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Title: Investigation of the potential role of serine proteases in apoptosis
Author: Moffitt, Kelly Louise
ISNI:       0000 0001 3413 067X
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2007
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Proteases playa critical role in the regulation of cell survival and execution of cell death, particularly apoptosis. Increasingly research has implicated serine proteases within this process. The identification of novel serine proteases in this study utilised a library of 'in-house' and commercially available inhibitors. Veril'ication of apoptotic death was established by measurement of cell viability, caspase-3 activation, PARP cleavage, phosphatidylserine translocation and upregulation of GAPDH. A chymotrypsin-like protease targeted by the cell-permeable inhibitor, tetramethylrhodaminephenylalanine- diphenyl phosphonate (TMR-Phe-DPP) was detected. Incubation with this peptide reduced I:ell viability to 9% of control values in both HeLa and U25 I mg cell-lines. This reduced cell viability correlated with a time-dependent increase in caspase-3 activity, PARP cleavage and phosphatidylserine translocation. Uptake and localisation of TMR-Phe-DPP in cells occurred through receptor-mediated endocytosis. Following SDS-PAGE, a fluorescent band was 'detected between 52 and 60 kDa, however sequencing this labelled protease proved problematic. Immunoprecipitation methods were unsuccessful, potentially due to the close coupling of the tetramethylrhodamine label to the phenylalanine residue. Alternative identificatioi1 methods utilizing a biotin-labelled phenylalanine diphenyl phosphonate (BPSPhe- DPP) also proved unsuccessful although it was possible to demonstrate that this peptide binds to the active site of our targe.t protease. Traditionally chloromethyl ketones have been used as inhibitors of serine proteases; however. they ~xhibit broad speciticity and nre unstable in the presence of serum. Three diphenyl phosphonates were inv~stigated for their ability to inhibit the serine protease TPPII- Ala-Ala-Phe-DPP, Ala-Ala-Thiophcne- Drp and Ala-Ala-Ferrocene-DPP. These inhibitors proved to be both potent and stable inh~bitors of TPPII activity I:ompared to the traditional chloromethyl ketone AAF-CMK. In particular. AAD-DPP demonstrated improved stability towards serum, selectivity of action, lack orovert toxicity and could be delivered intracellularly using liposomes. These properties make it an attractive candidate for probing the cellular role of TPPl1 in vitro and in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available