Abstract
Abstract
Purpose : Cystatin C is a cysteine protease inhibitor abundantly secreted by the retinal pigment epithelium (RPE) cells. A single point mutation in the cystatin C gene results in an A25T amino acid substitution and is a known risk factor for age-related macular degeneration (AMD) and Alzheimer’s Disease. This variant B form of cystatin C displays intracellular mistrafficking with partial mitochondrial retention. The purpose of this study was to characterise variant B cystatin C with regard to its interactome and downstream effect on mitochondrial function.
Methods : Halo-tagged fusion constructs encoding wild type or variant B cystatin C were expressed in ARPE19 cells. Pull down assays were performed with eluates submitted for nanoLC-ESI-MS/MS analysis. Cellular localisation of variant B cystatin C was examined via subcellular fractionation, as well as expression of eGFP-tagged proteins in live cells and immunofluorescence analysis. Quantification of mitochondrial reactive oxygen species (ROS) was performed using a mitochondrial ROS detection assay kit. Mitochondrial membrane potential was investigated using Mitotracker Red FM dye and flow cytometry analysis.
Results : Among the 28 proteins identified as interacting with at least one of cystatin C variants, 8 were exclusively pulled down by variant B protein. These included proteins abundantly distributed in mitochondria and localised to the mitochondrial outer membrane, such as 18 kDa translocator protein (TSPO) and cytochrome B5 type B. Subcellular fractionation and immunofluorescence experiments confirmed mitochondrial enrichment of variant B cystatin C. RPE cells expressing variant B cystatin C were significantly more susceptible to damage induced by mitochondrial ROS production (p ≤ 0.05), and displayed a significant increase in mitochondrial membrane potential (p ≤ 0.05).
Conclusions : Variant B cystatin C displays mistrafficking with partial mitochondrial retention, interacts with outer membrane mitochondrial proteins and have a functional effect on mitochondria. The findings substantiate the contribution of such molecular mechanisms to the pathogenesis of AMD and possibly other age-related neurodegenerative conditions.
Purpose : Cystatin C is a cysteine protease inhibitor abundantly secreted by the retinal pigment epithelium (RPE) cells. A single point mutation in the cystatin C gene results in an A25T amino acid substitution and is a known risk factor for age-related macular degeneration (AMD) and Alzheimer’s Disease. This variant B form of cystatin C displays intracellular mistrafficking with partial mitochondrial retention. The purpose of this study was to characterise variant B cystatin C with regard to its interactome and downstream effect on mitochondrial function.
Methods : Halo-tagged fusion constructs encoding wild type or variant B cystatin C were expressed in ARPE19 cells. Pull down assays were performed with eluates submitted for nanoLC-ESI-MS/MS analysis. Cellular localisation of variant B cystatin C was examined via subcellular fractionation, as well as expression of eGFP-tagged proteins in live cells and immunofluorescence analysis. Quantification of mitochondrial reactive oxygen species (ROS) was performed using a mitochondrial ROS detection assay kit. Mitochondrial membrane potential was investigated using Mitotracker Red FM dye and flow cytometry analysis.
Results : Among the 28 proteins identified as interacting with at least one of cystatin C variants, 8 were exclusively pulled down by variant B protein. These included proteins abundantly distributed in mitochondria and localised to the mitochondrial outer membrane, such as 18 kDa translocator protein (TSPO) and cytochrome B5 type B. Subcellular fractionation and immunofluorescence experiments confirmed mitochondrial enrichment of variant B cystatin C. RPE cells expressing variant B cystatin C were significantly more susceptible to damage induced by mitochondrial ROS production (p ≤ 0.05), and displayed a significant increase in mitochondrial membrane potential (p ≤ 0.05).
Conclusions : Variant B cystatin C displays mistrafficking with partial mitochondrial retention, interacts with outer membrane mitochondrial proteins and have a functional effect on mitochondria. The findings substantiate the contribution of such molecular mechanisms to the pathogenesis of AMD and possibly other age-related neurodegenerative conditions.
| Original language | English |
|---|---|
| Pages | 426 |
| Publication status | Published - 30 Jun 2023 |
| Event | Association for Research in Vision and Ophthalmology - Louisiana Convention Centre New Orleans, New Orleans, United States Duration: 23 Apr 2023 → 28 Apr 2023 |
Conference
| Conference | Association for Research in Vision and Ophthalmology |
|---|---|
| Country/Territory | United States |
| City | New Orleans |
| Period | 23/04/23 → 28/04/23 |
Keywords
- Cystatin C
- variant B
- Mitochondrial proteins
- retinal pigment epithelium (RPE)
- Age-related macular degeneration AMD
- Mitochondrial dysfunction
- Reactive oxygen species ROS
- Mitochondrial membrane potential
- neurodegenerative diseases
- Proteomics