title: Chymotrypsin-like serine proteinases are involved in the maintenance of cell viability.
abstract: An increasing number of studies have implicated serine proteinases in the development of apoptosis. In this study, we assessed the ability of a set of highly specific irreversible inhibitors (activity probes), incorporating an α-amino alkane diphenyl phosphonate moiety, to modulate cell death. In an initial assessment of the cellular toxicity of these activity probes, we discovered that one example, N-α-tetramethylrhodamine phenylalanine diphenylphosphonate {TMR-Phe(P)(OPh)(2)} caused a concentration-dependent decrease in the viability of HeLa and U251 mg cells. This reduced cell viability was associated with a time-dependent increase in caspase-3 activity, PARP cleavage and phosphatidylserine translocation, establishing apoptosis as the mechanism of cell death. SDS-PAGE analysis of cell lysates prepared from the HeLa cells treated with TMR-Phe(P)(OPh)(2), revealed the presence of a fluorescent band of molecular weight 58 kDa. Given that we have previously reported on the use of this type of activity probe to reveal active proteolytic species, we believe that we have identified a chymotrypsin-like serine proteinase activity integral to the maintenance of cell viability.
authors.affiliation: Biomolecular Sciences Research Group, McClay Research Centre, School of Pharmacy, Queen's University, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom. k.moffitt@qub.ac.uk
authors.forename: K L
authors.initials: KL
authors.lastname: Moffitt
authors.forename: B
authors.initials: B
authors.lastname: Walker
authors.forename: S L
authors.initials: SL
authors.lastname: Martin
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D063065:Organophosphonates / Q000737:chemistry / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry / Q000494:pharmacology; D057057:Serine Proteases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology
chemical_list: D063065:Organophosphonates; D010718:Phosphatidylserines; D012235:Rhodamines; D015842:Serine Proteinase Inhibitors; D010649:Phenylalanine; D011065:Poly(ADP-ribose) Polymerases; D057057:Serine Proteases; D002918:Chymotrypsin; D053148:Caspase 3