The study of the interfacial catalysis and therapeutic potential of PTEN- L

项目摘要 PTEN是PI3K途径的关键成员，通过直接拮抗PI3K途径发挥其主要的肿瘤抑制作用 PI3K作为一种界面脂质磷酸酶，通过使膜上的PIP3去磷酸化，导致 降低AKT活性。PTEN在许多类型的癌症中都没有表达，包括乳腺癌、前列腺癌和 胶质母细胞瘤，导致AKT活性增强，有利于细胞生长。帕森斯实验室最近的工作领导了 发现了PTEN-L，一种分泌的PTEN翻译异构体，可以重新进入细胞并去磷酸化 受体细胞中的PIP3。帕森斯研究小组和其他研究人员之前的研究表明，外源 PTEN-L可在PTEN缺失的异种移植模型中进入肿瘤细胞，并导致肿瘤的消退和衰减 PI3K信号。这些工作突出了PTEN-L作为患者靶向治疗的巨大潜力 伴有PTEN缺失的肿瘤。尽管PTEN-L具有PTEN的所有结构域，包括磷酸酶 结构域、C2结构域和C-尾，PTEN-L在N-端还有173个氨基酸残基， 其功能仍在调查中。PTEN和PTEN-L均存在于细胞质和胞浆中。 膜，但必须被招募到膜，以脱磷PIP3。出版的作品表明 PTEN-L的N端延伸，即膜结合螺旋(MBH)结构域，导致细胞数量增加 部分降低了磷酸酶活性的膜的亲和力。这个项目的目标是研究 PTEN-L的界面催化作用以确定其磷酸酶需要哪些PTEN-L结构域 纯化的PTEN-L对膜PIP3的活性及其对天然蛋白改变的成功与否 PI3K信号异常的肿瘤治疗领域。在第一个目标中，我们将确定PTEN的结构域- L对膜的定位及其随后的脂磷酸酶活性具有重要作用；这两种功能都是 对于膜上的PIP3去磷酸化至关重要。为了测试域的需求，我们将变异 PTEN-L的膜定位结构域我们将在PTEN缺失细胞中测试这些突变体对 PI3K下游信号转导和定位到内生膜的体外和生化分析。 在第二个目标中，我们将确定外源PTEN-L结构域突变蛋白是否能够在 用异常的PI3K信号治疗癌症以及这些PTEN-L结构域的改变是否会改善 治疗效果。我们将首先测试结构域突变体在PTEN缺失细胞中抑制细胞生长的能力 在试管中。我们将通过以下方法确定纯化的PTEN-L结构域突变体保持生长抑制的有效性 测量下游PI3K信号和经处理的PTEN空细胞系的生长速度，并通过使用 PTEN缺失型肿瘤的异种移植和同种移植模型。接下来，我们将确定是否有任何检测到的外源 突变蛋白PTEN-L在小鼠体内具有良好的药代动力学、药效学和毒性特征。 这项拟议研究的发现有可能改进对PI3K异常肿瘤的治疗 信号转导，也对促进治疗性蛋白质研究领域的发展具有重要意义。

Project Summary PTEN, a key member of the PI3K pathway, exerts its main effect as a tumor suppressor by directly antagonizing the activity of PI3K as an interfacial lipid phosphatase, via dephosphorylating membranous PIP3, resulting in lower AKT activation. Expression of PTEN is lost in many cancer types, including breast, prostate and glioblastoma, resulting in heightened AKT activity, which favors cell growth. Recent work in the Parsons lab led to the discovery of PTEN-L, a secreted PTEN translational isoform, which can re-enter cells and dephosphorylate PIP3 in the recipient cells. Previous studies from the Parsons group and others have indicated that exogenous PTEN-L can enter tumor cells in PTEN null xenograft models and cause tumor regression and attenuation of PI3K signaling. These works highlight the great potential for PTEN-L to be used as a targeted therapy for patients with tumors bearing loss of PTEN. Although PTEN-L shares all domains of PTEN, including the phosphatase domain, C2 domains, and the C-tail, PTEN-L also has an additional 173 amino acids at the N-terminus, the function of which is still under investigation. Both PTEN and PTEN-L can be found in the cytoplasm and at membranes but must be recruited to membranes in order to dephosphorylate PIP3. Published work indicates that the N-terminal extension of PTEN-L, namely the membrane binding helix (MBH) domain, causes increased affinity for the membrane with partially diminished phosphatase activity. The goal of this project is to study the interfacial catalysis of PTEN-L in order to determine which PTEN-L domains are required for its phosphatase activity against membranous PIP3 and determine the success of purified PTEN-L with alterations to the native domains in treating tumors with aberrant PI3K signaling. In the first aim, we will determine the domains of PTEN- L that are important for membrane localization and its subsequent lipid phosphatase activity; both functions are vital for dephosphorylation of membranous PIP3. To test the requirement of the domains, we will mutate the membrane localization domains of PTEN-L. We will test the effects of these mutants in PTEN null cells on downstream PI3K signaling and localization to endogenous membranes in vitro and using biochemical assays. In the second aim, we will determine whether exogenous PTEN-L domain mutant proteins can be effective in treating cancer with aberrant PI3K signaling and whether these alterations to PTEN-L domains will improve therapeutic efficacy. We will first test the ability of the domain mutants to suppress cell growth in PTEN null cells in vitro. We will determine efficacy of purified PTEN-L domain mutants that retain growth suppression by measuring downstream PI3K signaling and growth rates of treated PTEN null cell lines, and through the use of xenograft and allograft models of PTEN null cancers. Next, we will determine whether any tested exogenous PTEN-L mutant proteins have favorable pharmacokinetics, pharmacodynamics, and toxicity profiles in mice. Findings from this proposed study have the potential to improve treatments for tumors with PI3K aberrant signaling, and also be valuable to advancement of the therapeutic proteins field of research.