The ligase activity of proteases: Do proteases make proteins?

众所周知，蛋白酶可以裂解多肽键，但在某些条件下，它们也可以催化反转反应，并能够形成多肽键。例如，已有研究表明，在生理条件下，蛋白酶体可以通过蛋白质剪接产生从头开始的多肽。然而，蛋白酶催化的细胞蛋白嵌合体的产生从未被报道过。我们的假设是，细胞含有大量的蛋白质嵌合体，这些蛋白质嵌合体是由至少两个蛋白质片段融合而产生的，这些蛋白质嵌合体是由蛋白酶介导的连接酶活性产生的。这一假说得到了我们的观察的支持，即在体外实验中，蛋白质酶在生理pH下催化蛋白质与多肽探针之间的肽键连接酶反应，从而导致杂交蛋白质的产生。此外，我们最近已经证明，杂交蛋白的产生似乎是细胞中的一个常见过程。然而，目前尚不清楚这种蛋白质嵌合体是否具有任何生理功能。我们推测，通过连接酶活性定向的蛋白质结构域交换可能是将某些蛋白质功能从一种蛋白质转移到另一种蛋白质的一种经济的细胞机制。因此，我们提出了以下具体目标：1.我们将在模型蛋白底物和多肽探针的存在下，确定选定的蛋白酶催化蛋白质融合的体外特异性，这将使蛋白质片段的N-端或C-端融合成为可能。将根据已知的模型蛋白酶的亚位点特异性来设计探针。类似的体外实验将以两个模型蛋白质作为融合伙伴进行。2.我们将在His6标记和荧光素标记的多肽探针的存在下，使用成纤维细胞和树突状细胞系研究细胞内蛋白融合，这与AIM 1中使用的类似。Ni-NTA-琼脂糖层析将分离连接产物用于MS分析。使用特定类型的蛋白水解酶抑制剂将描述连接酶反应中涉及的蛋白水解酶类型。使用针对目标2中确定的特定蛋白融合候选的抗体，我们将发现不依赖于肽探针的融合蛋白。主要感兴趣的将是那些大小超过对亲本蛋白的预测的蛋白质，这可能表明连接酶事件。这些蛋白嵌合体将被分离出来，并通过免疫沉淀和MS分析进行鉴定。测序和结构域分析将阐明融合位点、蛋白质稳定性和潜在功能。3.最后，根据确定的融合蛋白序列，利用杂交cDNA将选定的蛋白嵌合体生产为重组蛋白，并对其进行功能和结构研究。随着拟议的研究在蛋白质科学中开辟了新的领域，随着进展，将对项目进行修改。这一提议正在挑战RNA-DNA-蛋白质范式，使其成为在活细胞中产生功能蛋白质的唯一不同途径。

Proteases are known to cleave peptide bonds but under certain conditions, they can also catalyze the reverse reaction and are capable of forming peptide bonds. For example, it has been shown that under physiological conditions, proteasomes can generate de novo peptides by protein splicing. However, a protease-catalyzed generation of cellular protein chimeras has never been reported. Our hypothesis is that cells contain a multitude of protein chimeras generated by protease-mediated ligase activities that result from the fusion of at least two protein fragments. This hypothesis is supported by our observation that in in vitro experiments proteases catalyze peptide bond ligase reactions between proteins and peptide probes at physiological pH, leading to the generation of hybrid proteins. Moreover, we have recently shown that the generation of hybrid proteins appears to be a common process in cells. However, it is unknown whether such protein chimeras have any physiological functions. We speculate that directed protein domain exchanges by ligase activities could be an economic cellular mechanism to transfer certain protein functions from one protein to another. Therefore, we propose the following Specific Aims: 1.We will determine the in vitro specificity of selected proteases to catalyze protein fusion in the presence of model protein substrates and peptide probes, which will allow fusion at the N- or C-terminus of protein fragments. Probes will be designed based on the known subsite specificities of the model proteases. Similar in vitro experiments will be performed with two model proteins as fusion partners. 2.We will study intracellular protein fusion using a fibroblast and dendritic cell line in the presence of His6-tagged and fluorescein-labeled peptide probes similar to those used in Aim 1. Ni-NTA-agarose chromatography will separate the ligation products for MS analysis. The use of class-specific protease inhibitors will delineate the involvement of protease classes in the ligase reactions. Using antibodies against selected protein fusion candidates identified in Aim 2, we will discover peptide probe-independent fusion proteins. Of primary interest will be proteins that are larger in size than predicted for the parent protein, which may indicate a ligase event. These protein chimeras will be isolated and characterized by immunoprecipitation and MS analysis. Sequencing and domain analysis will shed light on the fusion sites, protein stability, and potential functions. 3.Finally, selected protein chimeras will be produced as recombinant proteins using hybrid cDNAs based on the determined fusion protein sequence and will be functionally and structurally investigated. As the proposed research opens new frontiers in protein science, modification of the project will be implemented as progress is achieved. This proposal is challenging the RNA-DNA-Protein paradigm as the only distinct pathway to generate functional proteins in living cells.