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Department of Basic Medical Sciences

Division of Protein Metabolism
タンパク質代謝制御分野
Professor
Yasushi Saeki, Ph.D.
Associate Professor Taeko Kobayash, Ph.D.
Assistant Professor Takuya Tomita, Ph.D.

教 授
准教授
助 教

博士（薬学）
博士（理学）
博士（薬科学）

佐
小冨伯林
田

妙拓泰子
哉

The ubiquitin-proteasome system and lysosomes are the major proteolytic
pathways and play a fundamental role in cellular protein homeostasis(proteostasis). Their dysregulation causes a plethora of diseases, including
neurodegenerative disorders, but the detailed pathogenic mechanisms remain
largely unknown. Our goal is to elucidate the basic molecular mechanisms
governing proteostasis-related diseases by analyzing the regulation of protein
metabolism, thereby providing the basis for therapeutic strategies.

1.  Generation and characterization of proteasomopathy model mice
Hikaru Tsuchiya1, Takuya Tomita, Hiromichi Yonekawa1, He Zhang, Taeko Kobayashi, Keiji Tanaka1,
Yasushi Saeki: 1Laboratory of Protein Metabolism,
Tokyo Metropolitan Institute of Medical Science
The proteasome plays a central role in many biological processes, including cell proliferation, transcriptional regulation, inflammation and proteostasis,by selectively degrading ubiquitinated proteins. Nevertheless, proteasome research at the whole-body level has lagged far behind because the proteasome isessential for the viability of all cells and knockout
mice of the constitutive subunits are embryonic lethal. Recently, heterozygous mutations in PSMD12, a19S subunit gene of the proteasome, have been identified in patients with developmental disorders and autism. Based on this finding, we generated three linesof heterozygous mutant mice lacking the 3’ end ofPsmd12 at different lengths. The most deficient Psmd12
mutant mice exhibited growth retardation, impaired
liver function, and diaphragmatic hernia. Further histological analysis revealed hepatocyte shedding andaccumulation of ubiquitinated substrates in the cerebellum. The other two lines of mutant mice had mild

or no phenotypes. Next, we performed a deep proteomic analysis of the liver and unexpectedly foundthat the mildest Psmd12 mutant mice showed thelargest proteome changes, with accumulation of proteins involved in mitochondrial dysfunction, DNAdamage, and oxidative stress responses. Thus, thesystemic proteasome mutant mice revealed that proteasome dysfunction exhibits diverse phenotypes, including impaired liver function, and that a slight decrease of the proteasome activity can trigger abnormalproteostasis.
2.  A quality control system to monitor the integrity of protein complex
Takuya Tomita, Masato Kanemaki2, Akinori Endo1,Hikaru Tsuchiya1, Keiji Tanaka1, Yasushi Saeki:
2
Department of Chromosome Science, National Institute of Genetics
Approximately half of all proteins form complexes to achieve functional expression in cells. The stoichiometry of the subunits that form a complex mustbe properly maintained, and failure of assembly ordisruption of this balance due to causes such as aneuploidy results in the accumulation of misassembled
intermediates and orphan subunits. Recently, multi-59
ple protein quality control mechanisms have been
discovered that target these incorrectly assembled
proteins for degradation. For example, the E3 ligases
HUWE1 and STUB1/CHIP are well established as
general quality control factors, and some E3 ligasesthat are dedicated to specific complexes have been
identified, such as UBE2O for ribosomal proteins,
HERC1 for proteasome intermediates and HERC2 for
CCT/TRiC chaperonin. However, it has not been described whether cells have quality control mechanisms that specifically monitors the integrity protein
complexes.
We have discovered a quality control system forthe disintegrated minichromosome maintenance(MCM) complex generated by depletion of a single
MCM subunit using auxin-inducible degron (AID)
system. Rapid depletion of MCM2-mAID destabilized the remaining MCM subunits, and we identified
the ubiquitin ligase CRL4 (DCAF X) for selective removal of MCM5 by the proteasome. Mechanistically,
the CRL4 substrate adaptor DCAF X recognized theburied region inside the MCM complex and ubiquitinated only after the disintegration. This mechanism
appeared to be distinct from the elimination pathwayof the orphan subunit during assembly. Thus, CRL4
(DCAF X) is an E3 ligase that monitors the integrity of
the MCM complex, thus providing insights into thesurveillance mechanisms of complex integrity.
3.  Liquid-to-solid phase transition of ubiquitylated proteins triggered by ATP depletion
Akinori Endo1, Chikage Takahashi1, Hikaru Tsuchiya1, Keiji Tanaka1, Yasushi Saeki
Ubiquitin-positive inclusions are a hallmark of almost all the neurodegenerative diseases such as
Alzheimer’s disease and amyotrophic lateral sclerosis. Dysregulation of ubiquitin-dependent phase separation has been implicated in the disease pathogenesis, but little is known about the molecular mechanisms
involved. We have shown that proteasomes undergo
liquid-liquid phase separation (LLPS) together withubiquitinated proteins in response to changes of in
the cellular environments (Yasuda et al, Nature 2020;
Iriki et al, Cell Rep 2023). Recently, we found that ATP
depletion also induces LLPS of the proteasome andubiquitinated proteins. The decrease in ATP levels
caused a massive remodeling of the ubiquitin proteome, especially in ATP-binding proteins and components of certain protein complexes, and condensation in a RAD23B- and UBQLN-dependent manner.
Restoration of ATP levels resolved this condensation,
which required p97 activity and proteasome deubiquitination activity, suggesting that the primary function of the condensates is sequestration of the ubiquitinated proteins rather than their degradation.
Interestingly, inhibition of p97 or prolonged ATP depletion resulted in insolubilization of the ubiquitinat-

