Targeting protein-DNA interactions in prokaryotic systems

原核系统中靶向蛋白质-DNA 相互作用

• 批准号: 10014752
• 负责人: Federico Bernal
• 金额: $ 11.11万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014752
• 关键词: ATP phosphohydrolase; Acinetobacter baumannii; Active Biological Transport; Affect; Affinity; Alanine; Amino Acids; Antibiotic Resistance; Antibiotics; Assimilations; Bacteria; Bacterial Infections; Base Pairing; Binding; Binding Sites; Biological Assay; C-terminal; Campylobacter jejuni; Cell Survival; Cell Wall; Cell division; Cell membrane; Cells; Cessation of life; Charge; Chemistry; Circular Dichroism Spectroscopy; Clinic; Clinical; Confocal Microscopy; Cryoelectron Microscopy; Cytoplasm; DNA; DNA Binding; DNA Footprint; DNA-Directed RNA Polymerase; DNA-Protein Interaction; DNase protection assay; Data; Detection; Development; Diffusion; Drug resistance; Effectiveness; Elements; Ensure; Enterobacter; Enterococcus faecium; Environment; Enzymes; Escherichia coli; Eukaryotic Cell; Event; Exposure to; Fibroblasts; Flow Cytometry; Fluorescence; Genes; Genetic Transcription; Gram-Negative Bacteria; Growth; Helix-Turn-Helix Motifs; Human body; Hydrogen Bonding; Hydrophobicity; Individual; Infection; Ion Transport; Klebsiella pneumonia bacterium; Life; Major Groove; Masks; Mediating; Membrane; Metabolic; Methods; Microbial Biofilms; Minimum Inhibitory Concentration measurement; Minor; Modeling; Multi-Drug Resistance; Nitrogen; Normal Cell; PAX3 gene; Pathogenicity; Penetrance; Penetration; Peptides; Permeability; Pharmaceutical Preparations; Play; Population; Promoter Regions; Property; Proteins; Protons; Pseudomonas aeruginosa; Pump; RNA; Resolution; Role; Sampling; Sigma Factor; Site; Sodium Azide; Specificity; Stains; Structure; Surface; System; Transcription Initiation Site; Untranslated RNA; Virulence; Visual; Water; Work; alpha helix; antimicrobial; bactericide; base; cell motility; cytotoxic; design; drug development; ds-DNA; imaging detection; inhibitor/antagonist; interest; meetings; melting; multi-drug resistant pathogen; mutation screening; nitrogen metabolism; novel therapeutics; passive transport; pathogen; peptide analog; pressure; promoter; protein aminoacid sequence; protein expression; quorum sensing; resistance mechanism; response; small molecule; uptake

A number of structural studies have confirmed the binding of sigma54 to RNAP and DNA. Sigma54 binds DNA -24 and -12 base pairs from the transcription start site to initiate DNA melting, an essential step in transcription. A substantial body of work, including DNA footprinting, alanine scanning mutation studies, low resolution cryoelectron microscopy of sigma54-RNA-polyermase bound to DNA in the closed configuration, and high resolution NMR structure has shown that sigma54 binds specifically and tightly to the major groove of DNA. For example the Wemmer lab used NMR to show that the 66 amino acid long helix-turn-helix (HTH) motif from in Aquifex aeolicus binds DNA. Within this C-terminal HTH motif, a single alpha-helix at residues 377 to 386 (ARRTVAKYRE), termed the RpoN box, is responsible for binding to the major groove of DNA (PBD: 2O8K). Replacement of Arg378, Arg379, Tyr384 and Arg385 with Ala decreased DNA binding substantially. The protein NMR structure shows that this helix interacts selectively with the -24 region (5'-TGGCACG-3') of the promoter. In particular, Arg378 