Targeting protein-DNA interactions in prokaryotic systems

原核系统中蛋白质-DNA 相互作用的靶向

• 批准号: 9556660
• 负责人: Federico Bernal
• 金额: $ 30.74万
• 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/9556660
• 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 Count; 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; Effectiveness; Elements; Ensure; Enterobacter; Enterococcus faecium; Environment; Enzymes; Escherichia coli; Eukaryotic Cell; Event; 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; Resistance development; Resolution; Role; Sampling; Sigma Factor; Site; Sodium Azide; Specificity; Staining method; Stains; Structure; Surface; System; Transcription Initiation Site; Untranslated RNA; Virulence; Visual; Water; 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.

许多结构研究已经证实了sigma 54与RNAP和DNA的结合。Sigma 54结合来自转录起始位点的DNA -24和-12碱基对以启动DNA解链，这是转录中的必要步骤。大量的工作，包括DNA足迹，丙氨酸扫描突变研究，低分辨率低温电子显微镜的sigma 54-RNA-聚合酶结合到DNA的封闭配置，和高分辨率NMR结构显示，sigma 54特异性和紧密结合到DNA的大沟。例如，Wemmer实验室使用NMR显示来自Aquifex aeolicus的66个氨基酸长的螺旋-转角-螺旋（HTH）基序结合DNA。在该C-末端HTH基序内，在残基377至386处的单个α-螺旋（ARRTVAKYRE），称为RpoN盒，负责结合DNA的大沟（PBD：2 O 8 K）。用Ala取代Arg 378、Arg 379、Tyr 384和Arg 385显著降低DNA结合。蛋白质NMR结构显示该螺旋选择性地与启动子的-24区域（5 '-TGGCACG-3'）相互作用。特别地，Arg 378和Arg 379定位于相互作用的-24元素，并且基于15 N-HSQC光谱的显著谱线增宽，与DNA的非编码链上的Gua-25和Gua-26进行多重氢键和离子相互作用。这导致66-mer HTH基序和启动子区域之间的高亲和力相互作用（Kd = 114 nM）。尽管肽被建模为抑制转录，但有必要评估细胞活力以确保下游抗毒力效应是可观察的。通过使用标准肉汤微量稀释法，24评估所有钉合肽类似物的抗微生物活性。所有肽的最小抑制浓度（MIC）值为32 μ g/mL或更高。由于与许多常规抗生素相比，该值较高，因此可以在低浓度下评估该肽以研究其对革兰氏阴性菌毒力特性的影响。为了确定化合物是否对真核细胞具有细胞毒性，将WS 1成纤维细胞暴露于每种化合物的连续稀释液。数据表明，在峰值为10 μ M的浓度范围内，化合物对正常细胞没有毒性。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二色光谱。虽然野生型σ 54肽是非结构化的，但钉合肽1-4都具有α-螺旋二级结构的标志谱。流式细胞术用于获得关于每个钉合肽在大细菌群体中的有效性的高通量观点。通过利用其单个事件检测，确定肽的细胞摄取百分比。在E.大肠杆菌和铜绿假单胞菌物种的革兰氏阴性细菌，所有四种肽类似物都能够渗透。与溶剂对照相比，野生型肽（其中不存在钉合的序列）在PA 01铜绿假单胞菌中显示出较小的渗透，在BW 25113 E中显示出不显著的渗透水平。杆菌对于每种肽，在每种肽样品中存在至少50%的细胞渗透。特别地，sigma 54 -2似乎具有最高的外显率，而sigma 54 -4具有最低的外显率。为了确定细胞内的转运模式，使用叠氮化钠来抑制氢离子转运中使用的ATP酶。所有的内部运输都将通过被动或易化扩散等非主动方法进行。对于这两种类型的治疗，可以确定钉合肽的摄取模式是通过主动还是被动转运。此外，在被动转运的情况下，由于缺乏促进细胞进入所必需的代谢手段，肽的进入应该比主动进入更快。大多数肽类似物在叠氮化钠处理后渗透增加。这可能是一个指标，表明肽能够被细菌泵出到一定程度。总体而言，该测定证明钉合肽能够以电荷非依赖性方式比其未钉合的对应物更好地穿透细胞。在共聚焦显微镜下进行了进一步的研究，以获得一个低通量的视觉方法来评估细胞的迁移率。这些肽与流式细胞术数据相关，因为一些细胞显示强荧光强度，而另一些细胞显示较低。大肠杆菌似乎聚集在远离膜的中心。在观察到的许多细胞中，许多细胞正在进行细胞分裂，但仍然大量摄取这些肽。对于细胞膜，可以看出，由于缺乏绿色或红色膜染色的掩蔽，肽本身不整合到膜中。这些肽必须穿过细胞壁和细胞膜才能留在细菌的细胞质中。通过进行DNA酶保护测定来检查σ 54 RpoN与glnA的-24位点的结合，该测定显示钉合的σ 54肽保护DNA免于降解。为了确定这些肽是否阻断了氮代谢基因的转录，E.用钉合肽处理在氮缺乏条件下生长的大肠杆菌细胞。分离RNA，并分析σ 54依赖性基因glnA、yeaG和nac。我们还研究了pspA，尽管它是一个σ 54依赖性基因，但对氮消耗不敏感。数据显示，钉合的σ 54-2和σ 54 - 3在阻断σ 54依赖性氮耗竭反应基因的转录方面是最好的。pspA未观察到影响。总之，这些结果表明，钉合肽可以被设计为靶向细菌系统以及具有高特异性水平的蛋白质-DNA相互作用。