1Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; 2Institute for Genomic Statistics and Bioinformatics Bonn, Deutschland; 3Core Facility Analytical Proteomics Bonn, Deutschland
Introduction:
Endoplasmic reticulum stress and the resulting unfolded protein response (UPRER) significantly contribute to endothelial dysfunction and atherosclerosis. Similarly, the activation of the mitochondrial unfolded protein response (UPRMito) in endothelial dysfunction remains poorly understood. Our study focuses on utilizing multi-omics approaches to characterize UPRMito activation and regulation. This exploration aims to provide a comprehensive understanding of the molecular dynamics involved.
Methods and Results:
We utilized RNA sequencing and quantitative proteomic analysis to investigate the impact of stressors of mitochondrial (MitoBlock-6, CDDO) and endoplasmic (Tunicamycin) protein homeostasis on the transcriptome and proteome of Human coronary artery endothelial cells (HCAEC).
The analysis identified 12,745 unique transcripts and 7,891 unique proteins. Hierarchical clustering and heatmap analysis showcased reproducibility while emphasizing distinct expression changes among treatments. Compound-specific effects revealed that CDDO dysregulated 31.4% transcripts and 11.9% proteins, MitoBlock-6 altered 26.5% transcripts and 11.4% proteins, and Tunicamycin induced changes in 38.2% transcripts and 13.8% proteins.
Regarding mitochondrial influence, CDDO and Tunicamycin showed a lower percentage of regulated mitochondrial transcripts compared to overall transcripts. In contrast, MitoBlock-6 had a higher impact on mitochondrial transcripts than non-mitochondrial ones. Analysis of protein expression changes unveiled that 21 proteins were upregulated by both CDDO and MB, including essential mitochondrial chaperones (HSP10 and HSP60). Additionally, 142 proteins were commonly downregulated, affecting crucial biological processes such as mitochondrial translation, mitochondrial gene expression and ribosome biogenesis. Importantly, the molecular function that was most downregulated was NAD+ ADP-ribosyltransferase activity, involving PARP proteins with roles in DNA damage repair and anti-viral activity.
In general, the influence of mitochondrial stressors was particularly evident in altering mitochondrial protein expression, while changes in transcription applied to both mitochondrial and non-mitochondrial transcripts.
Quantitative single-gene PCR confirmed upregulation of mitochondrial chaperones. On a functional level, we found mitochondrial stressors to reduce cytosolic reactive oxygen species (DCFDA staining). This effect was abrogated after siRNA-mediated down-regulation of activating transcription factor 5 (ATF5), the putative main moderator of the mitochondrial UPR.
Conclusion:
In conclusion, our study of the mitochondrial unfolded protein response (UPRMito) in endothelial cells reveals diverse responses to stressors. Our findings lay the groundwork for future research, providing insights into modulating cellular stress responses for therapeutic benefit in endothelial dysfunction.