Proteolytic cleavage of Livin (ML-IAP) in apoptotic melanoma cells potentially mediated by a non-canonical caspase.
BACKGROUND
Several inhibitor of apoptosis proteins (IAPs) are cleaved during apoptosis. Studies of the melanoma-associated IAP (ML-IAP) Livin, using recombinant molecules, have implicated both caspases 3/7 and the serine protease Omi/HtrA2 in its proteolytic cleavage.


OBJECTIVE
To characterize the apoptotic cleavage of Livin in melanocytic cells, and evaluate the role of known proteases.


METHODS
We assessed the capacity of a variety of stimuli to induce Livin cleavage in human melanoma cell lines and normal human melanocytes. The role of caspases and Omi was examined using caspase inhibitors and RNAi, respectively. A potential caspase substrate was further examined by site-directed mutagenesis. Deletion mapping was used to identify the cleavage site.


RESULTS
Livin cleavage was observed in multiple human melanoma cell lines in response to a variety of apoptotic stimuli (UVB, 4-TBP, cisplatin, TNF, Bax), and not affected by the addition of various protease inhibitors or RNAi-mediated silencing of Omi/HtrA2. Livin cleavage induced by 4-TBP, but not UVB or cisplatin, was blocked by the pan-caspase inhibitor zVAD-fmk. Mutation of Asp52 to Glu in Livin did not affect cleavage, while either mutation of Asp52 to Ala, deletion of Asp52, or deletion of the adjacent region (residues 53-61) abrogated cleavage.


CONCLUSIONS
Livin cleavage, induced by multiple apoptotic stimuli in melanoma cells, likely occurs in an Omi-independent fashion at residue 52 within its potential caspase substrate (DHVD52). However, relative insensitivity of the apoptotic cleavage to zVAD-fmk, or Asp52 to Glu mutation, suggests the involvement of a non-canonical caspase.
Yan
Hui
H
Huntsman Cancer Institute, University of Utah, Suite 5243, 2000 Circle of Hope, Salt Lake City, UT 84112, USA.
Brouha
Brook
B
Liu
Tong
T
Raj
Deepak
D
Biddle
Diana
D
Lee
Ray
R
Grossman
Douglas
D
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D019943:Amino Acid Substitution; D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D010450:Endopeptidases / Q000187:drug effects / Q000378:metabolism; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism*; D008544:Melanocytes / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism*; D024101:Mitochondrial Proteins; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D025941:Protein Interaction Mapping; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones; C419866:BIRC7 protein, human; D061945:Caspase Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D024101:Mitochondrial Proteins; D009363:Neoplasm Proteins; D011480:Protease Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D010450:Endopeptidases; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2; D020169:Caspases