The ligase activity of proteases: Do proteases make proteins?

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.