Regulation of ADP-ribosylation factor

adp -核糖基化因子（Arfs）是Ras超家族的成员，它协调膜和肌动蛋白重塑，这是许多细胞功能的组成部分，包括细胞运动、胞吞和胞吐以及有丝分裂，并且是肿瘤细胞侵袭和转移等病理过程的核心。在我们对Arfs调控的研究中，我们发现了Arf gtpase激活蛋白（GAPs），它可以促进与Arf结合的GTP水解，将Arf-GTP转化为Arf- gdp。我们发现的第一个GAP是ASAP1，它由BAR、PH、Arf GAP、Ankyrin重复、脯氨酸丰富、E/DLPPKP重复和SH3结构域组成。它调节肌动蛋白细胞骨架的重塑和相关的局灶粘连。与这些生化活动一致，它参与调节分化，也参与调节癌细胞的行为，包括侵袭和转移。此外，ASAP1在许多癌症中过表达，包括儿童横纹肌肉瘤，并且在许多癌症中过表达与预后不良相关，这促使我们最近关注ASAP1。我们研究了ASAP1生物化学和生物学的三个方面，在过去的一年里，这三个方面都取得了进展。首先，我们正在努力确定Arf GAP结构域调控催化的机制。在过去的一年里，我们发现PH结构域是催化口袋的一个组成部分，是Arf GAP结构域功能所必需的，并且与Dr. R. Andrew Byrd合作，发现PH结构域直接与底物Arf- gtp的n端延伸结合。我们目前正在扩展工作，以原子分辨率定义机制。在第二个研究领域，我们正在研究ASAP1结合的癌蛋白与它介导的肌动蛋白重塑之间的联系。在过去的一年中，我们发现ASAP1的BAR和PH结构域与f -肌动蛋白直接结合，从而驱动f -肌动蛋白的捆绑。在正在进行的研究中，我们正在确定asap1驱动的肌动蛋白捆绑对应力纤维、内凹和圆形背褶中肌动蛋白重塑的贡献。在与儿科肿瘤科的Marielle Yohe博士合作的第三个领域，我们正在研究ASAP1对融合阴性横纹肌肉瘤（FN-RMS）行为的贡献，这是ASAP1在癌细胞中功能的理想模型。首先，在其他癌症中，ASAP1是过表达的。其次，ASAP1既影响非转化细胞的分化，也影响癌细胞的增殖，而FN-RMS在成肌细胞分化方面存在缺陷。在过去的一年中，我们发现ASAP1调节成肌细胞和RMS细胞系的分化途径，并且对成肌细胞和FN-RMS的作用存在差异。在正在进行的研究中，我们正在研究这些差异如何影响分化、增殖、侵袭和转移。

ADP-ribosylation factors (Arfs) are members of the Ras superfamily that coordinated membrane and actin remodeling, which integral to a number of cellular functions, including cell movement, endocytosis and exocytosis, and mitosis and are central to pathological processes such as tumor cell invasion and metastasis. In our studies of the regulation of Arfs, we discovered the Arf GTPase-activating proteins (GAPs), which facilitate the hydrolysis of GTP bound to Arf, converting Arf-GTP to Arf-GDP. The first GAP we discovered, ASAP1, is composed of a BAR, PH, Arf GAP, Ankyrin repeat, proline rich, E/DLPPKP repeat and SH3 domains. It regulates remodeling of the actin cytoskeleton and associated focal adhesions. Consistent with these biochemical activities, it has been implicated in regulating differentiation and has also been implicated as a regulator of cancer cell behaviors, including invasion and metastasis. Furthermore, ASAP1 is overexpressed in a number of cancers, including childhood rhabdomyosarcomas and overexpression correlates with poor prognosis in a number of cancers, which has motivated our recent focus on ASAP1. We study three aspects of ASAP1 biochemistry and biology, with progress in all three areas in the past year. First, we are working towards determining the mechanism of regulated catalysis by the Arf GAP domain. In the past year, we have discovered that the PH domain is an integral part of the catalytic pocket, necessary for function of the Arf GAP domain and, in collaboration with Dr. R. Andrew Byrd, have discovered that the PH domain binds directly to an N-terminal extension of the substrate Arf-GTP. We are currently extending the work to define mechanism at atomic resolution. In the second area of study, we are examining the link between oncoproteins to which ASAP1 binds and the actin remodeling that it mediates. In the past year, we have discovered direct binding of the BAR and PH domain of ASAP1 to F-actin, which drives bundling of the F-actin. In ongoing studies, we are determining the contribution to ASAP1-driven actin bundling to remodeling of actin in stress fibers, invadopodia and circular dorsal ruffles. In a third area of work in collaboration with Dr. Marielle Yohe of Pediatric Oncology Branch, we are examining the contribution of ASAP1 to the behavior of fusion-negative rhabdomyosarcoma (FN-RMS), an ideal model for the function of ASAP1 in cancer cells. First, as for other cancer, ASAP1 is overexpressed. Second, while ASAP1 has been found to affect both differentiation of nontransformed cells and proliferation of cancer cells, FN-RMS has a defect in differentiation of myoblasts. In the past year, we have discovered that ASAP1 regulates differentiation pathways in both myoblasts and RMS cell lines and that there are differences in effects on myoblasts and FN-RMS. In ongoing studies, we are examining how these differences affect differentiation, proliferation, invasion and metastasis.