PROFILIN I--AN ESSENTIAL PROTEIN IN CELLS AND ANIMALS

肌动蛋白细胞骨架是细胞必需的关键超结构。 运动性和细胞与细胞外基质的粘附性。肌动蛋白网络 与其相关的蛋白质也影响信号通路，细胞 周期和细胞新陈代谢。因此，对 肌动蛋白及其结合蛋白之间的相互作用是细胞的基础 生物学。Profilin是一种普遍存在的15 kDa蛋白质，它有4个 体外建立的直接配体：(1)Profilin与单体肌动蛋白结合 (化学计量比；一对一)：(Ii)Profilin与多-L-脯氨酸(PLP，1)结合 Profilin结合的长度大于Proline)；(Iii)Profilin结合 含有多磷肌醇的膜(一个Profilin结合到一个簇上 5个磷脂分子)；(4)Profilin结合Vasp(血管扩张剂 刺激的磷蛋白，未知的化学计量)，可能通过其 与Vasp分子的一段富含脯氨酸的相互作用。尽管它 很明显，Profilin在体内调节肌动蛋白聚合物的形成， 来自培养细胞和各种生物体的数据被 观察到，根据系统的不同，Profilin可以促进 肌动蛋白组装，或抑制肌动蛋白聚合。尽管有这些结果， Profilin显然是多细胞生物体中生命所必需的，例如 果蝇和老鼠。因此，无论Profilin的作用是什么，细胞和 缺乏轮廓蛋白的生物体是不能存活的。特别是，小鼠对 Profilin I在缺乏Profilin的最早的生物体中无法存活 是不可行的。特别是，Profilin I为空的小鼠不会存活 超过最早的胚胎阶段(很少的细胞)。哪种功能 Profilin是至关重要的，因为它对生存的影响尚不清楚。至 刻画Profilin的基本功能(S)，我们建议结合 强大的基因技术与Profilin突变体的生化实验 由定点突变产生。我们已经使用了复制 无效腺病毒作为载体，其骨架包含人 同源重组克隆Profilin I基因，快速诱导 Profilin在主动脉内皮细胞中的过表达。我们观察到 Profilin过表达的作用是促进细胞的黏附 内皮细胞到细胞外基质蛋白，特别是到 纤维连接蛋白。我们对生物化学进行了工程化、提纯和研究 由单一氨基酸替换产生的Profilin突变体。我们有 体外鉴定出功能异常的突变体。特别是，突变型 88-R/L(88位氨基酸的精氨酸取代亮氨酸)不结合 肌动蛋白或增加肌动蛋白核苷酸交换减少了与 磷脂酰肌醇与PLP结合正常。突变体125-H/D已降低 与肌醇磷脂相互作用，但与肌动蛋白结合正常，正常 核苷酸交换功能，与PLP正常结合。突变体119-H/D 与磷脂酰肌醇结合正常，与PLP结合正常，但 减少与肌动蛋白的结合，缺乏增加核苷酸的能力 交换。其他突变体降低了PLP结合活性，但正常 与肌动蛋白和肌醇磷脂的相互作用，或增加对 肌醇磷脂与肌动蛋白和磷脂酶原蛋白结合正常。我们已经产生了 缺乏一个Profilin I等位基因的转基因小鼠，并表现出减少 这些动物细胞中的Profilin I浓度。对于这个项目， 我们建议使用Profilin的体外检测来鉴定突变体 显示配置文件功能的选择性缺陷。接下来，我们将 检测选定的Profilin突变体拯救转基因小鼠的能力 缺乏野生型Profilin I.结合生化研究 重组突变体体外、内皮细胞过表达实验 应用复制不能性腺病毒的细胞及其体内分析 转基因小鼠，我们将对体内的 Profilin在哺乳动物细胞和生物体中的功能。

The actin cytoskeleton is a key superstructure required for cellular motility and cell adhesion to the extracellular matrix. The actin network and its associated proteins also influences signaling pathways, the cell cycle and cellular metabolism. Hence, the characterization of the interaction between actin and its binding proteins is fundamental to cell biology. Profilin is an ubiquitous 15 kDa protein which has four established direct ligands in vitro: (1) profilin binds to monomeric actin (stoichiometry; one to one): (ii) profilin binds to poly-L-proline (PLP,one profilin binds to a stretch of greater than prolines); (iii) profilin binds membranes containing polyphosphoinositides (one profilin binds to a cluster of 5 phospholipid molecules); (iv) profilin binds VASP (vasodilator stimulated phosphoprotein, unknown stoichiometry), possibly through its interaction wit a proline-rich stretch of the VASP molecule. Although it is clear that profilin regulates the formation of actin polymers in vivo, data from cultured cells and various organisms are confounded by the observation that, depending on the system, profilin can either promote actin assembly, or inhibit actin polymerization. Despite these results, profilin is clearly essential for life in multicellular organisms such as Drosophila and mice. Thus, whatever the role of profilin may be, cells and organisms lacking profilin are not viable. In particular, mice null for profilin I do not survive beyond the earliest organisms lacking profilin are not viable. In particular, mice, null for profilin I do not survive beyond the earliest embryonic stages (few cells). Which function of profilin is critical for its effect on survival remains unknown. To characterize profilin's essential function(s), we propose to combine powerful genetic techniques with biochemical experiment on profilin mutants generated by site directed mutagenesis. We have used a replication incompetent adenovirus as vector, the backbone of which contains the human profilin I cDNA cloned by homologous recombination, to induce rapid overexpression of profilin in aortic endothelial cells. We observed that the effect of profilin overexpression is to promote the adhesion of endothelial cell to extracellular matrix proteins and in particular, to fibronectin. We have engineered purified and studied biochemically fifteen profilin mutants generated by single amino acid substitutions. We have identified mutants with abnormal function in vitro. In particular, mutant 88-R/L (arginine at amino acid 88 substituted for a leucine) does not bind actin nor increase actin nucleotide exchange has reduced interaction with phosphoinositides but normal binding to PLP. Mutant 125-H/D has reduced interaction with phosphoinositides but normal binding to actin, normal nucleotide exchange function, and normal binding to PLP. Mutant 119-H/D has normal binding to phosphoinositides and normal binding to PLP, but reduced binding to actin, and lacks the ability to increase nucleotide exchange. Other mutants have reduced PLP binding activity, but normal interaction with actin and phosphoinositides, or increased affinity for phosphoinositides but normal binding to actin and PLP. We have generated transgenic mice lacking one allele for profilin I, and shown reduced profilin I concentration in the cells of these animals. For this project, we propose to use in vitro assays of profilin to identify nutants displaying selective deficiencies of profilin functions. Next, we will examine the ability of selected profilin mutants to rescue transgenic mice lacking wild type profilin I. With a combination of biochemical studies on recombinant mutants in vitro, overexpression experiments in endothelial cells using replication incompetent adenoviruses and in vivo analysis in transgenic mice, we will gain much greater insight into the in vivo functions of profilin in mammalian cells and organisms.