Page 3 - T. Protein modification and regulation

“Our findings indicate that the quality control of proteasomes is

essentially mediated by aggresomal sequestration and subsequent

autophagic degradation in mammalian cells.”

Quality Control of Mammalian Proteasome via Figure 1. Inactive proteasomes are accumulated in the aggresome Figure 2. Inactive proteasomes are transported to the aggresome through
HDAC6-mediated retrograde transportation
Autophagy
Won Hoon Choi, Tae-rim Oh and Min Jae Lee *
Department of Biochemistry and Molecular Biology,
Seoul National University College of Medicine
Abstract
The 26S proteasome is a self-compartmentized protease complex with its crucial
function in protein quality control. Multiple layers of regulatory systems have been
identified to elaborately modulate proteasome activity for hydrolysis of
polyubiquitinated proteins. However, the destruction mechanism of mammalian
proteasomes responding cellular environments has been relatively poorly understood.
Here, we describe that inactive 26S proteasomes are sequestered into the insoluble
aggresome, a large perinuclear inclusion, via histone deacetylase 6 (HDAC6)-mediated
retrograde transport. The proteasomes were colocalized with the autophagic receptor
p62/SQSTM1 and cleared through a selective macroautophagic process. Chemical and
genetic inhibitions of autophagy resulted in elevated levels of proteasomes in insoluble
fraction and more scattered puncta in cytoplasm, indicating that the proteaphagy is
biochemically linked to aggresomal segregation. When the cells were replenished with
inhibitor-free media, the aggresomal inclusion became gradually smaller and
disappeared. Structural changes, association of diverse proteins, and
polyubiquitination on different subunits appeared to be involved in the targeting Figure 3. Autophagic degradation of inactive proteasomes in the aggresome
mechanism of the inactive proteasome to the aggresome. These data indicate that
both aggresomal sequestration and autophagic clearance are the essential process of
the proteasome quality control to get rid of nonfunctional proteasomes.

Figure 4. Proteasome inhibitor treatment led to significant transcriptional upregulation of
proteasome subunits
Results
The inactive 26S proteasome can be sequestrated in the perinuclear aggresome (Figure
1) through retrograde transport mediated by the HDAC6-dynein complex (Figure 2). To
approve the fate of inactive proteasome accumulated in the aggresome, MG132
contained media was replenished with proteasome inhibitor-free media or autophagy
inhibitor contained media. Consequently, the proteasome-containing aggresome was
partially broken down and gradually reduced in size during the MG132 wash-out using Figure 6. Direct ubiquitination on inactive proteasome subunits
normal media. On the other hand, MG132 wash-out progression in autophagy
inhibition condition showed that the aggresome near the MTOC was split in part
(Figure 3). The long-term treatment of mild proteasome inhibitors increases the Figure 5. Proteasome interacting proteins in the presence of proteasome inhibitor
transcription of mRNAs not only proteasome subunits but also ubiquitin genes and
autophagy-related genes (Figure 4). Mass spectrometry analysis using purified
proteasomes showed that the inactive proteasomes had altered interaction with
various proteins (chaperones, Ub shuttle proteins, Ub-like proteins, proteasome
activators, proteasome adapters, etc.) (Figure 5). It also confirmed that the RP subunits
increased about 2-fold on average on treatment with MG132. Direct ubiquitination of
the inactive proteasome subunit seems to be the starting point of proteasome quality
control (Figure 6).
Discussion and conclusion
Aggresome is strongly associated with a wide variety of inclusion bodies formed by
overexpression, accumulation, and aggregation of proteins. In addition, in
neurodegenerative diseases, the co-localization of the inclusion bodies of pathological
proteins with ubiquitin and proteasome, has been reported in many studies.
Considering a number of reports of proteasome inhibition in proteopathy, we could
assume that the mechanism of proteasome quality control is closely related to the
formation of the inclusion body. The purpose of this study was to investigate the References
mechanism of proteasome quality control by maintaining, both, the quantitative and 1. Marshall, R. S., Li, F., Gemperline, D. C., Book, A. J. & Vierstra, R. D. Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in
active homeostasis of intracellular proteasomes. These observations will help us to Arabidopsis. Mol Cell 58, 1053-1066,doi:10.1016/j.molcel.2015.04.023 (2015).
establish a pathological causality relationship between various diseases and 2. Cohen-Kaplan, V. et al. p62- and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome. Proc Natl Acad Sci U S A 113, E7490-E7499,
doi:10.1073/pnas.1615455113 (2016).
proteasome activity and could be further developed into tailored treatment strategies
that depending on the condition and progression of the disease. Acknowledgement
This work was supported by grants from the National Research Foundation (2016R1A2B2006507 to M.J.L.), and the Creative-Pioneering Researchers Program through Seoul National University
(800-20160281to M.J.L.).
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