Quantitative analysis of the ubiquitin-proteasome system under proteolytic and folding stressors
Posted on: 27 March 2020 , updated on: 30 March 2020
Preprint posted on 22 November 2019
Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.201912041
Different types of stress require different responses: knowing the difference help cells adapt to changing environments
Selected by Sandra Malmgren Hill
Categories: cell biology, molecular biology
Context
Proteins are the building blocks of a living cell, and also responsible for carrying out all essential processes to ensure survival. Therefore, cells have evolved an intricate system to make sure that all proteins are properly produced, folded and maintained – thereby ensuring protein homeostasis, or “proteostasis”1. A faulty protein will lose its native structure and can be degraded, either by the ubiquitin-proteasome-system (UPS) or by lysosome-dependent autophagy, or rescued and refolded with the help of cellular chaperones to regain its original structure and function. If misfolded proteins are not resolved and begin to accumulate, they begin to coalesce and form larger insoluble aggregates. This process has been shown to be partly protective by spatially limiting potentially dangerous interactions of misfolded proteins2, but in the long-term this becomes detrimental as aggregates clog up the system.
The balance of proteostasis could be disrupted by intracellular or environmental factors that inhibit the capacity of the UPS to degrade proteins (proteolytic stress) and factors that increase protein misfolding (folding stress). A major regulator of proteasome activity in yeast is the transcription factor Rpn43. Upon proteotoxic stress, cells can increase proteolytic capacity by increasing transcriptional activity of Rpn4 which increases the expression of proteasomal genes by the so-called proteasome stress response (PSR).
In this preprint, the authors seek to investigate the how the cell responds to folding versus proteolytic stress to protects its proteome, how important the PSR is for different types of stress, and how different modes of responses might help cells adapt.
Approach
- To analyze the activity of the UPS, the authors usedba yeast model system expressing two different fluorescent reporters. The reporters contain a cluster of hydrophobic residues and a degron sequence targeting the protein for proteasomal degradation.
The GFP reporters Cyto-Deg (cytosolic) and ERm-Deg (localized to ER membrane) are expressed together with an mCherry reporter, so proteasome activity can be measured while still accounting for any changes in translation. - To induce proteolytic stress, the authors impair proteasomal degradation by treatment with Bortezomib.
- To induce protein misfolding, amino acid analogs Canavanine and AZC are used. The analogs get incorporated into newly translated proteins and destabilizes their structure.
- Rpn4 transcriptional activity (PSR) is measured using a GFP reporter gene fused to the Rpn4-targeted PACE (proteasome-associated control element) sequence.
Main findings
Impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) activates the PSR through separate mechanisms and leads to different strategies for adaptation of the UPS (Figure 1):
- Proteolytic stress induces PSR mainly by stabilizing Rpn4 protein levels, not by affecting its activity as a transcription factor.
- Folding stress induces SPR via aggregation rather than by straining the capacity of the proteasome. Aggregation sequesters cellular chaperones and induces PSR via an Hsf1-dependent increase in RPN4 expression.
- Adaption to proteolytic stress is near perfect at low doses of Bortezomib, and gradually declines with increasing concentrations, while the adaption to misfolding stress seems to be different for different substrates: Cyto-Deg degradation showed near-perfect adaption even with increasing doses of amino acid analogs, while degradation of ERm-Deg showed only partial adaption with a higher dose.
Figure 1; Model for how the UPS system can be modulated by the PSR. (Left) Proteolytic stressors increase the levels of proteasome substrates, including Rpn4 which activates PSR. (Right) Folding stress induces aggregation, which sequesters chaperones and activates the heatshock response via Hsf1. Hsf1 upregulates transcription of RPN4, leading to PSR activation. Reproduced from figure 6D of the preprint, made available under a CC-BY 4.0 International license.
Why this work is important
Proteostasis has been shown to be disrupted in several types of cancers and neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease. The disruption can come from either a congenital mutation, leading to expression of a faulty protein, or by a decline in the proteolytic and refolding capacity of the cell, induced by aging or environmental changes. Understanding how the cellular proteostasis system works is beneficial for us to understand what goes wrong, and how to potentially intervene in the context of disease. In this work, the authors highlight a diversity in the cellular response to different kinds of stress, indicating that the response to adapt and maintain proper protein degradation could be adapted for different substrates.
Open questions
- The authors studied the effects and adaptation of acutely induced proteotoxic stress. It will be interesting to see what pathways and adaptions occur in a physiological setting during prolonged stress, such as in the case of neurodegenerative diseases where there is a slow accumulation of misfolded proteins that eventually also hinders proteasomal degradation.
- Since the substrates that was used to measure proteasomal activity themselves are likely to misfold due to hydrophobic stretches, would this not result in a combined proteolytic and misfolding stress? Will the two types of stress not always go together, as decreased degradation leads to increased misfolding, and misfolding and aggregation eventually clogs the proteasomes, causing proteolytic stress4.
- What are the substrate characteristics that determine how well protein levels can be controlled, as in the case of different adaption of Cyro-Deg and Erm-Deg degradation: is it mainly a consequence of the peptide itself (protein size, fold and stability) or is it possible that it is regulated by toxicity of the protein and the interaction with different types of chaperones and/or proteasome-associated proteins?
- Is there a difference in response time to different types of stress, such that stabilizing Rpn4 levels by proteasomal inhibition might yield a faster response due to it being a more urgent stress compared to a misfolding stress?
- How are these adaptive systems linked to other protective systems of the cell, such as autophagy? In the scenario presented within this preprint, proteolytic stress is induced by inhibiting proteasomal function, which potentially would redirect many proteasomal substrates towards autophagic degradation. What role does autophagy play in the cells ability to adapt to proteolytic stress?
References
1 Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324-332, doi:10.1038/nature10317 (2011).
2 Hill, S. M., Hanzen, S. & Nystrom, T. Restricted access: spatial sequestration of damaged proteins during stress and aging. EMBO Rep 18, 377-391, doi:10.15252/embr.201643458 (2017).
3 Mannhaupt, G., Schnall, R., Karpov, V., Vetter, I. & Feldmann, H. Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast. FEBS letters 450, 27-34, doi:10.1016/s0014-5793(99)00467-6 (1999).
4 Andersson, V., Hanzen, S., Liu, B., Molin, M. & Nystrom, T. Enhancing protein disaggregation restores proteasome activity in aged cells. Aging (Albany NY) 5, 802-812, doi:10.18632/aging.100613 (2013).
doi: https://doi.org/10.1242/prelights.17898
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