Publications by authors named "Yasushi Saeki"

88 Publications

RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells.

Genes Cells 2021 Feb 20. Epub 2021 Feb 20.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.

In eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC-CB-HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC-HLBs with CBs and nucleation of PCs near preformed CB-HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs.
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http://dx.doi.org/10.1111/gtc.12840DOI Listing
February 2021

TRIP12 promotes small-molecule-induced degradation through K29/K48-branched ubiquitin chains.

Mol Cell 2021 04 9;81(7):1411-1424.e7. Epub 2021 Feb 9.

Institute for Advanced Life Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan; School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan; Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. Electronic address:

Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2 substrate HIF-1α. TRIP12 associates with BRD4 via CRL2 and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.
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http://dx.doi.org/10.1016/j.molcel.2021.01.023DOI Listing
April 2021

MIND bomb 2 prevents RIPK1 kinase activity-dependent and -independent apoptosis through ubiquitylation of cFLIP.

Commun Biol 2021 Jan 19;4(1):80. Epub 2021 Jan 19.

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo, 143-8540, Japan.

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIP), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIP mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIP, respectively.
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http://dx.doi.org/10.1038/s42003-020-01603-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815719PMC
January 2021

Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome.

Sci Rep 2020 11 12;10(1):19669. Epub 2020 Nov 12.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan.

eIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.
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http://dx.doi.org/10.1038/s41598-020-76239-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661504PMC
November 2020

A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.

Commun Biol 2020 Oct 20;3(1):592. Epub 2020 Oct 20.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Kita 15, Nishi 7, Kita-Ku, Sapporo, Hokkaido, 060-8638, Japan.

The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.
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http://dx.doi.org/10.1038/s42003-020-01328-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576197PMC
October 2020

α-synuclein strains that cause distinct pathologies differentially inhibit proteasome.

Elife 2020 07 22;9. Epub 2020 Jul 22.

Dementia Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.

Abnormal α-synuclein aggregation has been implicated in several diseases and is known to spread in a prion-like manner. There is a relationship between protein aggregate structure (strain) and clinical phenotype in prion diseases, however, whether differences in the strains of α-synuclein aggregates account for the different pathologies remained unclear. Here, we generated two types of α-synuclein fibrils from identical monomer and investigated their seeding and propagation ability in mice and primary-cultured neurons. One α-synuclein fibril induced marked accumulation of phosphorylated α-synuclein and ubiquitinated protein aggregates, while the other did not, indicating the formation of α-synuclein two strains. Notably, the former α-synuclein strain inhibited proteasome activity and co-precipitated with 26S proteasome complex. Further examination indicated that structural differences in the C-terminal region of α-synuclein strains lead to different effects on proteasome activity. These results provide a possible molecular mechanism to account for the different pathologies induced by different α-synuclein strains.
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http://dx.doi.org/10.7554/eLife.56825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406352PMC
July 2020

Multi-Step Ubiquitin Decoding Mechanism for Proteasomal Degradation.

Pharmaceuticals (Basel) 2020 Jun 23;13(6). Epub 2020 Jun 23.

Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.

The 26S proteasome is a 2.5-MDa protease complex responsible for the selective and ATP-dependent degradation of ubiquitylated proteins in eukaryotic cells. Proteasome-mediated protein degradation accounts for ~70% of all cellular proteolysis under basal conditions, and thereby any dysfunction can lead to drastic changes in cell homeostasis. A major function of ubiquitylation is to target proteins for proteasomal degradation. Accompanied by deciphering the structural diversity of ubiquitin chains with eight linkages and chain lengths, the ubiquitin code for proteasomal degradation has been expanding beyond the best-characterized Lys48-linked ubiquitin chains. Whereas polyubiquitylated proteins can be directly recognized by the proteasome, in several cases, these proteins need to be extracted or segregated by the conserved ATPases associated with diverse cellular activities (AAA)-family ATPase p97/valosin-containing protein (VCP) complex and escorted to the proteasome by ubiquitin-like (UBL)-ubiquitin associated (UBA) proteins; these are called substrate-shuttling factors. Furthermore, proteasomes are highly mobile and are appropriately spatiotemporally regulated in response to different cellular environments and stresses. In this review, we highlight an emerging key link between p97, shuttling factors, and proteasome for efficient proteasomal degradation. We also present evidence that proteasome-containing nuclear foci form by liquid-liquid phase separation under acute hyperosmotic stress.
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http://dx.doi.org/10.3390/ph13060128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344625PMC
June 2020

The HOIL-1L ligase modulates immune signalling and cell death via monoubiquitination of LUBAC.

Nat Cell Biol 2020 06 11;22(6):663-673. Epub 2020 May 11.

Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.
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http://dx.doi.org/10.1038/s41556-020-0517-9DOI Listing
June 2020

Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses.

Commun Biol 2020 04 3;3(1):163. Epub 2020 Apr 3.

Department of Pathobiochemistry, Graduate School of Medicine, Osaka City University, Osaka, Japan.

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.
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http://dx.doi.org/10.1038/s42003-020-0882-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125101PMC
April 2020

RQT complex dissociates ribosomes collided on endogenous RQC substrate SDD1.

Nat Struct Mol Biol 2020 04 23;27(4):323-332. Epub 2020 Mar 23.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
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http://dx.doi.org/10.1038/s41594-020-0393-9DOI Listing
April 2020

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Nat Commun 2020 03 6;11(1):1222. Epub 2020 Mar 6.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
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http://dx.doi.org/10.1038/s41467-020-15006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060239PMC
March 2020

Stress- and ubiquitylation-dependent phase separation of the proteasome.

Nature 2020 02 5;578(7794):296-300. Epub 2020 Feb 5.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.

The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.
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http://dx.doi.org/10.1038/s41586-020-1982-9DOI Listing
February 2020

Structural insights into ubiquitin recognition and Ufd1 interaction of Npl4.

Nat Commun 2019 12 13;10(1):5708. Epub 2019 Dec 13.

Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, 113-0032, Japan.

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48-UN complex and its assembly.
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http://dx.doi.org/10.1038/s41467-019-13697-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910952PMC
December 2019

Stepwise multipolyubiquitination of p53 by the E6AP-E6 ubiquitin ligase complex.

J Biol Chem 2019 10 6;294(41):14860-14875. Epub 2019 Sep 6.

Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.

The human papillomavirus (HPV) oncoprotein E6 specifically binds to E6AP (E6-associated protein), a HECT (homologous to the E6AP C terminus)-type ubiquitin ligase, and directs its ligase activity toward the tumor suppressor p53. To examine the biochemical reaction , we established an efficient reconstitution system for the polyubiquitination of p53 by the E6AP-E6 complex. We demonstrate that E6AP-E6 formed a stable ternary complex with p53, which underwent extensive polyubiquitination when the isolated ternary complex was incubated with E1, E2, and ubiquitin. Mass spectrometry and biochemical analysis of the reaction products identified lysine residues as p53 ubiquitination sites. A p53 mutant with arginine substitutions of its 18 lysine residues was not ubiquitinated. Analysis of additional p53 mutants retaining only one or two intact ubiquitination sites revealed that chain elongation at each of these sites was limited to 5-6-mers. We also determined the size distribution of ubiquitin chains released by cleavage from polyubiquitinated p53 to be 2-6-mers. Taken together, these results strongly suggest that p53 is multipolyubiquitinated with short chains by E6AP-E6. In addition, analysis of growing chains provided strong evidence for step-by-step chain elongation. Thus, we hypothesize that p53 is polyubiquitinated in a stepwise manner through the back-and-forth movement of the C-lobe, and the permissive distance for the movement of the C-lobe restricts the length of the chains in the E6AP-E6-p53 ternary complex. Finally, we show that multipolyubiquitination at different sites provides a signal for proteasomal degradation.
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http://dx.doi.org/10.1074/jbc.RA119.008374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791319PMC
October 2019

Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE.

Methods Enzymol 2019 1;618:135-147. Epub 2019 Feb 1.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Electronic address:

Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).
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http://dx.doi.org/10.1016/bs.mie.2018.12.032DOI Listing
November 2019

Methods to measure ubiquitin chain length and linkage.

Methods Enzymol 2019 15;618:105-133. Epub 2019 Feb 15.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Electronic address:

To understand the biological roles of different ubiquitin chains, it is important to determine the types of ubiquitin linkages, the lengths of the polymers, and the combinations of ubiquitin chains attached to substrates. In this chapter, we describe a mass spectrometry-based quantification method of ubiquitin chains, named Ub-AQUA/PRM (ubiquitin-absolute quantification/parallel reaction monitoring), for direct and highly sensitive measurement of the stoichiometry of all eight ubiquitin-ubiquitin linkage types simultaneously. We also show a method to quantify the K48/K63 branched ubiquitin chain, a recently identified ubiquitin signal with a complex topology. In addition, we describe a method to measure ubiquitin chain length of ubiquitylated substrates using a chain protector and limited digestion of ubiquitin chains, named Ub-ProT (ubiquitin chain protection from trypsinization). These strategies will contribute to our knowledge of polymeric ubiquitin signals and permit investigation of new mechanisms concerning the ubiquitin code.
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http://dx.doi.org/10.1016/bs.mie.2018.12.019DOI Listing
November 2019

Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways.

