Proteolytic cleavage of Livin (ML-IAP) in apoptotic melanoma cells potentially mediated by a non-canonical caspase.

Abstract

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.

CONCLUSION:

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.

• Authors: Hui Yan, Brook Brouha, Tong Liu, Deepak Raj, Diana Biddle, Ray Lee, Douglas Grossman
• Journal: Journal of dermatological science (J Dermatol Sci) Vol. 43 Issue 3 Pg. 189-200 (Sep 2006) ISSN: 0923-1811 [Print] Netherlands
• PMID: 16806840 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Adaptor Proteins, Signal Transducing
• Amino Acid Chloromethyl Ketones
• BIRC7 protein, human
• Caspase Inhibitors
• Inhibitor of Apoptosis Proteins
• Mitochondrial Proteins
• Neoplasm Proteins
• Protease Inhibitors
• benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
• Endopeptidases
• Serine Endopeptidases
• HTRA2 protein, human
• High-Temperature Requirement A Serine Peptidase 2
• Caspases

Topics

• Adaptor Proteins, Signal Transducing (genetics, metabolism)
• Amino Acid Chloromethyl Ketones (pharmacology)
• Amino Acid Substitution
• Apoptosis
• Caspase Inhibitors
• Caspases (metabolism)
• Endopeptidases (drug effects, metabolism)
• High-Temperature Requirement A Serine Peptidase 2
• Humans
• Inhibitor of Apoptosis Proteins (genetics, metabolism)
• Melanocytes (metabolism)
• Melanoma (metabolism)
• Mitochondrial Proteins
• Mutation
• Neoplasm Proteins (genetics, metabolism)
• Protease Inhibitors (pharmacology)
• Protein Interaction Mapping
• Serine Endopeptidases (genetics, metabolism)
• Substrate Specificity