Journal
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
Volume 1868, Issue 7, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.bbamcr.2021.119019
Keywords
Calpain; Calcium; Cell culture; Immunofluorescence; Membrane; Plasma membrane; Protease; Protein-lipid interaction
Categories
Funding
- National Institutes of Health [R01NS095229]
- Honors College INBRE thesis fellowship by an Institutional Development Award (IDeA) from NIGMS [P20GM103423]
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CAPN5 is a ubiquitous enzyme highly expressed in the central nervous system and associated with eye development and disease. The study revealed that CAPN5 undergoes calcium-activated autoproteolytic processing, and membrane association enhances CAPN5 autolysis.
The enzymatic characteristics of the ubiquitous calpain 5 (CAPN5) remain undescribed despite its high expression in the central nervous system and links to eye development and disease. CAPN5 contains the typical protease core domains but lacks the C terminal penta-EF hand domain of classical calpains, and instead contains a putative C2 domain. This study used the SH-SY5Y neuroblastoma cell line stably transfected with CAPN5-3xFLAG variants to assess the potential roles of the CAPN5 C2 domain in Ca2+ regulated enzyme activity and intracellular localization. Calcium dependent autoproteolysis of CAPN5 was documented and characterized. Mutation of the catalytic Cys81 to Ala or addition of EGTA prevented autolysis. Eighty mu M Ca2+ was sufficient to stimulate half-maximal CAPN5 autolysis in cellular lysates. CAPN5 autolysis was inhibited by tri-leucine peptidyl aldehydes, but less effectively by di-Leu aldehydes, consistent with a more open conformation of the protease core relative to classical calpains. In silico modeling revealed a type II topology C2 domain including loops with the potential to bind calcium. Mutation of the acidic amino acid residues predicted to participate in Ca2+ binding, particularly Asp531 and Asp589, resulted in a decrease of CAPN5 membrane association. These residues were also found to be invariant in several genomes. The autolytic fragment of CAPN5 was prevalent in membrane-enriched fractions, but not in cytosolic fractions, suggesting that membrane association facilitates the autoproteolytic activity of CAPN5. Together, these results demonstrate that CAPN5 undergoes Ca2+-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.
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