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

项目摘要 PTEN是PI 3 K信号通路的关键成员，其主要作用是通过直接拮抗PI 3 K信号通路中的信号通路， PI 3 K作为界面脂质磷酸酶的活性，通过使膜PIP 3去磷酸化，导致 降低AKT激活。PTEN的表达在许多癌症类型中丢失，包括乳腺癌、前列腺癌和乳腺癌。 胶质母细胞瘤，导致AKT活性升高，这有利于细胞生长。帕森斯实验室最近的工作 PTEN-L的发现，一种分泌型PTEN翻译亚型，可以重新进入细胞和去磷酸化 受体细胞中的PIP 3。帕森斯小组和其他人以前的研究表明，外源性 PTEN-L可以进入PTEN无效异种移植物模型中的肿瘤细胞，并引起肿瘤消退和肿瘤细胞增殖的减弱。 PI 3 K信号。这些工作凸显了PTEN-L用作患者靶向治疗的巨大潜力 肿瘤中PTEN缺失。尽管PTEN-L共享PTEN的所有结构域，包括磷酸酶， 除了PTEN-L的N-末端具有C2结构域、C2结构域和C-尾之外，PTEN-L还在N-末端具有另外的173个氨基酸， 其功能仍在调查中。PTEN和PTEN-L均可见于细胞质中，且位于 PIP 3可以被膜吸收，但必须被募集到膜上以使PIP 3去磷酸化。发表的作品表明 PTEN-L的N-末端延伸，即膜结合螺旋（MBH）结构域，导致PTEN-L的N-末端延伸增加， 对膜的亲和性与部分减弱的磷酸酶活性。该项目的目标是研究 PTEN-L的界面催化，以确定其磷酸酶需要哪些PTEN-L结构域 针对膜PIP 3的活性，并确定纯化的PTEN-L与天然PIP 3的改变的成功。 结构域在治疗具有异常PI 3 K信号传导的肿瘤中的作用。在第一个目标中，我们将确定PTEN的结构域- L，对于膜定位及其随后的脂质磷酸酶活性很重要;这两种功能都是 对膜PIP 3的去磷酸化至关重要。为了测试域的需求，我们将改变 PTEN-L的膜定位结构域。我们将测试这些突变体在PTEN缺失细胞中的作用， 下游PI 3 K信号传导和定位到内源性膜的体外和使用生物化学测定。 在第二个目标中，我们将确定外源性PTEN-L结构域突变蛋白是否可以有效地在细胞中表达。 用异常PI 3 K信号传导治疗癌症以及这些对PTEN-L结构域的改变是否会改善 疗效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 体外我们将通过以下方法确定保留生长抑制的纯化的PTEN-L结构域突变体的功效： 测量经处理的PTEN缺失细胞系的下游PI 3 K信号传导和生长速率，并通过使用 PTEN缺失癌症的异种移植和同种异体移植模型。接下来，我们将确定是否有任何测试的外源性 PTEN-L突变蛋白在小鼠中具有良好的药代动力学、药效学和毒性特征。 这项研究的结果有可能改善PI 3 K异常肿瘤的治疗。 信号转导，也是有价值的治疗性蛋白质领域的研究进展。

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.