Elucidating ‘design’ principles for engineering synthetic protein networks

阐明工程合成蛋白质网络的“设计”原则

• 批准号: RGPIN-2014-05322
• 负责人: Truong, Kevin
• 金额: $ 1.82万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588342
• 关键词: Elucidating; design; principles; engineering; synthetic

Through natural selection, cells have evolved protein networks to perform essential functions such as cell growth, differentiation and death. Evolution has converged on general network 'designs' capable of functioning precisely in the context of variability in intracellular and extracellular conditions (e.g. availability of metabolites and the concentrations and specificity of proteins). The uncovering of these ‘design’ principles will allow first, a deeper understanding of natural biological systems and second, insight into engineering synthetic protein networks for programming cells to perform a wide variety of tasks (e.g. biosensor and bioremediation applications). While synthetic biologists have uncovered many ‘design’ principles in transcriptional networks, protein networks differ by operating at faster time scales (e.g. ms to s) and wider spatial distribution (e.g. membrane morphologies), requiring unique ‘design’ principles. My long term research objective is to elucidate general ‘design’ principles for engineering protein networks through combined computational and experimental approaches. The first part of the proposal focuses on the development of computational tools needed for the molecular design of protein switches and the simulation of their interactions with natural protein networks. For molecular design, we will further develop our computational tool for generating protein conformational spaces to include protein backbone flexibility and thematic functions. The tool rapidly samples the conformational space of protein switches in the relevant time scales (e.g. ms to s) by deferring expensive energy calculations. For formulation of ‘design’ principles, we will further develop our computational tool for simulating protein networks to include millions of molecules in spatially distinct regions (e.g. membrane morphologies). The tool will simulate large scale biomolecular interactions within the constraints of complex spatially distinct regions that will be defined using a polygon-based model. In the second part, we will use these tools to first model and then experimentally validate 'design' principles related to accurate signal propagation in protein networks. Protein networks accurately propagate their signals despite the fact that many proteins have low specificity to their substrates. The caspase protein network for cell death is a good model system because caspases are notoriously non-specific to their target substrates, but yet distinct paths to cell death are achieved. Furthermore, caspases are curiously found in many spatially distinct regions. To explain the caspase mystery, we will formulate and test a ‘design’ principle for accurate signal propagation that uses non-specific local activity on certain local substrates to drive specific global activity. Spatiotemporally localized caspase signals will be generated experimentally by light-activated caspases invented by our group. Our discoveries of 'design' principles for protein networks will allow their application in engineering synthetic networks for reliably programming cells to perform a diverse set of tasks. Imagine programming a cell that detects, encapsulates and consumes environmental toxins. Once completed, the cell dies leaving no genetic trace. This proposal will develop the computational and experimental tools for modeling and testing 'design' principles, respectively, that will help realize this dream.

通过自然选择，细胞进化出了蛋白质网络来执行细胞生长、分化和死亡等基本功能。进化已经集中在通用网络“设计”上，该网络“设计”能够在细胞内和细胞外条件（例如代谢物的可用性以及蛋白质的浓度和特异性）的可变性背景下精确地发挥作用。揭示这些“设计”原理首先将有助于更深入地了解自然生物系统，其次将有助于深入了解工程合成蛋白质网络，以便对细胞进行编程以执行各种任务（例如生物传感器和生物修复应用）。虽然合成生物学家已经发现了转录网络中的许多“设计”原则，但蛋白质网络的不同之处在于以更快的时间尺度（例如毫秒到秒）和更广泛的空间分布（例如膜形态）运行，需要独特的“设计”原则。 我的长期研究目标是通过结合计算和实验方法阐明工程蛋白质网络的一般“设计”原则。 该提案的第一部分重点是开发蛋白质开关的分子设计及其与天然蛋白质网络相互作用的模拟所需的计算工具。对于分子设计，我们将进一步开发用于生成蛋白质构象空间的计算工具，以包括蛋白质主链灵活性和主题功能。该工具通过推迟昂贵的能量计算，在相关时间尺度（例如毫秒到秒）中快速采样蛋白质开关的构象空间。为了制定“设计”原则，我们将进一步开发用于模拟蛋白质网络的计算工具，以包含空间不同区域（例如膜形态）中的数百万个分子。 该工具将在复杂的空间不同区域的约束内模拟大规模生物分子相互作用，这些区域将使用基于多边形的模型定义。 在第二部分中，我们将使用这些工具首先进行建模，然后通过实验验证与蛋白质网络中准确信号传播相关的“设计”原理。尽管许多蛋白质对其底物的特异性较低，但蛋白质网络仍能准确地传播其信号。用于细胞死亡的半胱天冬酶蛋白网络是一个很好的模型系统，因为众所周知，半胱天冬酶对其靶底物不具有特异性，但仍实现了不同的细胞死亡途径。此外，在许多空间上不同的区域中奇怪地发现了半胱天冬酶。为了解释半胱天冬酶之谜，我们将制定并测试精确信号传播的“设计”原则，该原则使用某些局部底物上的非特异性局部活性来驱动特定的全局活性。我们小组发明的光激活半胱天冬酶将通过实验产生时空局部半胱天冬酶信号。 我们对蛋白质网络“设计”原理的发现将使其能够应用于工程合成网络，从而可靠地对细胞进行编程以执行各种任务。想象一下，对一个细胞进行编程，使其能够检测、封装和消耗环境毒素。一旦完成，细胞就会死亡，不留下任何遗传痕迹。该提案将开发分别用于建模和测试“设计”原理的计算和实验工具，这将有助于实现这一梦想。