Mechanism of PMT-Induced Anchorage-Independent Growth and mTOR Signaling

PMT 诱导锚定非依赖性生长和 mTOR 信号转导的机制

• 批准号: 9157396
• 负责人: P. BOON Chock
• 金额: $ 113.62万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/9157396
• 关键词: Agreement; Anchorage-Independent Growth; Animals; Antibodies; Apoptosis; Bacterial Infections; Bacterial Proteins; Base Pairing; Binding; Bioinformatics; Biological Assay; Bladder; Cell Line; Cell Proliferation; Cell Separation; Cell division; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Conditioned Culture Media; DNA; DNA Sequence; Deletion Mutation; Detection; Development; Distal; Engineering; Enzyme-Linked Immunosorbent Assay; Epithelium; Fibroblasts; Genes; Genetic Transcription; Genomic DNA; Genomics; Genotype; Guanosine Triphosphate Phosphohydrolases; Guide RNA; Heterotrimeric GTP-Binding Proteins; Hour; In Vitro; Incubated; Induced Mutation; Infection; Injection of therapeutic agent; Integrase; Intercistronic Region; Interleukin-6; Interleukins; Length; Link; MAP Kinase Gene; Mediating; Membrane; Mitogens; Modification; Molecular; Monocyte Chemoattractant Protein-1; Mus; Mutation; Neoplasm Metastasis; Normal Cell; Nucleotides; Pasteurella multocida toxin; Pathway interactions; Phenotype; Plasmids; Play; Point Mutation; Printing; Process; Property; Proteins; Puromycin; Reagent; Recombinants; Reporting; Resources; Restriction fragment length polymorphism; Role; Sampling; Serum; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Specificity; Streptavidin; Streptococcus pyogenes; Systems Integration; Techniques; Technology; Testing; Transfection; Transgenes; Ureter; Variant; Virulence Factors; autocrine; cell motility; cell type; chemokine; cofactor; cytokine; deamidation; design; ds-DNA; homologous recombination; human FRAP1 protein; in vitro Assay; in vivo; keratinocyte; nuclease; prevent; promoter; protein expression; repaired; research study; tumor

Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage-independent growth for certain type of cells. These properties suggest that PMT may have the potential to act as a tumor promotter especially in the case of chronic infections. The detailed mechanism behind mitogenic properties of PMT is unknown. We previously revealed that rPMT hijacks cellular signal transduction pathways via deamidation of heterotrimeric G-proteins and leads to a sustained activation of mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, which in part, leads to cell proliferation and migration. In addition, showed that rPMT treated cells secrete an autocrine/pracrine factor(s) that is responsible of mTOR and MAPK signaling but not the RTK pathway activation in a manner independent of Gαq/11 deamidation. This observation is in agreement with the report showing that experimental injection of rPMT in animal causes cell proliferation at distal sites, including the epithelium of the bladder and ureter. In an effort to find other diffusible factors, we used the mouse cytokine array. Each membrane array contains 40 different anti-cytokine antibodies, three positive controls, and one negative control printed in duplicate. Conditioned media from control nontreated cells and cells treated with rPMT for 24 h were diluted and mixed with a cocktail of biotinylated detection antibodies. The samples/antibody mixture was then incubated with the membranes. Any cytokine/detection antibody complex present was bound by its cognate immobilized capture antibody on the membrane. The membranes were revealed using streptavidin-HRP and chemiluminescent detection reagent. We found that three cytokines including interleukin IL-6, keratinocyte-derived chemokine (KC) and monocyte chemotactic protein-1 (MCP1) are significantly upregulated in rPMT treated cells. This result was validated using ELISA technique. Importantly, IL-6 was able to activate MAPK in serum starved fibroblast cells. Since rPMT is known to activate other signaling pathways independently of Gαq. To determine the specific role of Gαq deamidation induced by rPMT treatment, we choose to establish a cell line that harbor Gαq mutation of the key catalytic residue of the inherent GTPase activity leading to the synthesis of the active form of Gαq protein using clustered regularly interspaced short palindromic repeats (CRISPR) technology. The Cas9 nuclease from Streptococcus pyogenes can be directed by a chimeric single-guided RNA (sgRNA) to any genomic locus followed by a 5-NGG (where N can be any nucleotide.) protospacer adjacent motif (PAM). A 20 nucleotide guide sequence within the sgRNA directs Cas9 to genomic DNA target via Watson-crick base pairing. The resulting complex leads to site-specific double strand break (DSB) three base pairs upstream of PAM sequence by Cas9. The DSBs are repaired by the cell either by homologous recombination (HR) in the presence of a DNA donor template or by nonhomologous end joining (NHEJ) repair mechanisms. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HR-mediated repair, however, can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Five 20 bp-long Gαq sgRNAs, designed using several online bioinformatics resources, were cloned in CRISPR/cas9 plasmid downstream of U6 promoter. To test the cleavage efficiency of these gRNAs, we used an in vitro assay. We prepare the in vitro cleavage assay by mixing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed Gαq sgRNAs that are complementary to the genomic DNA sequence of Gαq. We found that 4 out of 5 Gαq sgRNAs are efficient at cuttting Gαq genomic DNA. To test the specificity of Gαq sgRNA-mediated DSBs, we introduced a mutation in the PAM sequences. These mutations prevent all Gαq sgRNAs to cut DNA. This result shows that Gαq sgRNAs-induced DNA cleavage is very specific for each sgRNA. We next used these 4 Gαq sgRNAs in subsequent experiment to test their ability to induce genomic DNA editing in the cell leading to a mutation in Gαq gene. Cells were transfected with constructs that express both Gαq sgRNA and Cas9 protein along with a DNA donor template that have the desired mutation. Forty eight hours post transfection, genomic DNA samples isolated from mock and Gαq sgRNAs transfected cells were used for PCR amplifications. The PCR products were subjected to Restriction fragment length polymorphism (RFLP), CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs), and the surveyor assays to detect Cas9-induced mutations and the cuting efficiency of each sgRNA. Using all these assays, we found that only one Gαq sgRNA out of four is efficient to induce a modification in cellular Gαq genomic DNA. This difference in Gαq sgRNAs efficiency is not due to a difference in sgRNAs or Cas9 protein expression levels. Currently, we are in the process of isolating the clones either by cell sorting or puromycin selection. In addition, we used targeted integration system that uses ϕC31 integrase that can integrate a donor plasmid of any size into an intergenic region with high transcription activity, as a single copy, and requires no cofactors. The integrated transgenes with a desired mutation are stably expressed and heritable. This level of targeting control allows for the study of phenotypic effects free from context and positional variations, which results in more accurate genotype to phenotype correlations.

