Periodic Reporting for period 4 - mitoUPR (Cellular modulation by the mitochondrial unfolded protein response)
Reporting period: 2023-08-01 to 2024-07-31
The mitochondrial unfolded protein response (UPRmt) activates upon protein misfolding in mitochondria. It is a highly conserved pathway that fulfills central quality control tasks in cells to maintain their function and health. In mammalian cells, the molecular events underlying UPRmt signaling and how it affects cells remain poorly understood. This has prevented a better understanding of the numerous diseases that lead to mitochondrial damage or dysfunction. Here, we aim to understand the results of UPRmt activation and its potential role in disease. We will characterize the effects of the UPRmt on the cellular environment and neighboring cells. In addition, we will examine the effect of mitochondrial stress and the UPRmt on mitochondrial structures that are important for the processing of RNAs in mitochondria. Ultimately, we will attempt to develop disease-relevant mutations in core quality control proteins within mitochondria to test their capacity to induce the UPRmt and to study the consequences. We have been able to address the different aims and have manuscripts prepared or published based on the outcomes of these objectives.
We carried out extensive analyses into the effects of the UPRmt on cellular function and towards neighboring cells. We established the required assays to monitor specific responses in the cytosol, mitochondrial and recipient cells and employed these to monitor effects of UPRmt activation. One key requirement was the development of a novel proteomics method that allows monitoring protein translation changes upon acute stress conditions. We developed such a method, termed mePROD, that includes a booster signal to specifically increase the signal of newly synthesized peptides/proteins to allow their detection and quantification. mePROD has been published and used extensively to study cellular stress responses including the UPRmt. We also employed this method to monitor cellular changes causes by SARS-CoV-2 infection. This novel method has a lot of potential and will allow introducing a novel layer of information (that is translation) to a wide range of biological questions. To better understand the UPRmt, we continue to use mePROD in combination with a variety of other methods. We determined how mitochondria communicate with their immediate environment and identified the molecular signaling mechanism of the UPRmt.
With the development of the mePROD method, we were able to monitor translation changes globally under stress conditions. We used this method extensively to characterize different cellular stress responses. Key outcomes of our work was to reveal that the integrated stress response and mTOR cause inhibition of a largely overlapping set of proteins and that the mRNA of proteins, whose translation is controlled by the integrated stress response and mTOR, contains some intrinsic factors that drive their sensitivity to these pathways. Importantly, these results revealed how cellular stress reshapes the mitochondrial proteome and proteostasis. We are further examined these results and identified how UPRmt activation leads to changes in the mitochondrial proteome via these pathways. To understand the direct effects on mitochondria, we defined the architecture, based on protein-protein interactions, of the RNA-processing structures within mitochondria. We gained extensive insight in these and revealed changes in these structures during stress and to define their contribution to proteostasis. In addition, identified signaling events of the UPRmt toneighboring cells and identified underlying mechanisms. Finally, we established new models for the UPRmt and are defining its role in different pathological conditions.