Protein kinase D-mediated balance between lipid homeostasis and secretory activity in mammalian cells - A systemic appoach

在哺乳动物细胞中，不同细胞器之间的脂类代谢、分选和运输受到严格的调控，因为脂类的动态平衡对维持细胞膜的完整性至关重要。在高尔基复合体，脂质成分决定了运输小泡的形成，运输小泡是将分泌蛋白从反高尔基网络(TGN)转移到细胞膜上所必需的。蛋白激酶D(PKD)已被证明在这一调节中发挥重要作用：活性PKD直接参与TGN转运小泡的分裂。此外，它还通过两种相反的方式调节神经酰胺转移蛋白(CERT)的活性，从而对神经酰胺从内质网(ER)到TGN的运输具有双重作用。PKD和CERT在一个相互关联的反馈电路的复杂结构中相互调节，使这个系统成为整体建模方法的一个有吸引力的目标。此外，最近实验技术的进步导致了对TGN分子相互作用的新见解，理论分泌模型中尚未考虑分泌调节。这个项目的目的是增加我们对PKD在平衡脂质平衡和分泌中的作用的理解。这将通过以化学反应动力学为基础的数据和知识驱动的建模方法来实现，同时将伴随着用于模型识别的扰动实验。我们将特别关注用于参数估计和模型选择的统计方法，可辨识性分析，以及研究耦合反馈的系统理论方法。我们项目的结果将与生物技术应用高度相关：我们可能揭示用于治疗性蛋白质生产的哺乳动物宿主细胞系的优化目标，其中TGN的调控过程最近被确定为分泌途径的瓶颈。

Lipid metabolism, sorting and transport between different organelles are tightly regulated in mammalian cells, since lipid homeostasis is critical for maintaining the integrities of cellular membranes. At the Golgi complex the lipid composition determines the formation of transport vesicles, which are required to shuffle secretory proteins from the trans-Golgi network (TGN) to the cell membrane. Protein kinase D (PKD) has been shown to play an important role in this regulation: Active PKD is directly involved in the fission of transport vesicles at the TGN. Furthermore, it has a dual function for the ceramide transport from the endoplasmic reticulum (ER) to the TGN by regulating the activity of the ceramide transfer protein (CERT) in two opposite ways. PKD and CERT mutually regulate each other in a complex structure of interrelated feedback circuits, making this system an attractive target for a holistic modeling approach. Moreover, recent advances in experimental techniques have led to new insights about molecular interactions at the TGN, and regulation of secretion has not yet been taken into account in theoretical secretion models. The goal of this project is to increase our understanding about the role of PKD in the balance between lipid homeostasis and secretion. This will be achieved by a data- and knowledge-driven modeling approach based on chemical reaction kinetics, which will be accompanied by perturbation experiments that are used for model identification. We will particularly focus on statistical methods for parameter estimation and model selection, identifiability analysis, and systems-theoretic methods to investigate coupled feedback. The results of our project will be highly relevant for biotechnological applications: We may reveal targets for optimization of mammalian host cell lines used for therapeutic protein production, where regulation processes at the TGN have recently been identified as a bottleneck in the secretory pathways.