Proteolytic activation of CREB3L1 in treating cancers and tissue fibrosis

 描述(申请人提供)：目前的项目继续专注于一种名为CREB3L1的转录因子，它是作为膜结合前体合成的，并通过一种称为调节膜内蛋白分解(RIP)的过程激活。该蛋白质含有一个跨膜螺旋，N-末端结构域面向胞浆。在上一个授予周期中，我们已经确定了转化生长因子-β诱导了CREB3L1的裂解，允许蛋白质的N-末端结构域进入细胞核，在那里它激活了刺激含有胶原的细胞外基质组装的基因的转录。由于转化生长因子诱导的含有胶原的基质过度沉积导致组织纤维化，抑制CREB3L1的蛋白分解激活可能有助于治疗纤维化疾病。这一假设将在该提案的目标1中得到验证，在该提案中，我们将通过在脂肪细胞中选择性地去除CREB3L1的小鼠来确定CREB3L1在肥胖诱导的脂肪组织纤维化中的作用。实现这一目标可能决定CREB3L1的蛋白水解性激活是否可以作为一种新的药物靶点，通过抑制脂肪组织的纤维化来治疗脂肪毒性。除了转化生长因子，我们已经确定阿霉素也可以刺激CREB3L1的裂解，允许蛋白质的N-末端结构域激活抑制细胞增殖的基因。我们证明阿霉素通过激活CREB3L1的RIP来抑制癌细胞的增殖。这一观察使我们提出了目标2，在该目标中，我们将确定CREB3L1的表达是否可以作为以阿霉素为基础的化疗的生物标志物。实现这一目标将通过识别可能从药物治疗中受益的患者来显著提高阿霉素的应答率。我们进一步确定阿霉素通过诱导神经酰胺的合成来激活CREB3L1的裂解。神经酰胺激活CREB3L1裂解的关键步骤是通过阻断跨膜蛋白TM4SF20的信号肽插入膜来逆转其膜取向。我们将这种新的调控机制命名为“选择性易位”。该项目的目的3是描述跨膜蛋白和分泌蛋白通过交替易位进行调节的机制。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特定的生理条件下发挥细胞内的功能。这项研究应该会深刻地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.