The Expression and Proteolysis of BIGH3 in Various Cancer and Non-Cancer Cells

Date
2022
Authors
Matta, Heansika
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Abstract

BIGH3 is an extracellular matrix protein expressed in almost all human tissues. The BIGH3 gene is upregulated by the cytokine, Transforming growth factor beta-1 gene. The human BIGH3 protein comprises 683 amino acids and has a predicted molecular weight of 68 kDa. This protein has been found to undergo Carboxy-terminal proteolysis (CTP) by an unknown protease, generating peptides encoding integrin binding motifs: EPDIM and RGD. These soluble peptides are released into the ECM. When the concentration of the released soluble peptides in the ECM is high enough, they interfere with the integrin-binding of the cells, disrupting the focal adhesions. Focal adhesions are the sites where integrins attach the cell to the substratum, sending pro-survival signals. A specific set of integrins, αVβ3, α3β1, and αVβ5, recognize the RGD sequence of the soluble peptides and bind to free RGD, destroying the cell attachment and triggering apoptosis. These effects of apoptosis on different cancer cell lines can be used in cancer research. In a 2009 study, Zamilpa et al. showed that MG63 cells express BIGH3 protein, and the protein undergoes CTP via integrin interference, which eventually leads to apoptosis. Furthermore, the recombinant BIGH3 lacking the C-terminal RGD/EPDIM integrin ligand peptides failed to induce apoptosis, suggesting RGD/EPDIM fragments of the C-terminus BIGH3 is imperative for apoptosis. Which led to the hypothesis that different cancer cells and non-cancer cells undergo C-terminal proteolysis, which eventually stimulates apoptosis. Our specific aim was to determine if the C-terminal proteolytic processing of BIGH3 occurs in distinct cell lines, including cancer cell lines, and non-cancer cell lines. Followed by, whether this C-terminal post-translational modification of BIGH3 can induce apoptosis in cancer cell lines. We investigated this hypothesis by testing four cancer cell lines and two non-cancer cell lines for BIGH3 Carboxy terminal proteolysis (CTP). Using MG63 as our control, we retrieved different cell lines. The cells are grown in DMEM media until confluency. Later, we collected the media and tested using SDS-PAGE, and the western blot technique, for BIGH3 C-terminal proteolysis. All the cell lines examined were positive for the presence of BIGH3 at both full length and truncated length (cleaved). Additionally, we also observed intermediate bands, along with the lower band in STTG1 and U-87, and the aggregates in two other cell lines at ~100 and ~240 KDa, indicating the formation of trimers and tetramers of BIGH3. Suggesting that every cell line tested expresses and processes BIGH3. Further research is required to determine if the cleavage is characteristic for all cancer cell lines and, if RGD/EPDIM ligand recognition peptides can serve as a potential tumor suppressor for different cancers. If BIGH3 CTP is prominent in the varied cell line types and is responsible for programmed cell death, BIGH3 can be used as a potential therapeutic target for different cancers.

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Keywords
Apoptosis, BIGH3, C-terminal proteolysis, Cancer cells
Citation
Department
Integrative Biology