New Function for the Serpin PAI-2 as a Regulator of pRb

描述(申请人提供)：信号转导分子的蛋白水解性切割是控制细胞生长、死亡和分化的重要机制，影响广泛的生理和病理过程。细胞内的蛋白分解必须受到内源性抑制物的严格调控。纤溶酶原激活物抑制物2(PAL-2)在结构和功能上是丝氨酸蛋白酶抑制物或蛇毒的一个大家族的成员。Serpins是补体激活、纤溶、凝血、细胞分化、肿瘤抑制、细胞凋亡和细胞运动等重要生物学过程的关键调节因子。PAL-2最初被认为是胞外尿激酶型纤溶酶原激活物的抑制物，然而PAL-2是一种低效分泌的丝氨酸，呈现核质分布。我们先前已经发现，PAL-2的表达在体外可以抵抗细胞凋亡，保护细胞免受某些细胞病变病毒的侵袭。我们的初步数据表明，PAL-2具有细胞内活性，是视网膜母细胞瘤肿瘤抑制因子(PRB)结合蛋白，保护PRB免受蛋白降解。PRb家族蛋白是普遍存在的转录调节因子，在控制细胞增殖中起着关键作用。这一应用的中心假设是，细胞内PAL-2稳定pRb和p130，从而促进pRb介导的与细胞周期停滞和促进分化相关的活动，降低对E2F1依赖的凋亡、转录调节和肿瘤抑制的敏感性。需要检验的具体假设是：1)PAL-2结合pRb和相关的Pocket蛋白，p130；2)PAL-2抑制pRb的蛋白水解性切割，从而提高pRb水平和pRb介导的活性；3)PAL-2通过pRb介导的稳定促进角质形成细胞和内皮细胞的存活。这些假说将通过以下方式验证：1)利用特定功能被破坏的突变蛋白表征PAL-2和pRb之间的特定分子相互作用；2)确定PAL-2稳定pRb的分子机制并评估Calain样蛋白酶在介导pRb蛋白水解性切割中的作用；以及3)使用PAL-2-/-小鼠模型测试PAL-2在稳定pRb中的体内功能。分析将专门评估PAL-2在角质形成细胞分化、内皮细胞增殖和血管生成中的作用。

DESCRIPTION (provided by applicant): Proteolytic cleavage of signal transduction molecules is an important mechanism for controlling cell growth, death and differentiation affecting a wide range of physiological and pathological processes. Intracellular proteolysis must be tightly regulated by endogenous inhibitors. Plasminogen activator inhibitor type 2 (PAl-2) is structurally and functionally a member of a large family of serine protease inhibitors or serpins. Serpins are key regulators of important biological processes such as complement activation, fibrinolysis, coagulation, cellular differentiation, tumor suppression, apoptosis and cell motility. PAl-2 was originally characterised as an inhibitor of the extracellular urokinase-type plasminogen activator, however PAl-2 is an inefficiently secreted serpin that exhibits a nucleocytoplasmic distribution. We have previously found that PAl-2 expression confers resistance to apoptosis and protects cells from certain cytopathic viruses in vitro. Our preliminary data identifies an intracellular activity for PAl-2 as a retinoblastoma tumor suppressor (pRb) binding protein that protects pRb from proteolytic degradation. The pRb family of proteins is ubiquitous regulators of transcription and plays a critical role in controlling cell proliferation. The central hypothesis of this application is that intracellular PAl-2 stabilizes pRb and p130, and in doing so, promotes pRb mediated activities associated with cell cycle arrest and promotion of differentiation, decreased sensitivity to E2F1 dependent apoptosis, transcriptional regulation and tumor suppression. The specific hypotheses to be tested are: 1) that PAl-2 binds pRb and the related pocket protein, p130, 2) that PAl-2 inhibits proteolytic cleavage of pRb, thereby enhancing pRb levels and pRb mediated activities, and 3) that PAl-2 promotes survival of keratinocytes and endothelial cells via pRb mediated stabilization. These hypotheses will be tested by 1) characterising the specific molecular interactions between PAl-2 and pRb utilizing mutant proteins in which specific functions have been disrupted, 2) determining the molecular mechanism by which PAl-2 stabilizes pRb and evaluating the role of calpain-like proteases in mediating proteolytic cleavage of pRb, and 3) testing the in vivo function of PAl-2 in stabilizing pRb using a PAl-2-/- mouse model. Analyses will specifically evaluate the roles of PAl-2 in keratinocyte differentiation, and endothelial cell proliferation and angiogenesis.