| Summary: | 碩士 === 國立交通大學 === 生物科技學系 === 105 === Thermostable Direct Hemolysin from Grimontia hollisae (Gh-TDH) has been reported as a pore-forming toxin that results in diverse damage to different kinds of cells. Recently, studies have suggested that TDH is the tetrameric protein by nature, which means it can form pores on cell membranes leading to changing the osmotic pressure, disturbing cell homoeostasis and causing cell death. The N-terminal helix of Gh-TDH has indicated the correlation with binging activity on targeted cell membranes, contributing to hemolytic and cytotoxic activity afterwards. Therefore, the pore-forming toxic activity of Gh-TDH has been adjusted for the requisition of cancer treatment as a protein drug. In order to reduce and avoid ubiquitous toxicity of Gh-TDH, we choose to design several targeted fusion proteins under the Gh-TDH backbone for applying this pore-forming toxin in the treatment of few cancers. In previous research, our group indicated when Gh-TDH conjugated with a peptide at the N-terminal region, resulting in the decrease of hemolytic and cytotoxic activity. Furthermore, construction of mutant Gh-TDHR46E (monomeric form) with a targeting peptide such as epidermal growth factor receptor (EGFR) binding peptide (EB) to obtain a fusion protein, having significant anti-tumor effects on A431 cells1.
In my study, there are several Gh-TDH recombinant proteins are designed as the pro-drug by using the concepts of recognition peptide to cell membrane and/or the cleavage sequences for cellular protease activation. The selective target molecules for the designs are MMP-7, Furin and PSA. The recognition peptide is conjugated at the N-terminus of Gh-TDH which might cause toxicity decrease of the designed recombinant protein. Besides, there is a cleavage site at the recognition peptide, which can cleave by proteolytic proteins produced by cancer cells. Next, the recognition peptide contacts with the target cancer cells with the treatment of recombinant protein. After that, the proteolytic proteins secreted from cancer cells interact with recognition peptide resulting in excision at cleavage site. Gh-TDH resumes wild-type activity after peptide-conjugated TDH releases sections cleaved by enzymatic proteins. Afterwards, active Gh-TDH can cause nearby cancer cell death as expected.
The protein MMP7-TDHWT is the wild-type Gh-TDH (Gh-TDHWT) conjugates with eight amino acids of MMP-7 recognition and cleavage peptide. MMP7-TDHWT in comparison with Gh-TDHWT shows no significant difference of hemolytic and cytotoxic activity. Furin-PSA-TDHWT which is the Gh-TDHWT conjugates with six amino acids of Furin recognition peptide, and six amino acids of PSA cleavage site peptide. His-Furin-PSA-TDHWT is Furin-PSA-TDHWT conjugates with six His amino acids at the N-terminus. The result of hemolytic activity of His-Furin-PSA-TDHWT is weaker than Gh-TDHWT significantly. And the MTT assay suggests that cytotoxicity of Furin-PSA-TDHWT for LNCaP prostate cancer cells is weaker than Gh-TDHWT. However, the cytotoxicity of His-Furin-PSA-TDHWT is much weaker than Furin-PSA-TDHWT. The conditioned medium of LNCaP cells incubates with His-Furin-PSA-TDHWT for more than three days, which shows the enzymatic activity of PSA. The result indicates releasing of Gh-TDHWT from His-Furin-PSA-TDHWT by enzymatic cleavage.
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