The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under protein misfolding stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins, yet a global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, named Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge, we find that the global suppression of protein synthesis during UPR is dependent on subcellular localization, with more severe slowdown in lysosome vs. endoplasmic reticulum (ER) protein turnover. Most candidate translocation events affect pre-existing proteins and likely involve vesicular transport across endomembrane fractions including an expansion of an ER-derived vesicle (ERV) compartment containing RNA binding proteins and stress response proteins. In parallel, we observed specific translocations involving only newly synthesized protein pools that are indicative of endomembrane stalling. The translocation of a subclass of cell surface proteins to the endomembrane including EGFR and ITGAV upon UPR affects only heavy labeled proteins, which suggest their internalization is driven by nascent protein trafficking rather than ligand dependent endocytosis. The approach described here may be broadly useful for inferring the coordinations between spatial and temporal proteome regulations in normal and stressed cells.
Cellular senescence, induced by stress factors within a cell, is a state of cell growth arrest that has been found to contribute to age‐related diseases. Senescent cells exhibit a particular phenotype that is generically characterized by increased expression of cell‐cycle regulator proteins, increased expression of senescence associated beta‐galactosidase, and increased oxidative mitochondrial stress. Particularly in the cardiomyocyte, this state of growth arrest contributes to many prevalent cardiomyopathies. However, while it is broadly known that oxidative stress and DNA damage inducing conditions can contribute to a cell becoming senescent, methods to induce senescence in cardiomyocytes effectively have yet to be systematically investigated. In particular, the particular lesions and concentrations that can adequately induce senescence as well as the particular pathway through which senescence is induced in each induction method are unknown. Accordingly, the aim of this study is to optimizethe dosage and regimen of three pro‐senescence insults (hydrogen peroxide, doxorubicin, and UV‐C light) that will best induce senescence as measured by the associated senescent phenotype while gaining deeper understanding of differences in the pathways that are employed within a cell in the process of inducing senescence from different forms of stress. To do so, we used western blots and staining techniques in order to analyze relative expression of classic senescence markers in cells that were exposed to stress conditions as compared to healthy cells. The most relevant proteins that were compared were p16 and p21, cell cycle regulator proteins, and H2A.X, a double‐stranded DNA damage protein. We found that in conditions of 500 μM H2O2, 0.1 μM Dox, and 5 mJ/cm^2 UV‐C light, p21 and H2A.X were significantly upregulated and p16 was interestingly found to be significantly downregulated in all conditions. Treated samples were then filtered and tested via mass spectrometry in order to determine relative protein expressions. Preliminary mass spectrometry data used to compare the different insults showed some evidence of activation of different senescent pathways. Samples insulted with Dox showed an enrichment in the apoptotic pathway while those treated with UV‐C or H2O2 showed enrichment in translation elongation and nonsense mediated decay related pathways. Furthermore, we found that combinatorial treatment of H2O2, UV‐C light and doxorubicin increased expressed mitochondrial oxidative stress as seen by MitoSox staining techniques. In summary, our results showed the different concentrations that can induce senescent phenotypes in cardiomyocytes and the differences in the phenotypes through different senescence induction methods.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.