Caged aminoluciferin probe for bioluminescent immunoproteasome activity analysis†

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY RSC Chemical Biology Pub Date : 2024-07-16 DOI:10.1039/D4CB00148F
Cody A. Loy and Darci J. Trader
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Abstract

The immunoproteasome (iCP) can be expressed under inflammatory conditions, such as exposure to interferon-gamma (IFN-γ), that alerts the cell to begin generating iCP preferentially over the standard proteasome (sCP). With the iCP becoming a widely targeted isoform in a variety of diseases, there is a need to understand its activity and expression in cells and in vivo. Activity-based probes for the iCP have been developed but their application has been limited due to their difficult synthesis and cannot be used in tissues or whole animals. Our lab has previously demonstrated we can monitor iCP activity using a 4-mer peptide linked to a fluorophore and a peptoid. This was utilized in the development of the first cell-permeable iCP activity-based probe that did not include a covalent reactive moiety. Here, we demonstrate that this same peptide recognition sequence can be appended to aminoluciferin, caging it, until its interaction with the iCP. This probe should be applicable to monitor iCP activity in animal models where tumor or other tissue has been engineered to produce luciferase. We anticipate it could also be applied to observe iCP activity as tumors are formed in vivo.

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用于生物发光免疫蛋白酶体活性分析的笼式氨基荧光素探针
免疫蛋白酶体(iCP)可在炎症条件下表达,如暴露于干扰素-γ(IFN-γ),从而提醒细胞开始优先于标准蛋白酶体(sCP)生成 iCP。随着 iCP 成为多种疾病的广泛靶向异构体,有必要了解它在细胞和体内的活性和表达。基于活性的 iCP 探针已经开发出来,但由于其合成困难,不能用于组织或整个动物,其应用受到限制。我们的实验室之前已经证明,我们可以使用与荧光团和蛋白胨相连的 4-mer 肽来监测 iCP 活性。我们利用这种方法开发出了首个不含共价反应分子的基于 iCP 活性的细胞渗透性探针。在这里,我们证明了同样的肽识别序列可以附加到氨褐藻素上,将其笼住,直到与 iCP 发生作用。这种探针可用于监测动物模型中 iCP 的活性,在这种动物模型中,肿瘤或其他组织已被设计成能产生荧光素酶。我们预计它还可用于观察体内肿瘤形成时 iCP 的活性。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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