BPS4127 Report.docx

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Department
Biopharmaceutical sciences
Course Code
BPS4127
Professor
All

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Description
A mutant Fas ligandEGFP fusion protein does not induce apoptosis in HeLa cellsNAMEEMAILSTUDENT NUMBERPerformed at the University of OttawaOttawa OntariordDecember 3 2012BPS4127mtFasLEGFP FUSION PROTEIN DOES NOT INDUCE APOPTOSIS IN HeLa CELLS2AbstractFas ligand FasL is a typeII transmembrane protein and part of the tumor necrosis factor TNF family Upon binding to Fas receptor it triggers the activation of apoptotic machinery in the target cell a fact which has led to its study as a possible targeted antitumoral agent It features three key regulatory regions that may lower levels of expression or downregulate its action A mutant FasL mtFasL has been constructed lacking amino acids 869 containing a protein binding site as well as casein kinase substrate region both involved in regulating FasL expression and 111133 containing a regulatory cleavage site An mtFasLEGFP fusion protein was created by subcloning mtFasL into pEGFPN1 HeLa cells a cervical cancer cell line were transfected with the construct and shown to successfully express the mtFasLEGFP construct Cell death was measured 24 and 48 hours post transfection and in both cases mtFasL with a Cterminal bound EGFP protein does not show any apoptotic effect on cervical cancer cells in vitroIntroductionFas ligand FasL CD95L CD178 APO1L is a typeII membrane bound protein and one of the key signaling factors in the extrinsic apoptotic pathway reviewed by Strasser in 1 It binds as a homotrimer to its similarly homotrimerized receptor Fas receptor Fas CD95 APO1 and in doing so triggers the formation of the deathinducing signaling complex DISC and ultimately results in the apoptosis of the target cell 2 Biologically the main purpose of this pathway is in preserving homeostasis in the immune system 3 Therapeutically however researchers are seeking to take advantage of this pathway as a method to induce apoptosis in cancerous cells reviewed by VillaMorales in 4NAMESTUDENT NUMBER
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