Analysis of Wild Type and Variant B Cystatin C Interactome in Retinal Pigment Epithelium Cells Reveals Variant B Interacting Mitochondrial Proteins

• Published in: Cells (Basel, Switzerland) Switzerland), 2023-02, Vol.12 (5), p.713
• Main Authors: Carlsson, Emil, Sharif, Umar, Supharattanasitthi, Wasu, Paraoan, Luminita
• Format: Article
• Language: English
• Subjects: Age; aging; Alzheimer's disease; Amino acid sequence; Cell fusion; Cellular proteins; Cystatin C; Cysteine proteinase; Cytochrome b5; Epithelium; Genotypes; Health aspects; Health risk assessment; Humans; Macular degeneration; Macular Degeneration - metabolism; Mass spectrometry; Mass spectroscopy; Membrane potential; mistrafficking; Mitochondria; Mitochondria - metabolism; Mitochondrial DNA; Mitochondrial Proteins - metabolism; Mutation; Neurodegenerative diseases; Peptides; Plasmids; Protein sorting signals; Protein-protein interactions; Proteinase inhibitors; Proteins; Receptors, GABA - metabolism; Retina; Retinal pigment epithelium; Retinal Pigment Epithelium - metabolism; Scientific imaging; translocator protein; variant B
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells12050713

Cystatin C, a secreted cysteine protease inhibitor, is abundantly expressed in retinal pigment epithelium (RPE) cells. A mutation in the protein's leader sequence, corresponding to formation of an alternate variant B protein, has been linked with an increased risk for both age-related macular degeneration (AMD) and Alzheimer's disease (AD). Variant B cystatin C displays intracellular mistrafficking with partial mitochondrial association. We hypothesized that variant B cystatin C interacts with mitochondrial proteins and impacts mitochondrial function. We sought to determine how the interactome of the disease-related variant B cystatin C differs from that of the wild-type (WT) form. For this purpose, we expressed cystatin C Halo-tag fusion constructs in RPE cells to pull down proteins interacting with either the WT or variant B form, followed by identification and quantification by mass spectrometry. We identified a total of 28 interacting proteins, of which 8 were exclusively pulled down by variant B cystatin C. These included 18 kDa translocator protein (TSPO) and cytochrome B5 type B, both of which are localized to the mitochondrial outer membrane. Variant B cystatin C expression also affected RPE mitochondrial function with increased membrane potential and susceptibility to damage-induced ROS production. The findings help us to understand how variant B cystatin C differs functionally from the WT form and provide leads to RPE processes adversely affected by the variant B genotype.