Team:NYMU-Taipei/Project/Speedy protein degrader
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Abstract
Introduction
What is our goal?
Fluorescent proteins, for example, green fluorescent protein (GFP), are now widely used to visualize a protein’s distribution and dynamics in a subcellular compartment. The remarkable feature of the fluorescent proteins (FP) is that the fluorophore forms spontaneously (Chalfie et al., 1994) without the need of any substrate or cofactor (Heim, Prasher, Tsien, 1994). In addition, FP variants can be fused to virtually any protein of interest, thus, they can be used in many species for detection purposes within single living cells. FPs, together with modern microscopes, have become essential tools for studying spatial and temporal dynamics of cellular processes at high resolution.
The biophysical characteristics of fluorescent probes provide opportunities and, set limitations for fusion-protein studies. The fluorophore of GFP and its variants is contained within a barrel of beta-sheet protein (Ormo et al., 1996). This compact structure renders FPs with high photostability under a variety of conditions, even under treatment with protease (Chalfie et al., 1994). Once stable FPs formed, they are cleared from the cells in tens of hours (Anderson et al, 1998). This property allows prolonged imaging of cells, and easier detection when FPs accumulated. However, the stability of FPs limits its application in some studies which needs fast reporter turnover, for example, studies of transient gene expression. This also impede the measurements of temporal expression pattern and behavior of proteins, because proteins at different stages of their lifetime are being detected. To overcome this limitation, we tried to develop destablized FPs. There are several ways to generate FP variants with different spectral and expression properties. One approach is mutagenesis studies. Though this approach is fruitful (Heim, Cubitt, Tsien, 1995; Delagrave et al, 1995; Zacharias et al, 2002), it seems impractical to screen possible mutations of each FP in ever-increasing FP libraries. Alternatively, we planed to control proteolysis by adding degradation tags to FPs (Anderson et al, 1998). This approach can be easily generalized to several FPs; thus, it provides extensibility to future applications.
Controlled protein degradation in bacteria
The flow of genetic information can—and sometimes need to — depend not only on its controlled synthesis but equally on its controlled degradation. Indeed, protein degradation in natural systems is essential for cellular functions. Previous study showed that 2.7% of proteins are degraded per generation in Escherichia coli (E. coli.) growing in logarithmic phase (Fox and Brown 1961). Control of protein turnover is required for cell cycle progression, signal transduction, and rapid responses to environmental challenges(Grunenfelder et al., 2001, Hengge-Aronis, 2002, Neher et al., 2003). Therefore, regulated degradation is expected to be critical for the development of synthetic circuits.
To control the half-life of specific proteins, we utilized the regulated proteolysis machineries naturally employed to bacterial systems. Regulated degradation is ubiquitous for prokaryotic and eukaryotic cells (Jenal and Hengge-Aronis, 2003; Hershko and Ciechanover 1998), because they need to clear damaged or aberrant proteins while ignoring functional ones. A common strategy for substrate selection is adding degradation tags to target proteins. Then, certain proteases execute energy dependent degradation of those proteins. One prominent type of degradation tag in prokaryotes is the ssrA tag, a tmRNA encoded by ssrA gene. This tagging system is engaged in protein quality control throughout all eubacteria (Karzai et al. 2000). Though it was first described as a mechanism to clear obstructed ribosomes (Keiler et al., 1996), recent studies suggested ssrA-mediated tagging also plays a regulatory role in the expression of lac operon (Abo et al., 2000). This motivated us to control the protein expression via manipulating controlled proteolysis.
ssrA tag
SsrA tag, encoded by the ssrA RNA, is important for protein quality control in bacteria. SsrA RNA is recognized as tmRNA because it has characteristics of both tRNA and mRNA(Atkins and Gesteland, 1996; Keiler et al., 1996). When E. coli. ribosomes stall during translation due to absence of a proper stop codon, ssrA ribosome-rescue system mediates C-terminal modification by adding the sequence AANDENYALAA to the the incomplete peptide (Keiler et al, 1996). SsrA tagging frees these ribosomes for other mRNAs, and targets the defective polypeptides for degradation by ClpXP and other ATP-dependent proteases (Gottesman et al. 1998). Previous studies suggest that 0.5% of protein products in E. coli. receive ssrA tags (Lies and Maurizi 2008). Bacterial strains lacking functional ssrA gene show slower growth (Oh and Apirion, 1991), reduction in motility (Komine et al, 1994), and subsided pathogenesis (Julio et al, 2000). These results indicate that ssrA tagging system play a major role in bacterial physiology.
Measurement
Data analysis
Experiment overview
What are we doing?