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Degradation and sequestration : cellular strategies to counteract proteotoxic stress

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posted on 2024-09-02, 23:45 authored by Shanshan Xu

Maintenance of proteome homeostasis (proteostasis) is essential to preserve cellular function in response to intrinsic and extrinsic stress conditions. This is regulated by the proteostasis network, which is comprised of machineries for protein synthesis, folding, sequestering, and degradation. The collaboration of these machineries is to ensure the functionality, subcellular localization and appropriate protein abundance, thereby preventing proteotoxic stress.

Disturbances in proteostasis can be caused by gene mutations, temperature fluctuations, alterations in synthesis or degradation, chemical insult, etc. If a disbalance in proteostasis is not addressed in a timely and correctly manner, aberrant proteins can accumulate and form insoluble aggregates, which are the hallmarks of the majority of neurodegenerative diseases and other so-called proteinopathies. Therefore, extending our knowledge and developing techniques to modulate the proteostasis network are important for the development of new therapeutic strategies for these disorders.

The work presented in this thesis describes how the proteostasis network responds to different proteotoxic stress conditions, and how its modulation preserves proteome integrity. In paper Ⅰ, we describe that the sequestration of aberrant, newly synthesized proteins in cellular stress granules prevent impairment of the ubiquitin-proteasome system in the nuclear compartment in response to thermal stress. In stress granuledeficient cells, these newly synthesized proteins passively diffuse into the nucleus instead of being sequestered in cytoplasmic granules. The newly synthesized proteins translocate to nucleoli in an HSP70-dependent manner. Under stress, HSP70 interacts with newly synthesized proteins to maintain their conformation. Our data suggest that heat shock factor 1 is released from HSP70, thereby prematurely activating the heat shock response while recovering from thermal stress. In line with a premature heat shock response, we found enhanced SUMO2/3-dependent degradation of aggregation-prone proteins, and impairs proteasomal degradation in the nuclear compartment.

In paper Ⅱ, we characterize the effect of the integrated stress response inhibitor ISRIB on the ubiquitin-proteasome system in response to thermal stress. During thermal stress, the integrated stress response is activated to inhibit protein translation, thereby preventing overloading of the proteostasis network with misfolded, newly synthesized proteins. However, ISRIB restores protein translation during stress, resulting in an increased amount of newly synthesized proteins. Part of the newly synthesized proteins under stress are dysfunctional and therefore polyubiquitinated targeted as substrates for proteasomal degradation. Meanwhile, we show that a large fraction of polyubiquitinated proteasome substrates converts to a detergent insoluble state. We propose that a limitation of ubiquitin availability results in the attenuation of ubiquitin proteasome system.

In paper Ⅲ, we studied the effect of a protein aggregation-preventing tag, the NT* domain, on an aggregation-prone protein. The NT* domain is a solubility tag derived from a spider silk protein. The fusion of this solubility tag with an aggregation-prone reporter protein prevented protein aggregation in mammalian cells in the cytosolic and nuclear compartments. This finding provides the possibility to reduce the burden if aggregation-prone proteins on proteostasis with natural anti-aggregation domains.

In paper Ⅳ, we characterized CBK79 as a novel proteostasis inhibitor that impairs both proteolytic pathways: the ubiquitin-proteasome system and autophagy. As a consequence of the proteostasis collapse caused by CBK79, the compound activates the heat shock response and induces aggresome formation. Intriguingly, preconditioning of cells by thermal stress relieves the negative effect of CBK79 on ubiquitin-proteasome system but not on autophagy.

List of scientific papers

I. Xu SS, Gierisch MG, Schellhaus AK, Poser I, Alberti S, Salomons FA, and Dantuma NP. Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment. [Manuscript]

II. Xu SS, Gierisch MG, Poser I, Alberti S, Salomons FA, and Dantuma NP. Chemical inhibition of the integrated stress response impairs the ubiquitin proteasome system. [Manuscript]

III. Schellhaus AK, Xu SS, Gierisch MG, Vornberger J, Johansson J and Dantuma NP. A spider silk-derived solubility domain inhibits nuclear and cytosolic protein aggregation in human cells. Communications Biology. 2022, 5:505.
https://doi.org/10.1038/s42003-022-03442-5

IV. Giovannucci TA, Salomons FA, Stoy H, Herzog LK, Xu SS, Qian WX, Merino LG, Gierisch MG, Haraldsson M, Lystad AH, Uvell H, Simonsen A, Gustavsson, Vallin M and Dantuma NP. Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy. Autophagy. 2022, 18: 1486-1502.
https://doi.org/10.1080/15548627.2021.1988359

History

Defence date

2022-10-28

Department

  • Department of Cell and Molecular Biology

Publisher/Institution

Karolinska Institutet

Main supervisor

Dantuma, Nico

Co-supervisors

Salomons, Florian; Farnebo, Marianne

Publication year

2022

Thesis type

  • Doctoral thesis

ISBN

978-91-8016-806-9

Number of supporting papers

4

Language

  • eng

Original publication date

2022-10-06

Author name in thesis

Xu, Shanshan

Original department name

Department of Cell and Molecular Biology

Place of publication

Stockholm

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