The ligase activity of proteases: Do proteases make proteins?

众所周知，蛋白酶可以裂解肽键，但在一定条件下，蛋白酶也可以催化逆反应，形成肽键。例如，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新生肽。然而，蛋白酶催化的细胞蛋白嵌合体的生成从未被报道过。我们的假设是，细胞中含有大量的蛋白质嵌合体，这些蛋白质嵌合体是由蛋白酶介导的连接酶活性产生的，这种酶活性是由至少两个蛋白质片段融合产生的。我们的观察支持了这一假设，在体外实验中，蛋白酶在生理pH下催化蛋白质和肽探针之间的肽键连接酶反应，导致杂交蛋白的产生。此外，我们最近表明，杂交蛋白的产生似乎是细胞中的一个共同过程。然而，这种蛋白质嵌合体是否具有任何生理功能尚不清楚。我们推测，通过连接酶活性进行的定向蛋白质结构域交换可能是一种经济的细胞机制，可以将某些蛋白质功能从一种蛋白质转移到另一种蛋白质。因此，我们提出以下具体目标：我们将确定所选蛋白酶在模型蛋白底物和肽探针存在下催化蛋白融合的体外特异性，这将允许在蛋白片段的N端或c端融合。探针将根据已知的模型蛋白酶的亚位点特异性来设计。类似的体外实验将以两种模型蛋白作为融合伙伴进行。2.我们将研究细胞内蛋白融合，使用成纤维细胞和树突状细胞系，在his6标记和荧光素标记的肽探针存在下，类似于Aim 1中使用的探针。ni - nta -琼脂糖层析分离结扎产物进行质谱分析。类特异性蛋白酶抑制剂的使用将描述连接酶反应中蛋白酶类的参与。使用针对Aim 2中确定的选定蛋白融合候选物的抗体，我们将发现肽探针无关的融合蛋白。主要的兴趣将是蛋白质的大小比亲本蛋白预测的大，这可能表明连接酶事件。这些蛋白嵌合体将被分离并通过免疫沉淀和质谱分析进行表征。测序和结构域分析将揭示融合位点、蛋白质稳定性和潜在功能。3.最后，选择的蛋白质嵌合体将根据确定的融合蛋白序列使用杂交cdna作为重组蛋白生产，并将进行功能和结构研究。由于所提出的研究开辟了蛋白质科学的新领域，随着取得进展，将对项目进行修改。这一建议挑战了rna - dna -蛋白质范式作为在活细胞中产生功能性蛋白质的唯一独特途径。

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.