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- Site-specific O-GlcNAcylation of Psme3 maintains mouse stem cell pluripotency by impairing P-body homeostasis (Graduate School of Science and Engineering・Glycan and Life Systems Integration Center Director, Professor Shoko Nishihara)
2021/07/14
Site-specific O-GlcNAcylation of Psme3 maintains mouse stem cell pluripotency by impairing P-body homeostasis (Graduate School of Science and Engineering・Glycan and Life Systems Integration Center Director, Professor Shoko Nishihara)
Highlights
- Proteasome activator subunit 3 (Psme3) carries O-GlcNAc on Ser111 in mouse embryonic stem cells (mESCs).
- O-GlcNAc on Ser111 of Psme3 regulates processing bodies (P-bodies) homeostasis via DEAD box polypeptide 6 (Ddx6) degradation.
- Loss of O-GlcNAc on Ser111 of Psme3 primes mESCs for differentiation.
The research group of Professor Shoko Nishihara at Soka University (Director of the Glycan & Life System Integration Center, Soka University), in collaboration with Professor Kazuo Yamamoto's research group at the University of Tokyo, showed that O-GlcNAc sugar chain modification inhibits the formation of P-body, thus maintaining mESC pluripotency.
In recent years, it has become increasingly clear that glycans play an important role in determining the properties of stem cells. In this study, we revealed that O-GlcNAc of Ser111 on the proteasome activation subunit 3 (Psme3) is an important modification that maintains pluripotency in mouse embryonic stem cells (mESCs). O-GlcNAc on Ser111 of Psme3 promotes the degradation of DEAD box polypeptide 6 (Ddx6) required for P-body assembly, consequently reducing P-body amount. As a result, mRNAs encoding core transcription factors involved in the maintenance of pluripotency, such as Klf4 and Klf2, are translated and the pluripotency state is maintained. Conversely, when a mutation is introduced into Ser111 to inhibit O-GlcNAcylation of Psme3, Ddx6 is stabilized and the P body amount increases. Thus, the protein level of the pluripotent core transcription factor decreases, and mESCs are primed for differentiation. These findings establish O-GlcNAcylation at Ser111 of Psme3 as a critical switch that regulates mESC pluripotency via control of P-body homeostasis.
The results of this study were published in the scientific journal Cell Reports on July 13th at 11:00 AM (EST).Results Significance
Research Background
The pluripotency of mESCs is tightly controlled by a complex network of exogenous and endogenous factors. O-GlcNAc is the sole sugar chain modification found in cytoplasmic and nucleoproteins and plays a vital role in the regulation of basic cellular processes. O-GlcNAc is added to the protein Ser or Thr by O-GlcNAc transferase and removed by O-GlcNAcase. So far, several research groups, including our, have reported its functions in mESCs. However, due to the variety of O-GlcNAcylated proteins, the role of O-GlcNAc in the pluripotency network is still poorly understood. In the present study, we exploited the highly sensitive O-GlcNAc modification detection method developed by our collaborator Yamamoto et al. to identify O-GlcNAcylated proteins in mESCs and mouse epiblast-like cells (mEpiLCs). Interestingly, mEpiLCs, which are slightly more differentiated but retain their pluripotency, differed from mESCs in their pattern of O-GlcNAcylated proteins. Since the regulation of the proteasome by O-GlcNAc and the pluripotency network of mESCs was still unknown, we selected the proteasome activation subunit 3 (Psme3) from the detected proteins and proceeded with the analysis.
Psme3 interactome was analyzed by expressing a Psme3-FLAG tagged protein and immunoprecipitation followed by mass spectrometry. As a result, we observed that Psme3 interacts with DEAD box polypeptide 6 (Ddx6), Grbp2, Fxr1, Rpl7, etc... which have all been reported to interact with the P bodies. Overexpression of Psme3 resulted in degradation of Ddx6 and reduced P-bodies. Mass spectrometry and immunoprecipitation revealed that Ser111 of Psme3 was O-GlcNAcylated. Using a site-specific mutagenesis methodology, we introduced a mutation in the Ser111 and expressed in mESCs for analysis. In the Ser111 mutants, the O-GlcNAc modification and the interaction with Ddx6 was significantly reduced, indicating that the O-GlcNAc modification of Ser111 in Psme3 regulated the interaction with Ddx6 and its subsequent degradation. mESCs overexpressing Ser111 mutants, Ddx6 degradation was suppressed, the amount of P-body increased, and the protein level of pluripotent core transcription factors, such as Klf4 and Klf2 decreased, resulting in exit from the pluripotent state. These findings revealed that O-GlcNAc on Ser111 in Psme3 acts as a pivotal switch that regulates mESCs pluripotency through regulation of P-body homeostasis.Publication:Scientific journal “Cell reports 7/13 issue”
Title:Site-specific O-GlcNAcylation of Psme3 maintains mouse stem cell pluripotency by impairing P-body homeostasis
Authors: Federico Pecori, Nanako Kondo, Chika Ogura, Taichi Miura, Masahiko Kume, Youhei Minamijima, Kazuo Yamamoto, Shoko Nishihara