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