多杀性巴氏杆菌毒素（Pasteurella multocida toxin，PMT）是一种细胞内作用蛋白，在体内外均具有促有丝分裂作用，并能诱导某些类型细胞的非贴壁依赖性生长。这些特性表明，PMT可能具有作为肿瘤促进剂的潜力，特别是在慢性感染的情况下。PMT促有丝分裂特性背后的详细机制尚不清楚。我们以前发现，rPMT通过异源三聚体G蛋白的脱酰胺作用劫持细胞信号转导途径，并通过G q/11/PLC/PKC介导的途径导致mTOR信号转导的持续激活，这部分导致细胞增殖和迁移。 此外，显示rPMT处理的细胞分泌自分泌/外分泌因子，其负责mTOR和MAPK信号传导，但不以独立于G q/11脱酰胺的方式激活RTK途径。该观察结果与报告一致，该报告显示在动物中实验性注射rPMT导致远端部位的细胞增殖，包括膀胱和输尿管的上皮。为了寻找其他扩散因子，我们使用了小鼠细胞因子阵列。每个膜阵列含有40种不同的抗细胞因子抗体、三种阳性对照和一种阴性对照，一式两份打印。将来自对照未处理细胞和用rPMT处理24小时的细胞的条件培养基稀释并与生物素化检测抗体的混合物混合。然后将样品/抗体混合物与膜一起孵育。存在的任何细胞因子/检测抗体复合物通过其在膜上的同源固定捕获抗体结合。使用链霉亲和素-HRP和荧光检测试剂显示膜。我们发现，三种细胞因子，包括白细胞介素IL-6，角质形成细胞衍生的趋化因子（KC）和单核细胞趋化蛋白1（MCP 1）在rPMT处理的细胞显着上调。用ELISA技术验证了该结果。重要的是，IL-6能够在血清饥饿的成纤维细胞中激活MAPK。 由于已知rPMT独立于G q激活其他信号通路。为了确定rPMT处理诱导的G q脱酰胺的具体作用，我们选择建立一个细胞系，该细胞系具有固有GTdR活性的关键催化残基的G q突变，从而使用成簇规则间隔短回文重复序列（CRISPR）技术合成G q蛋白的活性形式。来自酿脓链球菌的Cas9核酸酶可以通过嵌合单引导RNA（sgRNA）引导至任何基因组基因座，随后是5-NGG（其中N可以是任何核苷酸）。原型间隔区邻近基序（PAM）。sgRNA内的20个核苷酸的指导序列通过沃森-克里克碱基配对将Cas9引导至基因组DNA靶标。所得到的复合物通过Cas9导致PAM序列上游三个碱基对的位点特异性双链断裂（DSB）。DSB由细胞在DNA供体模板存在下通过同源重组（HR）或通过非同源末端连接（NHEJ）修复机制修复。不精确的NHEJ介导的修复可以在DSB位点产生可变长度的插入和/或缺失突变。然而，HR介导的修复可以从单链或双链DNA供体模板引入精确的点突变或插入。 使用几种在线生物信息学资源设计的五种20 bp长的G q sgRNA被克隆到CRISPR/cas9质粒U6启动子下游。为了测试这些gRNA的切割效率，我们使用体外测定。我们通过将来源于化脓性链球菌的重组Cas9蛋白与体外转录的与G q的基因组DNA序列互补的G q sgRNA混合来制备体外切割测定。我们发现5个G q sgRNA中有4个在切割G q基因组DNA方面是有效的。为了测试G q sgRNA介导的DSB的特异性，我们在PAM序列中引入突变。这些突变阻止所有G q sgRNA切割DNA。该结果表明，G q sgRNA诱导的DNA切割对于每种sgRNA都是非常特异的。 我们接下来在随后的实验中使用这些4个G q sgRNA来测试它们在细胞中诱导基因组DNA编辑的能力，从而导致G q基因突变。用表达G q sgRNA和Cas9蛋白的构建体连同具有所需突变的DNA供体模板一起转染细胞，所述构建体沿着。转染后48小时，将从模拟和G q sgRNA转染的细胞分离的基因组DNA样品用于PCR扩增。对PCR产物进行限制性片段长度多态性（RFLP）、CRISPR/Cas衍生的RNA引导的工程化核酸酶（RGEN）和surveyor测定以检测Cas9诱导的突变和每种sgRNA的切割效率。使用所有这些测定，我们发现四个中只有一个G q sgRNA有效地诱导细胞G q基因组DNA中的修饰。G q sgRNA效率的这种差异不是由于sgRNA或Cas9蛋白表达水平的差异。目前，我们正在通过细胞分选或嘌呤霉素选择来分离克隆。 此外，我们使用了靶向整合系统，该系统使用C31整合酶，该整合酶可以将任何大小的供体质粒整合到具有高转录活性的基因间区域中，作为单拷贝，并且不需要辅因子。具有所需突变的整合的转基因是稳定表达和可遗传的。这种水平的靶向控制允许研究不受背景和位置变化影响的表型效应，这导致更准确的基因型与表型相关性。