed by reperfusion for 24 h. The degree of BBB breakdown was evaluated by injection of fluorescein isothiocyanate (FITC)-dextran. Quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were performed to compare the expression of MMPs and claudin-5 and zonula occludens-1 (ZO-1). MCPIP1 deficiency in mice resulted in enhanced leakage of FITC-dextran, increased expression of MMP-9/3, and reduced expression of claudin-5 and ZO-1 in the brain compared to that seen in their wild-type littermates subjected to cerebral I/R. These results demonstrate that absence of MCPIP1 exacerbates cerebral I/R-induced BBB disruption by enhancing the expression of MMP-9/3 and the degradation of claudin-5 and ZO-1, providing novel insights into the mechanisms underlying BBB breakdown after cerebral ischemia/reperfusion.


Assuntos
Barreira Hematoencefálica/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Ribonucleases/metabolismo , Animais , Permeabilidade Capilar , Claudina-5/genética , Claudina-5/metabolismo , Infarto da Artéria Cerebral Média/genética , Masculino , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ribonucleases/genética , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
15.
Nucleic Acids Res ; 47(16): 8838-8859, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31329944

RESUMO

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.


Assuntos
Macrófagos Peritoneais/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Ribonucleases/genética , Transativadores/genética , Animais , Fibroblastos/citologia , Fibroblastos/imunologia , Células HEK293 , Células HeLa , Homeostase/genética , Homeostase/imunologia , Humanos , Imunidade Inata , Inflamação , Sequências Repetidas Invertidas , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Knockout , Mutação , Cultura Primária de Células , Ligação Proteica , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/imunologia , RNA Mensageiro/metabolismo , Ribonucleases/deficiência , Ribonucleases/imunologia , Imagem Individual de Molécula , Transativadores/imunologia
16.
Prep Biochem Biotechnol ; 49(9): 916-926, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31322478

RESUMO

A Gram-positive, rod-shaped, endospore-forming, and RNA-degrading bacterium RB-5 was isolated from a soil sample. Based on 16-rDNA gene sequence, the bacterium RB-5 was identified as Bacillus safensis (Accession number KX443714.1). The bacterium appeared to be related to Bacillus safensis KL-052, an other-member of genus Bacillus. One-factor-at-a-time (OFAT) and Response Surface Methodology (RSM) statistical approaches were used to optimize the fermentation broth to obtain an improved extracellular RNase production from B. safensis RB-5. These approaches improved RNase activity of B. safensis KL-052 from 4.26 to 7.85 U/mL. The OFAT approach was used to study the effects of supplementation of carbon, nitrogen and physical conditions, which included temperature, pH and agitation rate on extracellular RNase production by B. safensis KL-052. Five variables screened by Central Composite Design (CCD) were employed to evaluate their interactive effects on RNase production by the organism. CCD selected 25 factorial values obtained by the statistical approach were peptone 1.13% (w/v), sodium nitrate 1.13% (w/v), MgSO4 0.06% (w/v), pH 8.5, and temperature 35 °C for RNase production by B. safensis. The highest predicted value of RNase was 7.05 U/ml while actual obtained value was 7.85 U/ml that was ∼84% and 1.84-fold higher than OFAT approach.


Assuntos
Bacillus/enzimologia , Ribonucleases/metabolismo , Bacillus/genética , Bacillus/isolamento & purificação , Bacillus/metabolismo , Carbono/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Nitrogênio/metabolismo , Peptonas/metabolismo , Filogenia , Ribonucleases/genética , Microbiologia do Solo , Temperatura Ambiente
17.
Immunohorizons ; 3(5): 172-185, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31356171

RESUMO

Cytokine IL-17A (IL-17) acts on various cell types, including epidermal keratinocytes, and induces antimicrobial peptide and chemokine production to elicit antibacterial and antifungal defense responses. Excess IL-17 leads to inflammatory skin diseases such as psoriasis. The IκB family protein IκB-ζ mediates IL-17-induced responses. However, the mechanism controlling IκB-ζ expression in IL-17-stimulated cells remains elusive. In this study, we showed that JAK kinase TYK2 positively regulates IL-17-induced IκB-ζ expression. TYK2-deficient mice showed reduced inflammation and concomitant reduction of IκB-ζ mRNA compared with wild-type mice in imiquimod-induced skin inflammation. The analysis of the IκB-ζ promoter activity using human cell lines (HaCaT and HeLa) revealed that catalytic activity of TYK2 and its substrate transcription factor STAT3, but not IL-17, is required for IκB-ζ promoter activity. In contrast, IL-17-induced signaling, which did not activate STAT3, posttranscriptionally stabilized IκB-ζ mRNA via its 3'-untranslated region. IL-17 signaling protein ACT1 was required to counteract constitutive IκB-ζ mRNA degradation by RNase Regnase-1. These results suggested that transcriptional activation by TYK2-STAT3 pathway and mRNA stabilization by IL-17-mediated signals act separately from each other but complementarily to achieve IκB-ζ induction. Therefore, JAK/TYK2 inhibition might be of significance in regulation of IL-17-induced inflammatory reactions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interleucina-17/metabolismo , Estabilidade de RNA , Fator de Transcrição STAT3/metabolismo , TYK2 Quinase/metabolismo , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células HeLa , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Psoríase/induzido quimicamente , Psoríase/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , TYK2 Quinase/genética , Fatores de Transcrição/metabolismo
18.
Microb Pathog ; 135: 103648, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31356928

