Role of Phospholipid Scramblase in Interferon Action

干扰素是一种多效性细胞因子，具有多种免疫调节、抗肿瘤和抗病毒活性。虽然已经确定了几个具有不同抗增殖和抗病毒活性的干扰素调节基因，但干扰素这些重要生物效应背后的许多细胞机制仍不清楚。最近，我们发现磷脂扰乱酶-1(PLSCR1)是一种显著上调干扰素表达的基因，它是一种质膜蛋白，与钙离子依赖的质膜磷脂重组有关。我们的初步数据还表明，PLSCR1本身具有抗肿瘤和抗病毒活性，与正常情况下与干扰素反应相关的效果相似。在这项建议中，我们旨在阐明受干扰素调控的PLSCR基因家族成员在对干扰素的整体生物反应中的特定作用。我们的具体目标包括：(1)确定PLSCR基因家族中可能受干扰素调控的其他成员，以及确定由PLSCR1或同源蛋白直接介导的干扰素诱导的细胞变化。将评估细胞质膜磷脂在干扰素作用下的拓扑变化，并与新合成的PLSCR1多肽的脂肪酰化和细胞内分布的潜在变化有关。将探讨PLSCR1与病毒包膜蛋白在抗洗涤剂膜“筏”中共定位的可能性，并将确定PLSCR1共有的保守PPxY基序以及许多被包裹的病毒基质和GAG蛋白的功能意义；(2)阐明PLSCR1在干扰素抗肿瘤活性中的特异性贡献。将测定PLSCR1基因缺失/p53基因缺失小鼠的肿瘤发生率，并比较干扰素-α对PLSCR1基因缺失小鼠和野生型肿瘤生长的影响。将检测PLSCR1对细胞增殖和巨噬细胞激活/募集的潜在影响；以及(3)确定PLSCR1对干扰素抗病毒活性的贡献。水泡性口炎病毒(VSV)在野生型和PLSCR1缺失细胞中的复制将在有和没有干扰素的情况下进行比较。将确定细胞PLSCR1对病毒生命周期每个阶段的具体影响。PLSCR1在体内的潜在抗病毒活性将在野生型和PLSCR1缺失的小鼠中进行探索。我们认为，对PLSCR基因家族在干扰素生物学中的作用的详细了解可能会基于PLSCR蛋白的选择性诱导、激活或模仿而提出新的抗病毒和抗肿瘤策略。

Interferons (IFN) are pleiotropic cytokines with diverse immune regulatory, anti-tumor, and anti-viral activities. Whereas several IFN-regulated genes with distinct anti- proliferative and anti-viral activities have been identified, many of the cellular mechanisms underlying these biologically- important effects of IFN remain unknown. Recently, we identified phospholipid scramblase-1 (PLSCR1) a plasma membrane protein implicated in Ca2+-dependent reorganization of plasma membrane phospholipids as a markedly IFN-upregulated gene. Our preliminary data also suggest that PLSCR1 itself exhibits anti- tumor and anti-viral activities that mimic effects normally associated with the response to IFN. In this proposal we aim to elucidate the specific role of IFN-regulated members of the PLSCR gene family in the overall biologic response to IFN. Our Specific Aims include: (1) to identify other members of the PLSCR gene family that may be regulated by IFN, and to identify cellular changes induced by IFN that are directly mediated by PLSCR1 or homologous proteins. Changes in topology of plasma membrane phospholipids in response to IFN will be evaluated and related to potential changes in the fatty acylation and intracellular distribution of newly synthesized PLSCR1 polypeptide. The possibility that PLSCR1 co-localizes with viral envelope proteins in detergent-resistant membrane "rafts" will be explored, and the functional significance of conserved PPxY motifs common to PLSCR1 and many enveloped virus matrix and gag proteins will be determined; (2) to elucidate the specific contribution of PLSCR1 to IFN's anti-tumor activity. Tumor incidence in PLSCR1-null/p53-null mice will be determined and effects of IFN-alpha on the growth of PLSCR1-null and wild type tumors will be compared. Potential effects of PLSCR1 on cell proliferation and macrophage activation/recruitment will be examined; and (3) to determine the contribution of PLSCR1 to IFN's anti-viral activity. Vesicular stomatitis virus (VSV) replication in wild type and PLSCR1-null cells will be compared, both in presence and absence of IFN. Specific effects of cellular PLSCR1 on each stage of the viral life cycle will be identified. Potential anti-viral activity of PLSCR1 in vivo will be explored in wild type and PLSCR1-null mice. It is proposed that detailed understanding of the role of the PLSCR gene family in IFN biology may suggest novel anti-viral and anti-tumor strategies based on selective induction, activation, or mimicry of the PLSCR proteins.