Discipline: Technology and Engineering
Subcategory: Biomedical Engineering
Session: 1
Eva Gatune - Xavier University of Louisiana
Co-Author(s): W. Andrew Shockey, Manu O. Platt. Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, GA, USA.
Introduction: Cathepsins are a class of enzymes that are proteases and contain the most potent mammalian elastases and collagenases. Cathepsin expression and activity are regulated by endogenous protein inhibitors as well as proteolytic interactive networks that are not completely understood. Up to 50% of deaths in the United States are caused by diseases such as cancer metastasis, heart disease, stroke, and Alzheimer’s which all have implicated cathepsin activity in progression. To date, no cathepsin inhibitors have been passed by the FDA after clinical trials due to side effects and nonintuitive increases in cathepsin expression and activity. Here, we used mRNA transfection to gain a better understanding of the proteolytic regulation and interactions among cathepsins in macrophages after one cathepsin family member was overexpressed. Understanding these cellular feedback and regulatory interactions among cathepsins will lead to the development of effective and safe pharmaceuticals to treat fatal diseases such as metastatic cancer and atherosclerosis.
Materials and Methods: THP-1 macrophages were transfected with mRNA using Lipofectamine 2000. In order to validate our transfection protocol a Green Fluorescent Protein (GFP) mRNA transfection was done. The cells were transfected with 6 different proteins cathepsin K, Kmutant, V, L, S and cystatin C, a cathepsin inhibitor. Cells from the 6 different experimental groups were lysed 24 and 48 hours after transfection. Multiplex cathepsin zymography and Western blotting were the protein detection assays used on the lysate to assess the results of the transfection. Zymography was used to identify active cathepsins, and the western blot assay detected the presence of specific cathepsin expression.
Results and Discussion: THP-1 macrophages were transfected with GFP mRNA, and GFP was detectable by fluorescence microscopy in over 50 percent of cells after 24 hours (Figure 1A). Gelatin zymography showed active cathepsin L and S, which are native to the THP-1 cells line used (Figure 1B). Active transfected cathepsins were not detectable by gelatin zymography. In addition, compared to the controls an overexpression of cathepsin L and S was not seen, thus indicating that transfection was unsuccessful. Overexpression of target proteins was not detectable by western blots either. Endogenous cathepsins L and S were detectable by western blot (Figure 1C), however transfected cathepsins V, K and Kmutant were undetectable.
Conclusions: This experiment shows successful expression of GFP after mRNA transfection of THP-1 cells. However, cathepsin mRNA transfection was not successful. It is important to note that GFP has a much longer half-life than cathepsins, meaning even though the GFP was still present at 24 hours the cathepsins might have already been degraded or secreted. Lysing cells at earlier time points such as 4 and 8 hours may show presence of active transfected cathepsins. To further improve these results, the protocol will be conducted on HEK 293T cells which are optimized for transfection. Also, conditioned media will be examined for the target protein. Finally, quantitative real-time PCR will be used to quantify mRNA levels to confirm successful transfection.
Funder Acknowledgement(s): This work was funded by the NSF Research Center for Cell Manufacturing and Technologies (CMaT). I also would like to thank Platt Lab at Georgia Tech for allowing me to conduct this work.
Faculty Advisor: Manu O. Platt, manu.platt@bme.gatech.edu
Role: I participated directly or indirectly in all of the research stated in the abstract. All of the methods I performed myself after becoming proficient. The data obtained is a result of my own research.