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1.
Methods Mol Biol ; 2570: 223-234, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156786

RESUMO

RNA aptamers can be genetically encoded in cells to probe and manipulate cellular function. The usefulness of aptamers in mammalian cells is limited by low accumulation and degradation by ribonucleases. Expression of circular RNA aptamers using the Tornado expression system achieves high stability and an abundance of intracellular RNA aptamers. With this method, RNA aptamers with otherwise minimal activity become potent inhibitors. Here, we describe protocols to characterize circular RNA aptamers expressed using Tornado. Included are methods to assess stability, abundance, subcellular localization, and target binding by circular RNA aptamers.


Assuntos
Aptâmeros de Nucleotídeos , Animais , Aptâmeros de Nucleotídeos/química , Mamíferos/genética , RNA/química , RNA Circular , Ribonucleases/metabolismo
2.
Sci Rep ; 12(1): 15598, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114363

RESUMO

In angiosperms, self-incompatibility (SI) is a common and widespread mechanism for plant prevention of inbreeding, and late-acting self-incompatibility (LSI) may be ancestral in the group. In this work, we studied Schima superba, a species in Theaceae that is a commercially important timer and fire-resistant tree, and revealed its LSI mechanism. Hormones, enzymes, transcriptomes, and proteins were compared between self-pollination (SP) and outcross pollination (OP) in the styles and ovaries from 0 to 120 h after pollination. The self-pollen tubes grew to the bottom of the style and entered the ovary within 48 h but failed to penetrate the ovule. Meanwhile, the hormone and peroxidase levels dramatically changed. Transcriptome and proteome analyses explored the molecular mechanisms of LSI and candidate genes related to LSI in S. superba. Overall, 586.71 million reads were obtained, and 79,642 (39.08%) unigenes were annotated. KEGG and GO analysis showed that there were 4531 differentially expressed genes (DEGs) and 82 differentially expressed proteins (DEPs) at 48 h in self- (SP) versus outcross pollination (OP). Among these, 160 DEGs and 33 DEPs were involved in pollen-pistil interactions. "Pollen-pistil interaction," "signal recognition," and "component of membrane" were downregulated in SP, whereas "cell wall and membrane biosynthetic process," and "oxidoreductase activity" were upregulated. The DEGs involved with S-RNases and SCF during SP suggested that the LSI occurred at 48 h in the ovary and that the LSI in S. superba was under gametophyte control. Calcium ion increase and release, mitochondrial function loss, and ROS disruption further aggravated PCD progress and cell death. The LSI of S. superba, which happened 48 h after pollination, was a key time point. The incompatibility PT ceased growth in the ovary because of S-RNase recognition and PCD in this organ. This study highlights the LSI molecular mechanism in S. superba and provides a reference to other species in Theaceae.


Assuntos
Proteoma , Theaceae , Cálcio , Hormônios , Oxirredutases , Peroxidases , Pólen/genética , Espécies Reativas de Oxigênio , Ribonucleases
3.
PLoS One ; 17(9): e0271420, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36155485

RESUMO

Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C. acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C. acnes, but a system for evaluating the induction of innate immunity by C. acnes using melanization as an indicator has not yet been established. Here we demonstrated that C. acnes rapidly causes melanization of the silkworm hemolymph. On the other hand, Staphylococcus aureus, a gram-positive bacterium identical to C. acnes, does not cause immediate melanization. Even injection of heat-killed C. acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C. acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C. acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C. acnes and that C. acnes peptidoglycans are involved in the induction of innate immunity in silkworms.


Assuntos
Bombyx , Animais , Desoxirribonucleases , Hemolinfa/microbiologia , Humanos , Lisostafina , Melaninas , Monofenol Mono-Oxigenase , Muramidase , Peptidoglicano/farmacologia , Propionibacterium acnes , Ribonucleases
4.
Int J Mol Sci ; 23(17)2022 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-36077152

RESUMO

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.


Assuntos
Sistemas CRISPR-Cas , Ribonucleases , Sistemas CRISPR-Cas/genética , Endorribonucleases/genética , Edição de Genes , Técnicas de Inativação de Genes , Engenharia Genética , Humanos , Monócitos , RNA Mensageiro/genética , Ribonucleases/genética
5.
Viruses ; 14(9)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36146715

RESUMO

Members of the jingmenviruses group have been found in arthropods and mammals on all continents except Australia and Antarctica. Two viruses of this group were isolated from patients with fever after a tick bite. Using a nested RT-PCR assay targeting a jingmenvirus polymerase gene fragment, we screened ticks collected in seven regions of Russia and found that the abundant jingmenvirus-positive were of Ixodes ricinus species, with the prevalence ranging from 19.8% to 34.3%. In all cases, DNase/RNase treatment suggested that the detected molecule was DNA and subsequent next generation sequencing (NGS) proved that the viral polymerase gene was integrated in the I. ricinus genome. The copy number of the integrated polymerase gene was quantified by qPCR relative to the ITS2 gene and estimated as 1.32 copies per cell. At least three different genetic variants of the integrated polymerase gene were found in the territory of Russia. Phylogenetic analysis of the integrated jingmenvirus polymerase gene showed the highest similarity with the sequence of the correspondent gene obtained in Serbia from I. ricinus.


Assuntos
Ixodes , Animais , Desoxirribonucleases , Humanos , Mamíferos , Filogenia , Reação em Cadeia da Polimerase , Ribonucleases
6.
Mol Biol (Mosk) ; 56(5): 764-773, 2022.
Artigo em Russo | MEDLINE | ID: mdl-36165015

RESUMO

Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific tumor cells, it is necessary to understand the mechanisms of action of the drugs that make up the combination. Bacillus pumilus ribonuclease (binase) is considered as an adjuvant antitumor agent, and the sensitivity of malignant cells to the apoptogenic effect of binase depends on the presence of certain oncogenes. In the acute myelogenous leukemia cell line Kasumi-1, binase blocks the proliferation pathway mediated by the mutant tyrosine kinase KIT, which, as shown in our work, activates an alternative proliferation pathway through AKT kinase. In Kasumi-1 cells, binase in combination with an Akt1/2 inhibitor induces apoptosis, and their toxic effects add up: the Akt1/2 inhibitor blocks the binase-induced pathway after suppression of the KIT-dependent pathway. Thus, a combination of binase and AKT kinase inhibitors can effectively block various pathways of tumor cell proliferation and be used for their elimination.


Assuntos
Antineoplásicos , Proteínas Proto-Oncogênicas c-akt , Antineoplásicos/farmacologia , Apoptose , Endorribonucleases/metabolismo , Inibidores de Proteínas Quinases , Proteínas Tirosina Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Ribonucleases/genética , Ribonucleases/farmacologia
7.
Int J Mol Sci ; 23(18)2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36142343

RESUMO

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the 'XiangShui' lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in 'XiangShui' lemon are the same as those within other species. Expression pattern analysis revealed that S3-RNase and S4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S1-RNase, S2-RNase, S3-RNase and S4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S3-RNase interacted with TOPP4. Therefore, S3-RNase may play an important role in the SI of 'XiangShui' lemon.


Assuntos
Ácido Aspártico Proteases , Citrus , Autoincompatibilidade em Angiospermas , Citrus/metabolismo , Diacilglicerol O-Aciltransferase , Endorribonucleases , Fucose , Giberelinas , Fosfolipídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , RNA , RNA Ligase (ATP) , Ribonucleases/genética , Ribonucleases/metabolismo , Autoincompatibilidade em Angiospermas/genética , beta-Frutofuranosidase
8.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 9): 330-337, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36048083

RESUMO

Angiogenin is an unusual member of the RNase A family and is of great interest in multiple pathological contexts. Although it has been assigned various regulatory roles, its core catalytic function is that of an RNA endonuclease. However, its catalytic efficiency is comparatively low and this has been linked to a unique C-terminal helix which partially blocks its RNA-binding site. Assuming that binding to its RNA substrate could trigger a conformational rearrangement, much speculation has arisen on the topic of the interaction of angiogenin with RNA. To date, no structural data on angiogenin-RNA interactions have been available. Here, the structure of angiogenin bound to a double-stranded RNA duplex is reported. The RNA does not reach the active site of angiogenin and no structural arrangement of the C-terminal domain is observed. However, angiogenin forms a previously unobserved crystallographic dimer that makes several backbone interactions with the major and minor grooves of the RNA double helix.


Assuntos
RNA de Cadeia Dupla , Ribonuclease Pancreático , Sequência de Aminoácidos , Cristalografia por Raios X , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Ribonucleases/química , Ribonucleases/genética , Ribonucleases/metabolismo
9.
J Immunol ; 209(7): 1348-1358, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36165203

RESUMO

Endotoxin tolerance is a state of hyporesponsiveness to LPS, triggered by previous exposure to endotoxin. Such an immunosuppressive state enhances the risks of secondary infection and has been associated with the pathophysiology of sepsis. Although this phenomenon has been extensively studied, its molecular mechanism is not fully explained. Among candidates that play a crucial role in this process are negative regulators of TLR signaling, but the contribution of MCP-induced protein 1 (MCPIP1; Regnase-1) has not been studied yet. To examine whether macrophage expression of MCPIP1 participates in endotoxin tolerance, we used both murine and human primary macrophages devoid of MCPIP1 expression. In our study, we demonstrated that MCPIP1 contributes to LPS hyporesponsiveness induced by subsequent LPS stimulation and macrophage reprogramming. We proved that this mechanism revolves around the deubiquitinase activity of MCPIP1, which inhibits the phosphorylation of MAPK and NF-κB activation. Moreover, we showed that MCPIP1 controlled the level of proinflammatory transcripts in LPS-tolerized cells independently of its RNase activity. Finally, we confirmed these findings applying an in vivo endotoxin tolerance model in wild-type and myeloid MCPIP1-deficient mice. Taken together, this study describes for the first time, to our knowledge, that myeloid MCPIP1 participates in endotoxin tolerance and broadens the scope of known negative regulators of the TLR4 pathway crucial in this phenomenon.


Assuntos
Lipopolissacarídeos , Receptor 4 Toll-Like , Animais , Enzimas Desubiquitinantes , Endorribonucleases , Tolerância à Endotoxina , Endotoxinas , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Ribonucleases/genética , Receptor 4 Toll-Like/metabolismo
10.
Sci Adv ; 8(32): eabm0699, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35947655

RESUMO

Small RNAs regulate spermatogenesis across species ranging from Caenorhabditis elegans to humans. In C. elegans, two Argonaute proteins, ALG-3 and ALG-4, and their associated alg-3/4 26G-small RNAs are essential for spermiogenesis at 25°C. The alg-3/4 26G-small RNAs are antisense to their target mRNAs and produced by the RNA-dependent RNA polymerase, RRF-3. However, it remains unclear how the RNA templates for RRF-3 are generated and which cellular processes are affected by alg-3/4 26G-small RNAs. Here, we demonstrate a previously unidentified zc3h12a-like ribonuclease protein, NYN-3, in alg-3/4 26G-small RNAs biogenesis. NYN-3 is not only required for proper abundance of alg-3/4 26G-small RNAs but also crosslinked to their targeted mRNAs before RRF-3 from ePAR-CLIP-seq. Bioinformatics analysis was then used to parse the 26G-small RNA-targeted genes into functional subclasses. Collectively, these findings implicate NYN-3 as an initiator of alg-3/4 26G-small RNA generation.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endorribonucleases/metabolismo , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Espermatogênese/genética , Fatores de Transcrição/metabolismo
11.
Cell Rep ; 40(7): 111226, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977479

RESUMO

CRISPR-Cas13 RNA endonucleases show promise for programmable RNA knockdown. However, sequence-specific binding of Cas13 unleashes non-specific bystander RNA cleavage, or collateral activity, raising concerns for experiments and therapeutic applications. Although robust in cell-free and bacterial environments, collateral activity in mammalian cells remains disputed. We investigate Cas13d collateral activity in a therapeutic context for myotonic dystrophy type 1, caused by a transcribed CTG repeat expansion. We find that, when targeting CUGn RNA in mammalian cells, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis. Collateral effects also occur when targeting abundant endogenous transcripts. To minimize collateral activity for repeat-targeting approaches, we introduce GENO, an adeno-associated virus-compatible strategy that leverages guide RNA processing to control Cas13d expression. We argue that thorough assessment of collateral activity is necessary when applying Cas13 in mammalian cells and that GENO illustrates advantages of compact regulatory systems for Cas-based gene therapies.


Assuntos
Edição de Genes , Distrofia Miotônica , Animais , Sistemas CRISPR-Cas/genética , Homeostase , Humanos , Mamíferos/genética , Distrofia Miotônica/genética , RNA/genética , RNA Guia/genética , Ribonucleases/genética
12.
Sci Rep ; 12(1): 13785, 2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-35962056

RESUMO

Cell-free biosensors are promising tools for medical diagnostics, yet their performance can be affected by matrix effects arising from the sample itself or from external components. Here we systematically evaluate the performance and robustness of cell-free systems in serum, plasma, urine, and saliva using two reporter systems, sfGFP and luciferase. In all cases, clinical samples have a strong inhibitory effect. Of the different inhibitors, only RNase inhibitor mitigated matrix effects. However, we found that the recovery potential of RNase inhibitor was partially muted by interference from glycerol contained in the commercial buffer. We solved this issue by designing a strain producing an RNase inhibitor protein requiring no additional step in extract preparation. Furthermore, our new extract yielded higher reporter levels than previous conditions and tempered interpatient variability associated with matrix effects. This systematic evaluation and improvements of cell-free system robustness unified across many types of clinical samples is a significant step towards developing cell-free diagnostics for a wide range of conditions.


Assuntos
Ribonucleases , Saliva , Sistema Livre de Células
13.
Exp Oncol ; 44(2): 159-162, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35964653

RESUMO

AIM: To assess the inducible NO-synthase activity and the total RNase activity in tissue samples and blood neutrophils of the patients with prostate intraepithelial neoplasia (PIN) and prostate cancer (PCa) of different stages. MATERIALS AND METHODS: NO level was measured in tumor tissue and neutrophils of patients with PIN and PCa of different stages by electron paramagnetic resonance using the spin traps technology. RNase activity in tumor tissue of patients with PIN and PCa was measured by the method of zymography. RESULTS: We have found that NO levels in prostate tumor tissue were significantly higher than in the PIN and increased along with the disease progression. Analysis of NO level in neutrophils of the PCa patients demonstrated that the values were not dispersed and did not depend on the stage of disease. NO level in neutrophils of the PCa patients increased manifold as compared with that in healthy donors. At the same time, the RNase activity in the prostate tumor tissue gradually decreased with PCa progression. CONCLUSION: Activities of inducible NO-synthase and RNases change significantly with progression of PCa.


Assuntos
Neoplasia Prostática Intraepitelial , Neoplasias da Próstata , Humanos , Masculino , Próstata/patologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologia , Ribonucleases
14.
Int J Mol Sci ; 23(16)2022 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-36012339

RESUMO

Ovarian cancer represents one of the most malignant gynecological cancers worldwide, with an overall 5-year survival rate, being locked in the 25-30% range in the last decade. Cancer immunotherapy is currently one of the most intensively investigated and promising therapeutic strategy and as such, is expected to provide in the incoming years significant benefits for ovarian cancer treatment as well. Here, we provide a detailed survey on the highly pleiotropic oncosuppressive roles played by the human RNASET2 gene, whose protein product has been consistently reported to establish a functional crosstalk between ovarian cancer cells and key cellular effectors of the innate immune system (the monocyte/macrophages lineage), which is in turn able to promote the recruitment to the cancer tissue of M1-polarized, antitumoral macrophages. This feature, coupled with the ability of T2 ribonucleases to negatively affect several cancer-related parameters in a cell-autonomous manner on a wide range of ovarian cancer experimental models, makes human RNASET2 a very promising candidate to develop a "multitasking" therapeutic approach for innovative future applications for ovarian cancer treatment.


Assuntos
Neoplasias Ovarianas , Ribonucleases , Proteínas Supressoras de Tumor , Feminino , Genes Supressores de Tumor , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/genética
15.
Int J Mol Sci ; 23(16)2022 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-36012542

RESUMO

RNase H1s are associated with growth and development in both plants and animals, while the roles of RNase H1s in bryophytes have been rarely reported. Our previous data found that PpRNH1A, a member of the RNase H1 family, could regulate the development of Physcomitrium (Physcomitrella) patens by regulating the auxin. In this study, we further investigated the biological functions of PpRNH1A and found PpRNH1A may participate in response to heat stress by affecting the numbers and the mobilization of lipid droplets and regulating the expression of heat-related genes. The expression level of PpRNH1A was induced by heat stress (HS), and we found that the PpRNH1A overexpression plants (A-OE) were more sensitive to HS. At the same time, A-OE plants have a higher number of lipid droplets but with less mobility in cells. Consistent with the HS sensitivity phenotype in A-OE plants, transcriptomic analysis results indicated that PpRNH1A is involved in the regulation of expression of heat-related genes such as DNAJ and DNAJC. Taken together, these results provide novel insight into the functions of RNase H1s.


Assuntos
Bryopsida , Bryopsida/genética , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ribonucleases/metabolismo
16.
Nanoscale ; 14(33): 12153-12161, 2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-35968721

RESUMO

The potential for liquid biopsy samples to be used in place of more invasive tissue biopsies has become increasingly revalent as it has been found that nucleic acids (NAs) present in the blood of cancer patients originate from tumors. Nanomagnetic extraction has proven to be a highly effective means to rapidly prepare NA from clinical samples for molecular diagnostics. In this article, the lysis reaction used to extract RNA from the human epithelial melanoma cells have been optimized using silica coated superparamagnetic nanoparticles (SPM NP). The lysis buffer (LB) is composed of several agents that denature cells, i.e., surfactant and guanidinium isothiocyanate (GITC), and agents that inhibit the degradation of circulated nucleic acids (cfNAs). The surfactant Triton X-100 has been widely used in LB but has been placed on the European Union REACH list. We have compared the qRT-PCR sensitivity resulting from LBs composed of Triton X-100 to several sustainable surfactants, i.e., Tergitol 15-S-7, Tergitol 15-S-9 and Tween-20. Surprisingly, the inclusion of these surfactants in the LB was not found to significantly improve cell lysis, and subsequently the sensitivity of qRT-PCR. The role of the sample matrix was also examined by performing extractions from solutions containing up to 30 mg mL-1 serum albumin. The qRT-PCR sensitivity was found to decrease as the concentration of this protein was increased; however, this was linked to an increased RNase activity and not the concentration of the protein itself. These results lead us to recommend a reformulation of LB for clinical samples, and to conclude that sensitive qRT-PCR RNA analysis can be performed in serum with the timely addition of an RNase inhibitor.


Assuntos
Detergentes , Ácidos Nucleicos , RNA , Ribonucleases , Células Eucarióticas , Humanos , Melanoma , Octoxinol , Poloxaleno , RNA/isolamento & purificação , Ribonucleases/antagonistas & inibidores
17.
Int J Mol Sci ; 23(15)2022 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-35955913

RESUMO

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.


Assuntos
RNA de Transferência , Ribonucleases , Humanos , Queratinócitos/metabolismo , Estresse Oxidativo , RNA de Transferência/genética , Ribonuclease Pancreático/metabolismo
18.
Biochim Biophys Acta Gen Subj ; 1866(11): 130219, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35926731

RESUMO

HIV-1 transactivator (Tat) protein plays a critical role in neurological disorders resulting from viral infection, commonly known as HIV-1-associated neurocognitive disorders (HAND). Previous studies have shown that circulant Tat induces M1 microglial activation, one of the hallmark features of HAND, and this is coupled with ER stress and subsequent Unfolded Protein Response (UPR) triggering. Here, we demonstrate that bystander stimuli of Tat in microglial cells result in the simultaneous overexpression of IRE1-related markers and production of M1-typed proinflammatory mediators. We also show that blocking IRE1/XBP-1 signaling using 4µ8C diminishes such inflammatory response. These findings reinforce a role for the IRE1/XBP-1 pathway in HIV-1 Tat neuropathology and suggest that targeting IRE1 RNase activity using 4µ8C or analogue compounds may provide a therapeutic intervention to mitigate against neuroinflammation in HAND.


Assuntos
HIV-1 , Endorribonucleases , Microglia , Proteínas Serina-Treonina Quinases , Ribonucleases
19.
Bioorg Med Chem ; 71: 116888, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35944385

RESUMO

Ribonuclease A is used as a model enzyme system for the design of RNase inhibitors. Previous studies have established that the geometric nature of the active site cleft is an important feature for the accommodation of crescent-shaped compounds in the active site of RNase A. In the current research, benzene-based triazolylated semicircular hybrid molecules carrying different polar functionalities were synthesized and screened for their RNase A inhibitory potency. An additional carboxylic acid group at the C1-position of the 1,3,5-trisubstituted benzene ring enhanced the inhibitory properties significantly. Furthermore, the studies revealed that the reduced arm lengths of 3,5-substituents result in a better geometric complementarity that makes the molecules fit favorably in the semicircular cavity of the active site as visualized by docking studies. In a series of ten such new compounds, the 3,5-bis[4-(sulfomethyl)-1H-1,2,3-triazol-1-yl]benzoic acid exhibited, the highest inhibition efficiency with a Ki value of 12 ±â€¯0.9 µM. This study identifies a new class of non-nucleoside inhibitors which are competitive inhibitors of the ribonucleolytic activity of RNase A.


Assuntos
Ribonuclease Pancreático , Ribonucleases , Benzeno , Derivados de Benzeno , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia
20.
J Chem Inf Model ; 62(17): 4247-4260, 2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-35960929

RESUMO

A range of in silico methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1α (IRE1). Using Protein-RNA molecular docking along with a series of extensive restrained/unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid-general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation.


Assuntos
Inositol , Ribonucleases , Endorribonucleases/genética , Simulação de Acoplamento Molecular , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Ribonucleases/metabolismo
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