ed protein, that is, a phase transition from theliquid-to-solid phase transition. Thus, ubiquitin-dependent LLPS contributes to the correction of impaired proteostasis upon ATP stress and may be involved in the formation of ubiquitin-positiveinclusions.
4. USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting ubiquitin signals fromendosomes
Akinori Endo1, Toshiaki Fukushima3, Chikage
Takahashi1, Hikaru Tsuchiya1, Fumiaki Ohtake4,
Sayaka Ono1, Tony Ly3, Yukiko Yoshida1, Keiji
Tanaka1, Yasushi Saeki, Masayuki Komada3: 3Cell
Biology Center, Institute of Innovative Research,Tokyo Institute of Technology, 4Institute for Advanced Life Sciences, Hoshi University
K63-linked ubiquitin chains attached to plasma
membrane proteins serve as tags for endocytosis andendosome-to-lysosome sorting. USP8 is an essential
deubiquitinase for the maintenance of endosomal
functions. Prolonged depletion of USP8 leads to cell
death, but the major effects on cellular signaling pathways are poorly understood. Here, we show thatUSP8 depletion causes aberrant accumulation of K63linked ubiquitin chains on endosomes and induces
immune and stress responses. Upon USP8 depletion,
two different decoders for K63-linked ubiquitin
chains, TAB2/3 and p62, were recruited to endosomes
and activated the TAK1–NF-κB and Keap1–Nrf2
pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin
chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results
demonstrate that USP8 is a gatekeeper of misdirectedubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin
chains from endosomes.
5.  Dynamic changes of lysosomal protein degradation in neural stem cells of the postnatal
mouse brain
He Zhang, Karan Ishii5, Tatsuya Shibata5, Shunsuke
Ishii6, Marika Hirao5, Zhou Lu, Risa Takamura5,Satsuki Kitano7, Hitoshi Miyachi7, Ryoichiro Kageyama8, Eisuke Itakura6 and Taeko Kobayashi:
5
Graduate School of Biostudies, Kyoto University,
6
Graduate School of Science, Chiba University, 7Institute for Life and Medical Sciences, Kyoto University, 8RIKEN Center for Brain Science
Lysosomes are intracellular organelles responsible for degrading diverse macromolecules deliveredfrom several pathways, including the endo-lysosomal
and autophagic pathways. Recent reports have sug-

60
gested that lysosomes are essential for regulatingneural stem cells in developing, adult, and aged
brains. However, the activity of these lysosomes has
yet to be monitored in these brain tissues. Here, wereport the development of a new probe to measurelysosomal protein degradation in brain tissue by immunostaining. Our results indicate that lysosomal
protein degradation fluctuates in neural stem cells of
the hippocampal dentate gyrus, depending on age
and brain disorders. Neural stem cells increase theirlysosomal activity during hippocampal developmentin the dentate gyrus, but aging and aging-related disease reduce lysosomal activity. In addition, physicalexercise increases lysosomal activity in neural stem
cells and astrocytes in the dentate gyrus. We therefore
propose that three different stages of lysosomal activity exist: the state of increase during development, thestable state during adulthood, and the state of reduction due to damage caused by either age or disease.

Publications
Akizuki, Y., Morita, M., Mori, Y., Kaiho-Soma, A.,
Dixit, S., Endo, A., Shimogawa, M., Hayashi, G.,Naito, M., Okamoto, A., Tanaka, K., Saeki, Y.,
Ohtake, F. cIAP1-based degraders induce degradation via branched ubiquitin architectures. Nat.
Chem. Biol., 19:311-322, 2023.
Iriki, T., Iio, H., Yasuda, S., Matsuta, S., Kato, M., Kosako, H., Hirayama, S., Endo, A., Ohtake, F., Kamiya, M., Urano, Y., Saeki, Y., Hamazaki, J., and Murata S. Senescent Cells form nuclear foci that containthe 26S proteasome. Cell Rep. 42:112880, 2023.
Zhang, H., Ishii, K., Shibata, T., Ishii, S., Hirao, M., Lu,Z., Takamura, R., Kitano, S., Miyachi, H., Kageyama, R., Itakura, E. and *Kobayashi, T. Fluctuation of lysosomal protein degradation in neuralstem cells of postnatal mouse brain. Development,in press.
*Endo, A., Fukushima, T., Takahashi, C., Tsuchiya, H.,Ohtake, F., Ono ,S., Ly, T., Yoshida, Y., Tanaka, K.,*Saeki, Y., and *Komada, M. USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting
ubiquitin signals from endosomes. J. Cell Biol., inpress.
Kobayashi, T. Protein homeostasis and degradationin quiescent neural stem cells. J. of Biochem., inpress.
佐伯泰、土屋光、遠藤彬則、冨田拓哉．ユビキチン化プロテオーム解析とユビキチン鎖絶対定量．羊土社．実験医学別冊「決定版 質量分析活用スタン
ダード」．213-224, 2023.
小林妙子．神経幹細胞におけるリソソーム機能．羊土社．実験医学．41(11): 1743-1747, 2023.