Proteolytic activation of CREB3L1 in treating cancers and tissue fibrosis

 描述（由申请人提供）：目前的项目继续关注一种名为CREB 3L 1的转录因子，该转录因子作为膜结合前体合成，并通过一种称为调节性膜内蛋白水解（RIP）的过程激活。该蛋白含有一个单一的跨膜螺旋，N-末端结构域面向胞质溶胶。在最后一个授权周期中，我们已经确定TGF-β诱导CREB 3L 1的切割，允许蛋白质的N-末端结构域进入细胞核，在那里它激活基因的转录，刺激含胶原蛋白的细胞外基质的组装。由于TGF-β诱导的含胶原基质的过度沉积导致组织纤维化，抑制CREB 3L 1的蛋白水解活化可用于治疗纤维化疾病.该假设将在该提案的目标1中进行测试，其中我们将使用在脂肪细胞中选择性消融CREB 3L 1的小鼠来确定CREB 3L 1在肥胖诱导的脂肪组织纤维化中的作用。实现这一目标可以确定CREB 3L 1的蛋白水解激活是否可以成为通过抑制脂肪组织纤维化来治疗脂毒性的新药物靶点。 除了TGF-β，我们已经确定阿霉素也刺激CREB 3L 1的切割，允许蛋白质的N-末端结构域激活抑制细胞增殖的基因。我们证明了阿霉素通过激活CREB 3L 1的RIP来阻断癌细胞的增殖。这一观察结果使我们提出了目标2，其中我们将确定CREB 3L 1表达是否可以作为基于阿霉素的化疗的生物标志物。实现这一目标将显着提高阿霉素的反应率，允许识别患者谁可能受益于药物治疗。 我们已经进一步确定，阿霉素通过诱导神经酰胺的合成来激活CREB 3L 1裂解。神经酰胺激活CREB 3L 1切割的关键步骤是通过阻断其信号肽插入膜来逆转称为TM 4SF 20的跨膜蛋白的膜取向。我们将这种新的调控机制称为“交替易位”。该项目的目标3是阐明跨膜和分泌蛋白通过交替易位调节的机制。实现这一目标将展示膜蛋白如何采用不同的膜拓扑结构，以及分泌蛋白如何在某些生理条件下在细胞内发挥作用。这项研究将大大拓宽我们对这些蛋白质的看法。

 DESCRIPTION (provided by applicant): The current project continues to focus on a transcription factor called CREB3L1, which is synthesized as a membrane-bound precursor and activated through a process known as regulated intramembrane proteolysis (RIP). The protein contains a single transmembrane helix, with the N-terminal domain facing the cytosol. During the last grant cycle we have determined that TGF-ß induces cleavage of CREB3L1, allowing the N-terminal domain of the protein to enter nucleus where it activates transcription of genes stimulating assembly of collagen- containing extracellular matrix. Since TGF-ß-induced excess deposition of the collagen- containing matrix leads to tissue fibrosis, inhibiting proteolytic activation of CREB3L1 may be useful in treating fibrotic diseases. This hypothesis will be tested in Aim 1 of the proposal in which we will determine the roles of CREB3L1 in obesity-induced fibrosis of adipose tissue using mice in which CREB3L1 is selectively ablated in adipocytes. Achieving this aim may determine whether proteolytic activation of CREB3L1 could be a novel drug target to treat lipotoxicity by inhibiting fibrosis of adipose tissue. In addition to TGF-ß,we have determined that doxorubicin also stimulates cleavage of CREB3L1, allowing the N-terminal domain of the protein to activate genes that inhibit cell proliferation. We demonstrated that doxorubicin blocked proliferation of cancer cells through activating RIP of CREB3L1. This observation led us to propose Aim 2 in which we will determine whether CREB3L1 expression may serve as a biomarker for doxorubicin- based chemotherapy. Achieving this aim will markedly improve the response rate of doxorubicin by allowing identification of patients who are likely to benefit from the drug treatment. We have further determined that doxorubicin activates CREB3L1 cleavage by inducing synthesis of ceramide. A crucial step for ceramide to activate cleavage of CREB3L1 is to invert the membrane orientation of a transmembrane protein called TM4SF20 by blocking the insertion of its signal peptide into membranes. We designate this novel regulatory mechanism as "alternative translocation". Aim 3 of the project is proposed to delineate the mechanism through which transmembrane and secretory proteins are regulated by alternative translocation. Achieving this aim will demonstrate how membrane proteins can adopt different membrane topologies, and how secretory proteins can function intracellularly under certain physiological conditions. This study should profoundly broaden our views to these proteins.