and Arg379 are localized to the -24 element of the interaction and make multiple hydrogen bonding and ionic interactions with Gua-25 and Gua-26 on the non-coding strand of DNA based on significant line-broadening of a 15N-HSQC spectrum. This leads to a high affinity interaction between the 66-mer HTH motif and the promoter region (Kd = 114 nM) Although the peptides are modeled to inhibit transcription, it is necessary to assess cell viability to ensure that downstream antivirulence effects are observable. By using the standard broth microdilution method,24 the antimicrobial activity of all stapled peptide analogs was assessed. The minimum inhibitory concentration (MIC) value of all peptides is 32 microg/mL or higher. As this value is high compared to many conventional antibiotics, the peptide can be assessed at low concentrations to study its effects on virulence properties of Gram negative bacteria. To determine whether the compounds are cytotoxic to eukaryotic cells, WS1 fibroblasts were exposed to serial dilutions of each compound. The data demonstrate that the compounds are not toxic to normal cells over a concentration range peaking at 10 microM. In order to assess the helical structure of the synthesized peptides, circular dichroism spectroscopy was carried out by dissolving the compounds in water. While the wild type sigma54 peptide is unstructured, stapled peptides 1-4 all possess the hallmark spectrum of an alpha-helical secondary structure. Flow cytometry was used to gain a high-throughput perspective on the effectiveness of each stapled peptide in a large population of bacteria. By making use of its individual event detection, the percentage of cellular uptake of the peptides was determined. In both E. coli and P. aeruginosa species of Gram negative bacteria, all four peptide analogs are capable of penetration. Compared to the vehicle control, the wild type peptide, the sequence in which no staple is present, shows minor penetration in PA01 P. aeruginosa and insignificant levels of penetration in BW25113 E. coli. With each peptide, there is a penetration of at least 50% of cell in each peptide sample. In particular, sigma54-2 appears to have the highest penetrance whereas sigma54-4 has the least. To determine the mode of transport within the cell, sodium azide was used to inactivate ATPases used in hydrogen ion transport. All interior transport will be conducted through a non-active method such as passive or facilitated diffusion. With both types of treatments, it's possible to determine whether the mode of uptake of stapled peptides is through active or passive transport. Furthermore, with passive transport, the entry of the peptide should be faster than active due to the lack of metabolic means necessary to promote cell entry. The majority of peptide analogs increase in penetration upon sodium azide treatment. This may be an indicator that the peptide is able to be pumped out by bacteria to a degree. Overall, this assay demonstrates that stapled peptides are capable of penetrating cells better than their unstapled counterpart in a charge-independent manner. Further studies in confocal microscopy were conducted to gain a low-throughput visual method of assessing cell penetrance. The peptides correlate with the flow cytometry data in that some cells display strong fluorescence intensity and others display less. By