Designed Protein Degradation Method Based on
Trim21 And Nanobody -- Description
TRIM-AWAY, through introducing antibody and Trim21 protein into cells by microinjection or electroporation, represents a novel strategy which could rapidly remove unmodified native proteins in diverse cell types. However, the high complexity and low efficiency limited its application. Through combining TRIM-AWAY and ectopic expression of recombinant antibodies, we developed PR PREDATOR, a robust tool for degrading endogenous proteins in mammalian cells. Basically, parts for expression of Trim21 and recombinant antibodies were constructed and inserted in one single vector to realize the P2A-mediated bicistronic expression. GFP and ErbB-3, a member of the receptor tyrosine-protein kinases highly involved in the proliferation and metastasis of cancer cells, were chosen as targets of PR PREDATOR for the proof of concept and further demonstration of our design respectively. Our PR PREDATOR method shall provide not only novel tools for protein function study but also brand-new options for treating disease caused by aberrant protein aggregations.
Protein structure variation and polymerization aberrances been proved to be the reasons of a series of diseases related to proteopathies, including Alzheimer’s disease, Parkinson’s disease（PD）, amyloidosis and so on[1-3]. Besides, although genetic mutation and inherited genetics are the main causes of cancer, it is still because of the aberrant expression of certain proteins that directly lead to the variation of the original cells[4-10]. The idea that interfering these function or amount abnormal proteins intracellularly or extracellularly has made a potential idea to cure these protein-aberrance related diseases.
Traditionally, the most commonly used methods to interfer the function of a protein are ways that repress the producing process of these target proteins, including DNA knockout which has been even more heated after the CRISPR/Cas9 came out, and RNA interference. These methods works on the genetic or mRNA level, and surely have their advantages in repressing the target proteins such as high efficiency and consistent longevity. However, these ways are indirect and may even be inefficient to certain long-lived proteins.
Recently, by utilizing the ubiquitin-proteasome pathway, researchers have developed several tools for the direct degradation of endogenous protein, such as PROTAC, TRIM-AWAY et al. Among these methods, TRIM-AWAY, a novel strategy through microinjecting antibody and introducing Trim21 protein into cells, harnesses the cellular protein degradation machinery to remove unmodified native proteins and allows the study of protein function in diverse cell types . The core fragment of TRIM-AWAY is TRIM21 protein, an E3 ubiquitin ligase that binds with high affinity to the Fc domain of antibodies. After binding, TRIM21 recruits the ubiquitin-proteasome system to antibody-bound pathogens, leading to their destruction. This method makes a direct way to function on the protein level, but its poor cell viability after microinjection or electroporation, the high cost of antibody production, the short lifespan of injected-antibody inside the cell and the technique complexity limit its wide application.
This year through combining TRIM-AWAY strategy and ectopic expression of nanobody, we developed a new method for degrading endogenous proteins in mammalian cells. Basically, parts for expression Trim21 and nanobody-IgG Fc were constructed, and then were inserted in a single vector to realize P2A mediated bicistronic expression. To prove the concept, sequences for GFP nanobody and human IgG Fc fragment expression were ligated for fusion expression. To demonstrate our design, the receptor tyrosine-protein kinase erbB-3 is selected. This protein is able to bind and form the heterodimerization with proteins from the erbB family, which is highly involved in proliferation and metastasis of cancer cells, also implicated in chemotherapeutic resistance through multiple signaling pathways. It has been proved that the downregulation of erbB-3 slows down the process several cancers. In our demonstration, the erbB-3 inhibitory antibody would be expressed along with trim21 in cells and directly functions on erbB-3 to bring down its containment.
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