RESUMO

Norovirus is a highly infectious human pathogen that causes acute foodborne diseases worldwide. As global diet patterns have begun to incorporate a higher consumption of fresh agricultural products, the internalization of norovirus into plants has emerged as a potential threat to human health. Here, we demonstrated that murine norovirus (MNV1) was internalized into Arabidopsis in multiple phases, and this internalization was correlated with Arabidopsis innate immunity responses. Under hydroponic conditions, continuous treatment of MNV1 retarded root growth and facilitated flower development of Arabidopsis without causing necrotic lesions. Examination of viral titers and RNA levels revealed that MNV1 was internalized into Arabidopsis in at least three different phases. In response to MNV1 treatment, the Arabidopsis defensive marker PR1 (a salicylic acid signaling marker) was transiently up-regulated at the early stage. PDF1.2, a jasmonic acid signaling marker, exhibited a gradual induction over time. Noticeably, Arabidopsis RNS1 (T2 ribonuclease) was rapidly induced by MNV1 and exhibited anti-correlation with the internalization of MNV1. Exposure to recombinant Arabidopsis RNS1 protein reduced the viral titers and degraded MNV1 RNA in vitro. In conclusion, the internalization of MNV1 into Arabidopsis was fluctuated by mutual interactions that were potentially regulated by Arabidopsis immune systems containing RNS1.


Assuntos
Arabidopsis/imunologia , Arabidopsis/virologia , Norovirus/fisiologia , Plântula/imunologia , Plântula/virologia , Internalização do Vírus , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos , Defensinas/metabolismo , Doenças Transmitidas por Alimentos/virologia , Imunidade Inata , Camundongos , Oxilipinas , Desenvolvimento Vegetal , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/virologia , Proteínas Recombinantes , Ribonucleases/genética , Ribonucleases/metabolismo , Plântula/genética , Plântula/metabolismo , Regulação para Cima , Carga Viral
19.
Cell Host Microbe ; 25(6): 815-826.e4, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31155345

RESUMO

CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are integrated into the CRISPR locus, prompting transcription of CRISPR RNAs (crRNAs) that guide Cas12a cleavage of foreign complementary DNA. However, mobile genetic elements counteract Cas12a with inhibitors, notably type V-A anti-CRISPRs (AcrVAs). We present cryoelectron microscopy structures of Cas12a-crRNA bound to AcrVA1 and AcrVA4 at 3.5 and 3.3 Å resolutions, respectively. AcrVA1 is sandwiched between the recognition (REC) and nuclease (NUC) lobes of Cas12a and inserts into the binding pocket for the protospacer-adjacent motif (PAM), a short DNA sequence guiding Cas12a targeting. AcrVA1 cleaves crRNA in a Cas12a-dependent manner, inactivating Cas12a-crRNA complexes. The AcrVA4 dimer is anchored around the crRNA pseudoknot of Cas12a-crRNA, preventing required conformational changes for crRNA-DNA heteroduplex formation. These results uncover molecular mechanisms for CRISPR-Cas12a inhibition, providing insights into bacteria-phage dynamics.


Assuntos
Sistemas CRISPR-Cas , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/metabolismo , Inibidores Enzimáticos/metabolismo , RNA Guia/metabolismo , Ribonucleases/metabolismo , Microscopia Crioeletrônica , Endodesoxirribonucleases/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA Guia/ultraestrutura , Ribonucleases/ultraestrutura
20.
Nat Commun ; 10(1): 2544, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186424

RESUMO

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ribonucleases/metabolismo , Ruminococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Conformação de Ácido Nucleico , Domínios Proteicos , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Guia/genética , Ribonucleases/química , Ribonucleases/genética , Ruminococcus/enzimologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA