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1.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 46(1): 11-18, 2024 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-38433625

RESUMO

Objective To investigate the effect of staphylococcal nuclease and tudor domain containing 1(SND1) on the biological function of osteosarcoma cells and decipher the mechanism of SND1 in regulating ferroptosis in osteosarcoma cells via SLC7A11. Methods Human osteoblasts hFOB1.19 and osteosarcoma cell lines Saos-2,U2OS,HOS,and 143B were cultured,in which the expression level of SND1 was determined.Small interfering RNA was employed to knock down the expression of SND1(si-SND1) in the osteosarcoma cell line HOS and 143B.The CCK8 assay kit,colony formation assay,and Transwell assay were employed to examine the effect of SND1 expression on the biological function of osteosarcoma cells.Furthermore,we altered the expression of SND1 and SLC7A11 in osteosarcoma cells to investigate the effect of SND1 on osteosarcoma ferroptosis via SLC7A11. Results The mRNA and protein levels of SND1 in Saos-2,U2OS,HOS,and 143B cells were higher than those in hFOB1.19 cells(all P<0.01).Compared with the control group,transfection with si-SND1 down-regulated the expression level of SND1 in HOS and 143B cells(all P<0.01),decreased the viability of HOS and 143B cells,reduced the number of colony formation,and inhibited cell invasion and migration(all P<0.001).The ferroptosis inducer Erastin promoted the apoptosis of HOS and 143B cells,while the ferroptosis inhibitor Ferrostatin-1 improved the viability of HOS and 143B cells(all P<0.001).After SND-1 knockdown,Erastin reduced the viability of HOS and 143B cells,while Ferrostatin-1 restored the cell viability(all P<0.001).After treatment with Erastin in the si-SND1 group,the levels of iron and malondialdehyde were elevated,and the level of glutathione was lowered(all P<0.001).The results of in vivo experiments showed that SND1 knockdown inhibited the mass of the transplanted tumor in 143B tumor-bearing nude mice(P<0.001).Knocking down the expression of SND1 resulted in down-regulated SLC7A11 expression(all P<0.001) and increased ferroptosis in HOS and 143B cells(P<0.001,P=0.020). Conclusions SND1 presents up-regulated expression in osteosarcoma cells.It may inhibit ferroptosis by up-regulating the expression of SLC7A11,thereby improving the viability of osteosarcoma cells.


Assuntos
Neoplasias Ósseas , Cicloexilaminas , Eliptocitose Hereditária , Ferroptose , Osteossarcoma , Fenilenodiaminas , Animais , Humanos , Camundongos , Sistema y+ de Transporte de Aminoácidos , Endonucleases , Camundongos Nus , Nuclease do Micrococo , Domínio Tudor
2.
Nucleic Acids Res ; 52(4): 1575-1590, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38296834

RESUMO

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.


Assuntos
DNA Catalítico , DNA Forma Z , Quadruplex G , Animais , Camundongos , DNA Catalítico/metabolismo , Desoxirribonuclease I/metabolismo , Nuclease do Micrococo/genética , Cloreto de Sódio , Hemina , DNA Bacteriano/metabolismo , Biofilmes , Staphylococcus/genética , DNA , Polissacarídeos , Peroxidase/metabolismo , Mamíferos/genética
3.
Bioorg Chem ; 144: 107133, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38278047

RESUMO

The staphylococcal nuclease also referred as micrococcal nuclease (MNase) is a key drug target as the enzyme degrades the neutrophil extracellular trap (NET) and empowers the pathogen to subvert the host innate immune system. To this end, the current study presents a critical evaluation of MNase inhibition rendered by benzimidazole-based ligands (C1 and C2) and probes its therapeutic implications. A nuclease assay indicated that MNase inhibition rendered by C1 and C2 was âˆ¼ 55 % and âˆ¼ 72 %, respectively, at the highest tested concentration of 10 µM. Studies on enzyme kinetics revealed that C2 rendered non-competitive inhibition and significantly reduced MNase turnover number (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of âˆ¼ 1122 nM. In CD spectroscopy, a notable perturbation in the ß-sheet content of MNase was observed in presence of C2. Fluorescence-microscope analysis indicated that MNase inhibition by C2 could restore entrapment of methicillin-resistant Staphylococcus aureus (MRSA) in calf-thymus DNA (CT-DNA). Flow cytometry and confocal microscope analysis revealed that uptake of DNA-entrapped MRSA by activated THP-1 cells was reinstated by MNase inhibition rendered by C2. Inhibition of nuclease by the non-toxic ligand C2 holds therapeutic prospect as it has the potential to bolster the DNA-mediated entrapment machinery and mitigate MRSA infections.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Nuclease do Micrococo/análise , Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Ligantes , DNA/química , Macrófagos/metabolismo , Benzimidazóis/farmacologia
4.
Neoplasia ; 47: 100963, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38176295

RESUMO

Muscle-invasive and metastatic bladder cancer indicates extra worse prognosis. Accumulating evidence roots for the prominent role of circular RNAs(circRNAs) in bladder cancer, while the mechanisms linking circRNAs and bladder cancer metastasis remain limitedly investigated. Here, we identified a significantly upregulated circRNA candidate, hsa_circ_0001583, from online datasets. Validated by qRT-PCR, PCR, sanger sequencing, actinomycin D and RNase R digestion experiments, hsa_circ_0001583 was proved to be a genuine circular RNA with higher expression levels in bladder cancer tissue. Through gain and loss of function experiments, hsa_circ_0001583 exhibited potent migration and invasion powers both in vitro and in vivo. The staphylococcal nuclease and Tudor domain containing 1 (SND1) was identified as an authentic binding partner for hsa_circ_0001583 through RNA pulldown and RIP experiments. Elevated levels of hsa_circ_0001583 could bind more to SND1 and protect the latter from degradation. Rescue experiments demonstrated that such interaction-induced increased in SND1 levels in bladder cancer cells enabled the protein to pump its endonuclease activity, leading to the degradation of tumor-suppressing MicroRNAs (miRNAs) including miR-126-3p, the suppressor of Disintegrin And Metalloproteinase Domain-Containing Protein 9 (ADAM9), ultimately driving cells into a highly migrative and invasive state. In summary, our study is the first to highlight the upregulation of hsa_circ_0001583 in bladder cancer and its role in downregulating miR-126-3p by binding to and stabilizing the SND1 protein, thereby promoting bladder cancer cell migration and invasion. This study adds hsa_circ_0001583 to the pool of bladder cancer metastasis biomarkers and therapeutic targets.


Assuntos
MicroRNAs , Neoplasias da Bexiga Urinária , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Domínio Tudor , Biomarcadores Tumorais/genética , Neoplasias da Bexiga Urinária/genética , Proliferação de Células , Movimento Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Endonucleases/genética , Endonucleases/metabolismo
5.
J Vis Exp ; (201)2023 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-37982508

RESUMO

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.


Assuntos
Cromatina , Nucleossomos , Animais , Cromatina/genética , Mamíferos , Nuclease do Micrococo , Regiões Promotoras Genéticas
6.
PLoS One ; 18(11): e0293809, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37988351

RESUMO

In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain.


Assuntos
Nucleossomos , Trypanosoma , Nucleossomos/genética , Cromatina/genética , Histonas/genética , Trypanosoma/genética , DNA , Nuclease do Micrococo/metabolismo
7.
ACS Appl Mater Interfaces ; 15(31): 37174-37183, 2023 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-37525332

RESUMO

Periprosthetic infections are one of the most serious complications in orthopedic surgeries, and those caused by Staphylococcus aureus (S. aureus) are particularly hard to treat due to their tendency to form biofilms on implants and their notorious ability to invade the surrounding bones. The existing prophylactic local antibiotic deliveries involve excessive drug loading doses that could risk the development of drug resistance strains. Utilizing an oligonucleotide linker sensitive to micrococcal nuclease (MN) cleavage, we previously developed an implant coating capable of releasing covalently tethered vancomycin, triggered by S. aureus-secreted MN, to prevent periprosthetic infections in the mouse intramedullary (IM) canal. To further engineer this exciting platform to meet broader clinical needs, here, we chemically modified the oligonucleotide linker by a combination of 2'-O-methylation and phosphorothioate modification to achieve additional modulation of its stability/sensitivity to MN and the kinetics of MN-triggered on-demand release. We found that when all phosphodiester bonds within the oligonucleotide linker 5'-carboxy-mCmGTTmCmG-3-acrydite, except for the one between TT, were replaced by phosphorothioate, the oligonucleotide (6PS) stability significantly increased and enabled the most sustained release of tethered vancomycin from the coating. By contrast, when only the peripheral phosphodiester bonds at the 5'- and 3'-ends were replaced by phosphorothioate, the resulting oligonucleotide (2PS) linker was cleaved by MN more rapidly than that without any PS modifications (0PS). Using a rat femoral canal periprosthetic infection model where 1000 CFU S. aureus was inoculated at the time of IM pin insertion, we showed that the prophylactic implant coating containing either 0PS- or 2PS-modified oligonucleotide linker effectively eradicated the bacteria by enabling the rapid on-demand release of vancomycin. No bacteria were detected from the explanted pins, and no signs of cortical bone changes were detected in these treatment groups throughout the 3 month follow-ups. With an antibiotic tethering dose significantly lower than conventional antibiotic-bearing bone cements, these coatings also exhibited excellent biocompatibility. These chemically modified oligonucleotides could help tailor prophylactic anti-infective coating strategies to meet a range of clinical challenges where the risks for S. aureus prosthetic infections range from transient to long-lasting.


Assuntos
Infecções Estafilocócicas , Vancomicina , Ratos , Camundongos , Animais , Vancomicina/química , Nuclease do Micrococo/farmacologia , Staphylococcus aureus , Antibacterianos/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/prevenção & controle
8.
PeerJ ; 11: e15520, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37361042

RESUMO

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm's chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements.These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars.


Assuntos
Histonas , Nucleossomos , Masculino , Animais , Suínos/genética , Humanos , Histonas/genética , Nucleossomos/genética , Nuclease do Micrococo/genética , Análise do Sêmen , Sêmen/metabolismo , Cromatina/genética , Espermatozoides/metabolismo , Fatores de Transcrição/genética , Genômica , Desenvolvimento Embrionário/genética , Mamíferos/genética
9.
Biochemistry ; 62(11): 1670-1678, 2023 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-37227385

RESUMO

Several proteins have been shown to undergo a shift in the mechanism of ligand binding-induced folding from conformational selection (CS; folding precedes binding) to induced fit (IF; binding precedes folding) with increasing ligand concentration. In previous studies of the coupled folding/binding reaction of staphylococcal nuclease (SNase) in the presence of a substrate analogue, adenosine-3',5'-diphosphate (prAp), we found that the two phosphate groups make important energetic contributions toward stabilizing its complex with the native protein as well as transient conformational states encountered at high ligand concentrations favoring IF. However, the structural contributions of each phosphate group during the reaction remain unclear. To address this question, we relied on fluorescence, nuclear magnetic resonance (NMR), absorption, and isothermal titration calorimetry to study the effects of deletion of the phosphate groups of prAp on the kinetics of ligand-induced folding, using a strategy analogous to mutational ϕ-value analysis to interpret the results. Kinetic measurements over a wide range of ligand concentrations, together with structural characterization of a transient protein-ligand encounter complex using 2D NMR, indicated that, at high ligand concentrations favoring IF, (i) the 5'-phosphate group interacts weakly with denatured SNase during early stages of the reaction, resulting in loose docking of the two domains of SNase, and (ii) the 3'-phosphate group engages in some specific contacts with the polypeptide in the transition state prior to formation of the native SNase-prAp complex.


Assuntos
Nuclease do Micrococo , Dobramento de Proteína , Nuclease do Micrococo/metabolismo , Ligantes , Cinética , Conformação Proteica
10.
Clin Exp Rheumatol ; 41(11): 2182-2191, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37083155

RESUMO

OBJECTIVES: To investigate the role of fragile X mental retardation syndrome-related protein 1 (FXR1), an RNA binding protein, in the development of osteoarthritis (OA), to define its mechanism of action in cartilage, and to determine whether targeting FXR1 can prevent OA in mice. METHODS: Western blot analysis and quantitative polymerase chain reaction were performed using cartilage tissue from control and osteoarthritic mice. FXR1 expression was detected by immunofluorescence staining using cartilage tissue from mice. OA was induced by destabilising the medial meniscus in the mice. Infection of mouse chondrocytes with FXR1 lentivirus, as well as viral injection into the mouse knee joint cavity, resulted in high FXR1 protein expression. Chondrocyte apoptosis was detected by TUNEL assay and cell senescence was detected by SA-ß-gal staining assay. RESULTS: FXR1 expression was significantly reduced in cartilage and soft tissue from mice with OA compared with the controls. FXR1 overexpression reduced staphylococcal nuclease domain protein 1 (SND1) levels. Furthermore, FXR1 is able to inhibit apoptosis and senescence of chondrocytes via SND1 and hinder the development of OA in mice. CONCLUSIONS: FXR1 down-regulates SND1 expression, thereby alleviating osteoarthritic symptoms in mice. In summary, FXR1 may have a therapeutic approach to the treatment of OA.


Assuntos
Cartilagem Articular , Osteoartrite do Joelho , Osteoartrite , Camundongos , Animais , Nuclease do Micrococo/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Condrócitos/metabolismo , Apoptose
11.
Anticancer Drugs ; 34(5): 627-639, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36730541

RESUMO

Nasal-type natural killer/T-cell lymphoma (NKTCL) is a typical class of non-Hodgkin's lymphoma, which is quite malignant because of its high resistance to chemotherapy. N6-methyladenosine (m6A) modification, a prevalent modification of eukaryotic RNA, was emerging as an important regulatory mechanism in progression of various tumors. Here, we demonstrated that methyltransferase-like 3 (METTL3), an RNA methyltransferase, was obviously upregulated in human NKTCL cell lines (NK-92, YTS, SNT-8, and SNK-6) compared with normal NK cells. Knockdown of METTL3 noticeably repressed proliferation and facilitated apoptosis in SNT-8 cells, whereas overexpression of METTL3 showed opposite results in SNK-6 cells. In the mechanism exploration, we found that METTL3 stimulated the m6A modification of staphylococcal nuclease and Tudor domain-containing protein 1 (SND1) mRNA, recruited YTH m6A RNA binding protein 1 to recognize the m6A site, thereby enhancing its mRNA stability. Rescue experiments demonstrated that METTL3 significantly prohibited NKTCL cell chemotherapy sensitivity to cisplatin (DDP) through regulating SND1 expression. Furthermore, knockdown of SND1 suppressed tumor growth and reduced DDP resistance in vivo . Taken together, our findings uncovered the role of METTL3 in the regulation of chemotherapy resistance in NKTCL oncogenesis.


Assuntos
Cisplatino , Linfoma de Células T , Humanos , Proliferação de Células/genética , Endonucleases , Células Matadoras Naturais , Metiltransferases/genética , Metiltransferases/metabolismo , Nuclease do Micrococo , RNA Mensageiro/genética
12.
Curr Comput Aided Drug Des ; 19(4): 278-287, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36627784

RESUMO

AIMS: Cancer is a disease that takes lives of thousands of people each year. There are more than 100 different types of cancers known to man. This fatal disease is one of the leading causes of death today. BACKGROUND: Astrocyte elevated gene-1(AEG-1)/Metadherin (MTDH) activates multiple oncogenic signaling pathways and leads to different types of cancers. MTDH interacting with staphylococcal nuclease domain containing 1(SND1) supports the survival and growth of mammary epithelial cells under oncogenic conditions. OBJECTIVE: Silencing MTDH or SND1 individually or disrupting their interaction compromises the tumorigenic potential of tumor-initiating cells. The aim of our present study was to investigate novel interactions of staphylococcal nuclease domain containing 1 (SND1) binding domain of AEG-1/MTDH with different lead compounds through molecular docking approach using MOE software. METHODS: Molecular docking was done by docking the ChemBridge database against important residues of MTDH involved in interaction with SND1. After docking the whole ChemBridge database, the top 200 interactive compounds were selected based on docking scores. After applying Lipinski's rule, all the remaining chosen compounds were studied on the basis of binding affinity, binding energy, docking score and protein-ligand interactions. Finally, 10 compounds showing multiple interactions with different amino acid residues were selected as the top interacting compounds. RESULTS: Three compounds were selected for simulation studies after testing these compounds using topkat toxicity and ADMET studies. The simulation study indicated that compound 32538601 is a lead compound for inhibiting MTDH-SND1 complex formation. CONCLUSION: These novels, potent inhibitors of MTDH-SND1 complex can ultimately help us in controlling cancer up to some extent.


Assuntos
Nuclease do Micrococo , Neoplasias , Masculino , Humanos , Simulação de Acoplamento Molecular , Nuclease do Micrococo/metabolismo , Proteínas Oncogênicas/metabolismo , Endonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA
13.
Methods Mol Biol ; 2594: 29-43, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36264486

RESUMO

Functional cis-regulatory elements (CREs) act as precise transcriptional switches for fine-tuning gene transcription. Identification of CREs is critical for understanding regulatory mechanisms of gene expression associated with various biological processes in eukaryotes. It is well known that CREs reside in open chromatin that exhibits hypersensitivity to enzyme cleavage and physical shearing. Currently, high-throughput methodologies, such as DNase-seq, ATAC-seq, and FAIRE-seq, have been widely applied in mapping open chromatin in various eukaryotic genomes. More recently, differential MNase (micrococcal nuclease) treatment has been successfully employed to map open chromatin in addition to profiling nucleosome landscape in both mammalian and plant species. We have developed a MNase hypersensitivity sequencing (MH-seq) technique in plants. The MH-seq procedure includes plant nuclei fixation and purification, differential treatments of purified nuclei with MNase, specific recovery of MNase-trimmed small DNA fragments within 20~100 bp in length, and MH-seq library construction followed by Illumina sequencing and data analysis. MH-seq has been successfully applied for global identification of open chromatin in both Arabidopsis thaliana and maize. It has been proven to be an attractive alternative for profiling open chromatin. Thus, MH-seq is expected to be valuable in probing chromatin accessibility on a genome-wide scale for other plants with sequenced genomes. Moreover, MHS data allow to implement footprinting assays to unveil binding sites of transcription factors.


Assuntos
Arabidopsis , Cromatina , Animais , Cromatina/genética , Nucleossomos , Nuclease do Micrococo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Plantas/genética , Fatores de Transcrição/metabolismo , Mamíferos/genética
14.
Methods Mol Biol ; 2599: 59-68, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36427143

RESUMO

Genomic DNA wraps around core histones to form nucleosomes, which provides steric constraints on how transcription factors (TFs) can interact with gene regulatory sequences. It is increasingly apparent that well-positioned, accessible nucleosomes are an inherent feature of active enhancers and can facilitate cooperative TF binding, referred to as nucleosome-mediated cooperativity. Thus, profiling chromatin and nucleosome properties (accessibility, positioning, and occupancy) on the genome is crucial to understand cell-type-specific gene regulation. Here we describe a simplified protocol to profile accessible nucleosomes in the mammalian genome using low-level and high-level micrococcal nuclease (MNase) digestion followed by genome-wide sequencing.


Assuntos
Cromatina , Nucleossomos , Animais , Nucleossomos/genética , Cromatina/genética , Nuclease do Micrococo/metabolismo , Genoma , Histonas/genética , Histonas/metabolismo , Mamíferos/genética , Mamíferos/metabolismo
15.
Theranostics ; 12(16): 6898-6914, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36276642

RESUMO

Rationale: Protein palmitoylation is tightly related to tumorigenesis or tumor progression as many oncogenes or tumor suppressors are palmitoylated. AEG-1, an oncogene, is commonly elevated in a variety of human malignancies, including hepatocellular carcinoma (HCC). Although AEG-1 was suggested to be potentially modified by protein palmitoylation, the regulatory roles of AEG-1 palmitoylation in tumor progression of HCC has not been explored. Methods: Techniques as Acyl-RAC assay and point mutation were used to confirm that AEG-1 is indeed palmitoylated. Moreover, biochemical experiments and immunofluorescent microscopy were applied to examine the cellular functions of AEG-1 palmitoylation in several cell lines. Remarkably, genetically modified knock-in (AEG-1-C75A) and knockout (Zdhhc6-KO) mice were established and subjected to the treatment of DEN to induce the HCC mice model, through which the roles of AEG-1 palmitoylation in HCC is directly addressed. Last, HCQ, a chemical compound, was introduced to prove in principal that elevating the level of AEG-1 palmitoylation might benefit the treatment of HCC in xenograft mouse model. Results: We showed that AEG-1 undergoes palmitoylation on a conserved cysteine residue, Cys-75. Blocking AEG-1 palmitoylation exacerbates the progression of DEN-induced HCC in vivo. Moreover, it was demonstrated that AEG-1 palmitoylation is dynamically regulated by zDHHC6 and PPT1/2. Accordingly, suppressing the level of AEG-1 palmitoylation by the deletion of Zdhhc6 reproduces the enhanced tumor-progression phenotype in DEN-induced HCC mouse model. Mechanistically, we showed that AEG-1 palmitoylation adversely regulates its protein stability and weakens AEG-1 and staphylococcal nuclease and tudor domain containing 1 (SND1) interaction, which might contribute to the alterations of the RISC activity and the expression of tumor suppressors. For intervention, HCQ, an inhibitor of PPT1, was applied to augment the level of AEG-1 palmitoylation, which retards the tumor growth of HCC in xenograft model. Conclusion: Our study suggests an unknown mechanism that AEG-1 palmitoylation dynamically manipulates HCC progression and pinpoints that raising AEG-1 palmitoylation might confer beneficial effect on the treatment of HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Lipoilação , Cisteína/metabolismo , Nuclease do Micrococo/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Endonucleases/metabolismo
16.
J Immunol Res ; 2022: 5239006, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36213325

RESUMO

Staphylococcal nuclease domain-containing protein 1 (SND1) is an evolutionarily conserved multidomain protein, which has gained attention recently due to its positive regulation in several cancer progression and metastatic spread. However, the specific contribution of SND1 glycosylation in glioma remains uncertain. In the current study, we confirmed that SND1 was highly expressed in human glioma. Using site-directed mutagenesis, we created four predicted N-glycosylation site mutants for SND1 and provided the first evidence that SND1 undergoes N-glycosylation on its Asn50, Asn168, Asn283, and Asn416 residues in human glioma U87 cells. In addition, we found that removing the N-glycans on the Asn50 site destabilized SND1 and led to its endoplasmic reticulum-associated degradation. Furthermore, destabilized SND1 inhibits the glioma cell proliferation and metastasis. Collectively, our results reveal that N-glycosylation at Asn50 is essential for SND1 folding and trafficking, thus essential for the glioma process, providing new insights for SND1 as a potential disease biomarker for glioma.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Glioma , Biomarcadores/metabolismo , Proliferação de Células , Endonucleases/genética , Endonucleases/metabolismo , Glioma/metabolismo , Glicosilação , Humanos , Nuclease do Micrococo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
17.
J Phys Chem B ; 126(40): 7870-7882, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36190807

RESUMO

The impact of pH on proteins is significant but often neglected in molecular dynamics simulations. Constant-pH Molecular Dynamics (CpHMD) is the state-of-the-art methodology to deal with these effects. However, it still lacks widespread adoption by the scientific community. The stochastic titration CpHMD is one of such methods that, until now, only supported the GROMOS force field family. Here, we extend this method's implementation to include the CHARMM36m force field available in the GROMACS software package. We test this new implementation with a diverse group of proteins, namely, lysozyme, Staphylococcal nuclease, and human and E. coli thioredoxins. All proteins were conformationally stable in the simulations, even at extreme pH values. The RMSE values (pKa prediction vs experimental) obtained were very encouraging, in particular for lysozyme and human thioredoxin. We have also identified a few residues that challenged the CpHMD simulations, highlighting scenarios where the method still needs improvement independently of the force field. The CHARMM36m all-atom implementation was more computationally efficient when compared with the GROMOS 54A7, taking advantage of a shorter nonbonded interaction cutoff and a less frequent neighboring list update. The new extension will allow the study of pH effects in many systems for which this force field is particularly suited, i.e., proteins, membrane proteins, lipid bilayers, and nucleic acids.


Assuntos
Simulação de Dinâmica Molecular , Ácidos Nucleicos , Escherichia coli , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas , Proteínas de Membrana , Nuclease do Micrococo/química , Muramidase , Tiorredoxinas
18.
Nat Struct Mol Biol ; 29(10): 1011-1023, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36220894

RESUMO

The linear sequence of DNA provides invaluable information about genes and their regulatory elements along chromosomes. However, to fully understand gene function and regulation, we need to dissect how genes physically fold in the three-dimensional nuclear space. Here we describe immuno-OligoSTORM, an imaging strategy that reveals the distribution of nucleosomes within specific genes in super-resolution, through the simultaneous visualization of DNA and histones. We combine immuno-OligoSTORM with restraint-based and coarse-grained modeling approaches to integrate super-resolution imaging data with Hi-C contact frequencies and deconvoluted micrococcal nuclease-sequencing information. The resulting method, called Modeling immuno-OligoSTORM, allows quantitative modeling of genes with nucleosome resolution and provides information about chromatin accessibility for regulatory factors, such as RNA polymerase II. With Modeling immuno-OligoSTORM, we explore intercellular variability, transcriptional-dependent gene conformation, and folding of housekeeping and pluripotency-related genes in human pluripotent and differentiated cells, thereby obtaining the highest degree of data integration achieved so far to our knowledge.


Assuntos
Nuclease do Micrococo , Nucleossomos , Cromatina/genética , DNA/genética , Histonas/genética , Humanos , Nuclease do Micrococo/metabolismo , Nucleossomos/genética , RNA Polimerase II/genética
19.
J Phys Chem B ; 126(38): 7321-7330, 2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36106487

RESUMO

Classical molecular dynamics simulations are a versatile tool in the study of biomolecular systems, but they usually rely on a fixed bonding topology, precluding the explicit simulation of chemical reactivity. Certain modifications can permit the modeling of reactions. One such method, multiscale reactive molecular dynamics, makes use of a linear combination approach to describe condensed-phase free energy surfaces of reactive processes of biological interest. Before these simulations can be performed, models of the reactive moieties must first be parametrized using electronic structure data. A recent study demonstrated that gas-phase electronic structure data can be used to derive parameters for glutamate and lysine which reproduce experimental pKa values in both bulk water and the staphylococcal nuclease protein with remarkable accuracy and transferability between the water and protein environments. In this work, we first present a new model for aspartate derived in similar fashion and demonstrate that it too produces accurate pKa values in both bulk and protein contexts. We also describe a modification to the prior methodology, involving refitting some of the classical force field parameters to density functional theory calculations, which improves the transferability of the existing glutamate model. Finally and most importantly, this reactive molecular dynamics approach, based on rigorous statistical mechanics, allows one to specifically analyze the fundamental physical causes for the marked pKa shift of both aspartate and glutamate between bulk water and protein and also to demonstrate that local steric and electrostatic effects largely explain the observed differences.


Assuntos
Ácido Aspártico , Simulação de Dinâmica Molecular , Glutamatos , Lisina , Nuclease do Micrococo , Proteínas/química , Eletricidade Estática , Água/química
20.
Methods Mol Biol ; 2516: 29-38, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35922619

RESUMO

The digestion of chromosomes using micrococcal nuclease (MNase) enables the analysis of their fundamental structural units. For example, the digestion of eukaryotic chromatin using MNase results in laddered DNA fragments (~150 bp increment), which reflects the length of the DNA wrapped around regularly spaced nucleosomes. Here, we describe the application of MNase to examine the chromosome structure in Archaea. We used Thermococcus kodakarensis, a hyperthermophilic euryarchaeon that encodes proteins homologous to eukaryotic histones. Methods for chromosome extraction and agarose gel electrophoresis of MNase-digested DNA including small fragments (~30 bp) are also described.


Assuntos
Archaea , Nuclease do Micrococo , Archaea/genética , Archaea/metabolismo , Cromatina/genética , DNA/genética , Digestão , Nuclease do Micrococo/metabolismo , Nucleossomos
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