using an image-based detection method for permeability, we observe that the double-stranded DNA of E. coli appears to aggregate in the center away from the membrane. In many of the cells observed, a number of the cells were undergoing cell division but still had significant uptake of these peptides. With the cell membranes, it is seen that the peptide does not integrate itself into the membrane due to a lack of green or masking of the red membrane stain. These peptides must cross both the cell wall and cell membrane to remain in the cytoplasm of bacteria. The binding of sigma54 RpoN with the -24 site of glnA was examined by performing a DNase protection assay which showed that stapled sigma 54 peptides protected DNA from degradation. To determine whether the peptides blocked the transcription of nitrogen metabolism genes, E. coli cells grown under nitrogen deficient conditions were treated with stapled peptides. RNA was isolated, and the sigma 54 dependent genes glnA, yeaG, and nac were analyzed. We also examined pspA which, despite being a sigma54 dependent gene, is insensitive to nitrogen depletion. The data showed that stapled sigma 54-2 and -3 were the best at blocking the transcription of sigma 54-dependent nitrogen depletion response genes. No effects were seen with pspA. Taken together, these results demonstrate that stapled peptides can be designed to target bacterial systems as well as protein-DNA interactions with a great level of specificity. The work on this project concluded in April 2019

许多结构研究已经证实了Sigma54与RNAP和DNA的结合。 Sigma54结合了从转录起始位点的DNA -24和-12碱基对，以启动DNA熔化，这是转录的重要步骤。大量的工作，包括DNA足迹，丙氨酸扫描突变研究，低分辨率的低分辨率冷冻显微镜在封闭构型中与DNA结合的SIGMA54-RNA-溶质酶以及高分辨率NMR结构表明SIGMA54特异性地结合了DNA的主要groove。例如，Wemmer Lab使用NMR表明来自Aquifex Aeolicus中的66个氨基酸长螺旋 - 转螺螺旋（HTH）基序结合了DNA。在该C末端HTH基序中，称为RPON盒的残基377至386（Arrtvakyre）处的单个α-螺旋负责与DNA的主要凹槽（PBD：2O8K）结合。替代Ara 378，Arg379，Tyr384和Arg385的ALA大大降低了DNA的结合。蛋白质NMR结构表明，该螺旋与启动子的-24区（5'-TGGCACG-3'）有选择性相互作用。特别是，ARG378和ARG379位于相互作用的-24元素上，并基于15N-HSQC频谱的显着线路，在非编码DNA上与GUA-25和GUA-26进行多种氢键和离子相互作用。这导致66-mer HTH基序与启动子区域（KD = 114 nm）之间的高亲和力相互作用，尽管对肽进行了建模以抑制转录，但有必要评估细胞活力，以确保可观察到下游抗动力效应。通过使用标准的肉汤微稀释法，评估了所有钉肽类似物的抗菌活性。所有肽的最小抑制浓度（MIC）值为32 microg/ml或更高。由于该值与许多常规抗生素相比很高，因此可以在低浓度的情况下评估肽对革兰氏阴性细菌的毒力特性的影响。为了确定化合物是否对真核细胞具有细胞毒性，WS1成纤维细胞暴露于每种化合物的系列稀释液中。数据表明，在浓度范围内，这些化合物对正常细胞没有毒性，在10 microM处峰值。为了评估合成肽的螺旋结构，通过将化合物溶解在水中，进行圆形二分色谱法。虽然野生型Sigma54肽是非结构化的，但钉钉肽1-4都具有α-螺旋二级结构的标志性光谱。流式细胞仪用于对大量细菌中每个钉肽的有效性获得高通量的观点。通过利用其单个事件检测，确定了肽的细胞摄取百分比。在革兰氏阴性细菌的大肠杆菌和铜绿假单胞菌中，所有四个肽类似物均能够穿透。与媒介物对照相比，野生型肽（不存在主食的序列）在PA01 P中显示了少量的渗透。铜绿铜和BW25113大肠杆菌中的渗透水平微不足道的渗透水平。在每个肽中，每个肽样品中至少有50％的细胞穿透。特别是，Sigma54-2似乎具有最高的渗透率，而Sigma54-4的渗透率最少。为了确定细胞内的运输方式，使用叠氮化钠来灭活氢离子传输中使用的ATPases。所有内部运输将通过一种非活性方法（例如被动或促进的扩散）进行。通过两种类型的处理，可以确定钉钉肽的摄取方式是通过主动或被动转运的。此外，通过被动运输，由于缺乏促进细胞进入所需的代谢手段，肽的进入应比活性更快。叠氮化钠处理后，大多数肽类似物在渗透中的渗透增加。这可能表明该肽能够在一定程度上被细菌泵出。总体而言，该测定法表明，钉钉肽能够以无电荷的方式与无缝合的肽更好地穿透细胞。进行了共聚焦显微镜的进一步研究，以获得评估细胞渗透率的低通量视觉方法。肽与流式细胞仪数据相关，因为某些细胞表现出强烈的荧光强度，而另一些细胞显示较少。通过使用基于图像的检测方法进行渗透性，我们观察到大肠杆菌的双链DNA似乎在远离膜的中心聚集。在观察到的许多细胞中，许多细胞正在经历细胞分裂，但仍具有这些肽的明显摄取。使用细胞膜，可以看到由于缺乏红色膜染色的绿色或掩盖，肽不会将自身整合到膜中。这些肽必须越过细胞壁和细胞膜，以保留在细菌的细胞质中。通过进行DNase保护测定法检查了Sigma54 RPON与-24位点的结合，该测定法表明SIGMA 54肽可保护DNA免受降解。为了确定肽是否阻断了氮代谢基因的转录，用钉钉肽处理了在氮缺乏条件下生长的大肠杆菌细胞。分离RNA，并分析Sigma 54依赖性基因GLNA，YEA和NAC。我们还检查了PSPA，尽管它是SigMA54依赖性基因，但对氮的耗竭不敏感。数据表明，固定的Sigma 54-2和-3是阻止Sigma 54依赖性氮耗尽反应基因转录的最佳方法。 PSPA看不到效果。综上所述，这些结果表明，可以设计钉肽的固定肽以及具有很高水平的特异性的蛋白质-DNA相互作用。该项目的工作于2019年4月结束