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

Emil Carlsson, Umar Sharif, Wasu Supharattanasitthi, Luminita Paraoan

Research output: Contribution to journalArticle (journal)peer-review

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Abstract

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.
Original languageEnglish
Article number12050713
Pages (from-to)1-17
JournalCells
Volume12
Issue number5
Early online date23 Feb 2023
DOIs
Publication statusPublished - 23 Feb 2023

Keywords

  • Alzheimer’s disease
  • Article
  • age-related macular degeneration
  • aging
  • cystatin C
  • halo-tag
  • mistrafficking
  • mitochondria
  • translocator protein
  • variant B

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