PROFILIN I--AN ESSENTIAL PROTEIN IN CELLS AND ANIMALS

肌动蛋白细胞骨架是细胞生长所需的关键超结构， 运动性和细胞与细胞外基质的粘附。 肌动蛋白网络 及其相关蛋白质也会影响信号通路， 循环和细胞代谢。 因此， 肌动蛋白与其结合蛋白之间相互作用是细胞 生物学 Profilin是一种普遍存在的15 kDa蛋白，具有四个 体外建立的直接配体：（1）profilin与单体肌动蛋白结合 （化学计量;一比一）：（ii）profilin结合聚-L-脯氨酸（PLP，一比一） （iii）profilin结合大于脯氨酸的一段）; 含有多磷酸肌醇的膜（一个profilin结合到一个簇 （iv）profilin结合VASP（血管扩张剂 刺激磷蛋白，未知化学计量），可能通过其 与富含脯氨酸的VASP分子的相互作用。 虽然 很明显，profilin调节体内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，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， 在多细胞生物体中，profilin显然是生命所必需的， 果蝇和老鼠 因此，无论profilin的作用是什么，细胞和 缺乏profilin的生物体不能存活。 特别是，小鼠无效， profilin I不能在缺乏profilin的最早生物体中存活 是不可行的。 特别是，没有profilin I的小鼠无法存活 超过最早的胚胎阶段（细胞很少）。 的哪个功能 profilin是关键的，因为它对存活的影响仍然未知。 到 描述了轮廓蛋白的基本功能，我们建议将联合收割机 强大的遗传技术与生物化学实验的profilin突变体 通过定点诱变产生。 我们用了一个复制品 不合格的腺病毒作为载体，其骨架含有人 通过同源重组克隆profilin I cDNA，以诱导快速 主动脉内皮细胞中profilin的过度表达。 我们观察到 profilin过表达的作用是促进 内皮细胞与细胞外基质蛋白的结合，特别是， 纤连蛋白。 我们已经从生物化学的角度研究了15种 由单个氨基酸取代产生的profilin突变体。 我们有 在体外鉴定具有异常功能的突变体。 特别是突变体 88-R/L（氨基酸88处的精氨酸取代亮氨酸）不结合 肌动蛋白也不增加肌动蛋白核苷酸交换减少相互作用 磷酸肌醇，但与PLP的结合正常。 突变体125-H/D已减少 与磷酸肌醇相互作用，但与肌动蛋白正常结合，正常 核苷酸交换功能和与PLP的正常结合。 突变体119-H/D 与磷酸肌醇和PLP的结合正常，但 与肌动蛋白的结合减少，并且缺乏增加核苷酸 交易所 其他突变体具有降低的PLP结合活性，但正常 与肌动蛋白和磷酸肌醇的相互作用，或增加对 磷酸肌醇，但与肌动蛋白和PLP的结合正常。 我们已经生成 转基因小鼠缺乏一个等位基因的profilin I，并显示减少 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的体外测定来鉴定突变体， 表现出选择性的profilin功能缺陷。 接下来我们就 检测所选的profilin突变体拯救转基因小鼠的能力 缺乏野生型profilin I。 结合生物化学研究， 体外重组突变体，内皮细胞过度表达实验 使用无复制能力的腺病毒的细胞和在 转基因小鼠，我们将获得更大的洞察到体内 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.