# Candidate Interaction Partners of Calpain-5 Suggest Clues to Its Involvement in Neovascular Inflammatory Vitreoretinopathy

## TL;DR

Researchers identified 51 proteins that interact with calpain-5, suggesting roles in protein quality control and possibly contributing to an autoimmune eye disease.

## Contribution

The study identifies novel calpain-5 interaction partners and links them to protein quality control and a retinal disease.

## Key findings

- Fifty-one candidate interaction partners of calpain-5 were identified in neuroblastoma cells.
- Many of these interactors are associated with protein quality control systems like chaperones and proteasomes.
- Pathogenic CAPN5 variants showed impaired calcium-induced proteolysis of co-IP proteins.

## Abstract

What are the main findings?
Fifty-one candidate interaction partners of calpain-5/CAPN5 were identified in neuroblastoma cells.Many candidate CAPN5 interactors are associated with the chaperome and protein quality control complexes.

Fifty-one candidate interaction partners of calpain-5/CAPN5 were identified in neuroblastoma cells.

Many candidate CAPN5 interactors are associated with the chaperome and protein quality control complexes.

What is the implication of the main finding?
The findings provide hints regarding both the physiological and pathological roles of CAPN5.

The findings provide hints regarding both the physiological and pathological roles of CAPN5.

Although calpain-5/CAPN5 is widely expressed in mammals, little is known regarding its functions. Pathogenic mutations of CAPN5 are causal for a devastating autoimmune eye disease, neovascular inflammatory vitreoretinopathy (NIV). To provide insight into both the physiological and pathological roles of CAPN5, it is essential to identify candidate interaction partners and possible substrates. Human SH-SY5Y neuroblastoma cells, transfected with full-length catalytically dead (Cys81Ala) CAPN5-3×FLAG, were used for anti-FLAG co-immunoprecipitation (co-IP) and quantitative proteomics using Sequential Window Acquisition of all THeoretical mass spectra (SWATH-MS). Fifty-one proteins were enriched at least four-fold, p < 0.01, relative to cells transfected with an empty FLAG vector. A high proportion (24/51) of candidate CAPN5 interaction partners are associated with protein quality control, including components of the chaperonin, chaperone, and ubiquitin–proteasome systems. Additional candidate interactors include tubulins, kinases, phosphatases, G proteins, and mitochondrial proteins. CAPN5 interactions for 14 of the candidate proteins were confirmed by co-IP and immunoblotting. Of these 14 proteins, 11 exhibited in vitro calcium-induced proteolysis following co-IP with WT CAPN5-3×FLAG. Impaired calcium-induced proteolysis of co-IP proteins was observed for the pathogenic CAPN5 variants R243L and R289W. Further studies are needed to validate the association of candidate CAPN5 interactors with proteins and complexes suggested by the SWATH-MS and co-IP results, and the possible role of CAPN5 within such complexes. The possible involvement of CAPN5 in protein quality control is relevant to NIV, as defects in protein quality control have been implicated in inherited retinal disorders. Proteomic data are available via ProteomeXchange with identifier PXD068008.