Related Experiment Video
Updated: Aug 5, 2025
In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila
Published on: November 27, 2016
DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in
Petr Lapcik1, Barbora Vesela2, David Potesil3
1Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic.
Abstract:
Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.
Insights
Inhibiting caspase-9 in chondrocytes alters cell metabolism and extracellular matrix, revealing non-apoptotic roles for caspases in cartilage health.
Area of Science:
- Cell Biology
- Biochemistry
- Proteomics
Background:
- Caspase-9 is key to the intrinsic apoptotic pathway.
- Caspase-9 inhibition in chondrocytes causes unexpected morphological changes unrelated to apoptosis.
Purpose of the Study:
- To investigate the molecular mechanisms behind caspase-9 inhibition's effects on chondrogenic cultures.
- To identify proteins affected by caspase-9 inhibition using advanced proteomics.
Main Methods:
- Proteomics analysis using Liquid Chromatography-Mass Spectrometry (LC-MS).
- Comparison of data-independent acquisition (DIA) methods, highlighting diaPASEF on a timsTOF Pro system.
- Quantitative analysis of protein expression changes (fold change and Q value).
Main Results:
- DiaPASEF identified 7849 protein groups, surpassing other DIA methods.
- Caspase-9 inhibition significantly altered 236 downregulated and 551 upregulated proteins.
- Affected proteins involved extracellular matrix, collagen metabolism, and metabolic pathways (shift from glycolysis to lipid metabolism).
- No significant changes observed in apoptosis-related proteins.
Conclusions:
- Caspase-9 plays a significant role in chondrocyte homeostasis beyond apoptosis.
- These findings suggest novel non-apoptotic functions of caspases in cartilage biology.
- Potential therapeutic implications for cartilage pathophysiology.
