Pal Anupama, Rehemtulla Alnawaz
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA.
Division of Molecular Imaging, Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI, USA.
Methods Mol Biol. 2018;1731:247-260. doi: 10.1007/978-1-4939-7595-2_22.
Proteases are "protein-cleaving" enzymes, which, in addition to their non-specific degrading function, also catalyze the highly specific and regulated process of proteolytic processing, thus regulating multiple biological functions. Alterations in proteolytic activity occur during pathological conditions such as cancer. One of the major deregulated classes of proteases in cancer is caspases, the proteolytic initiators and mediators of the apoptotic machinery. The ability to image apoptosis noninvasively in living cells and animal models of cancer can not only provide new insight into the biological basis of the disease but can also be used as a quantitative tool to screen and evaluate novel therapeutic strategies. Optical molecular imaging such as bioluminescence-based genetically engineered biosensors has been developed in our laboratory and exploited to study protease activity in animal models with a high signal to noise. Using the circularly permuted form of firefly luciferase, we have developed a reporter for Caspase 3/7, referred to as Caspase 3/7 GloSensor. Here, we discuss the use of the Caspase 3/7 GloSensor for imaging apoptotic activity in mouse xenografts and genetically engineered mouse models of cancer and present the potential of this powerful platform technology to image the proteolytic activity of numerous other proteases.
蛋白酶是“蛋白裂解”酶,除了具有非特异性降解功能外,还催化蛋白水解加工这一高度特异性且受调控的过程,从而调节多种生物学功能。在诸如癌症等病理状况下会发生蛋白水解活性的改变。癌症中主要失调的蛋白酶类别之一是半胱天冬酶,它是凋亡机制的蛋白水解启动子和介质。在活细胞和癌症动物模型中对凋亡进行无创成像的能力不仅可以为该疾病的生物学基础提供新的见解,还可以用作筛选和评估新型治疗策略的定量工具。我们实验室已经开发了诸如基于生物发光的基因工程生物传感器等光学分子成像技术,并用于在信噪比高的动物模型中研究蛋白酶活性。利用萤火虫荧光素酶的环形排列形式,我们开发了一种半胱天冬酶3/7报告基因,称为半胱天冬酶3/7 GloSensor。在此,我们讨论了半胱天冬酶3/7 GloSensor在小鼠异种移植瘤和癌症基因工程小鼠模型中对凋亡活性进行成像的应用,并展示了这种强大平台技术对众多其他蛋白酶的蛋白水解活性进行成像的潜力。