EMBO J 2019 03 4;38(5). Epub 2019 Jan 4.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no-go decay) and the nascent polypeptide (RQC: ribosome-associated quality control). RQC requires Hel2-dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2-dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di-ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an translation reaction. Cryo-EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2-mediated cleavages upstream of the disome, governed by initial Not4-mediated monoubiquitination of eS7 and followed by Hel2-mediated K63-linked polyubiquitination. We propose that Hel2-mediated ribosome ubiquitination is required both for canonical NGD (NGD) and RQC coupled to the disome and that RQC-uncoupled NGD outside the disome (NGD) can occur in a Not4-dependent manner.
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http://dx.doi.org/10.15252/embj.2018100276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396155PMC
March 2019

Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice.

Mol Cell Biol 2019 01 11;39(1). Epub 2018 Dec 11.

Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified encoding the catalytic α' subunit of casein kinase II (CK2α') as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.
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http://dx.doi.org/10.1128/MCB.00426-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290375PMC
January 2019

HERC2 Facilitates BLM and WRN Helicase Complex Interaction with RPA to Suppress G-Quadruplex DNA.

Cancer Res 2018 11 2;78(22):6371-6385. Epub 2018 Oct 2.

Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.

BLM and WRN are RecQ DNA helicasesessential for genomic stability. Here, we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacted with BLM, WRN, and replication protein A (RPA) complexes during the S-phase of the cell cycle. Depletion of HERC2 dissociated RPA from BLM and WRN complexes and significantly increased G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4-suppressing function. , HERC2 released RPA onto single-stranded DNA (ssDNA) rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 inhibited ubiquitination of RPA2, caused RPA accumulation in the helicase complexes, and increased G4, indicating an essential role for E3 activity in the suppression of G4. Both depletion of HERC2 and inactivation of E3 sensitized cells to the G4-interacting compounds telomestatin and pyridostatin. Overall, these results indicate that HERC2 is a master regulator of G4 suppression that affects the sensitivity of cells to G4 stabilizers. Given that HERC2 expression is frequently reduced in many types of cancers, G4 accumulation as a result of HERC2 deficiency may provide a therapeutic target for G4 stabilizers. HERC2 is revealed as a master regulator of G-quadruplex, a DNA secondary structure that triggers genomic instability and may serve as a potential molecular target in cancer therapy. http://cancerres.aacrjournals.org/content/canres/78/22/6371/F1.large.jpg .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1877DOI Listing
November 2018

Deubiquitylases USP5 and USP13 are recruited to and regulate heat-induced stress granules through their deubiquitylating activities.

J Cell Sci 2018 04 12;131(8). Epub 2018 Apr 12.

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8501, Japan

Stress granules are transient cytoplasmic foci induced by various stresses that contain translation-stalled mRNAs and RNA-binding proteins. They are proposed to modulate mRNA translation and stress responses. Here, we show that the deubiquitylases USP5 and USP13 are recruited to heat-induced stress granules. Heat-induced stress granules also contained K48- and K63-linked ubiquitin chains. Depletion of USP5 or USP13 resulted in elevated ubiquitin chain levels and accelerated assembly of heat-induced stress granules, suggesting that these enzymes regulate the stability of the stress granules through their ubiquitin isopeptidase activity. Moreover, disassembly of heat-induced stress granules after returning the cells to normal temperatures was markedly repressed by individual depletion of USP5 or USP13. Finally, overexpression of a ubiquitin mutant lacking the C-terminal diglycine motif caused the accumulation of unanchored ubiquitin chains and the repression of the disassembly of heat-induced stress granules. As unanchored ubiquitin chains are preferred substrates for USP5, we suggest that USP5 regulates the assembly and disassembly of heat-induced stress granules by mediating the hydrolysis of unanchored ubiquitin chains while USP13 regulates stress granules through deubiquitylating protein-conjugated ubiquitin chains.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.210856DOI Listing
April 2018

Ub-ProT reveals global length and composition of protein ubiquitylation in cells.

Nat Commun 2018 02 6;9(1):524. Epub 2018 Feb 6.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.

Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.
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http://dx.doi.org/10.1038/s41467-018-02869-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802829PMC
February 2018

K63 ubiquitylation triggers proteasomal degradation by seeding branched ubiquitin chains.

Proc Natl Acad Sci U S A 2018 02 29;115(7):E1401-E1408. Epub 2018 Jan 29.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan

Different polyubiquitin chain linkages direct substrates toward distinct cellular pathways. K63-linked ubiquitylation is known to regulate proteasome-independent events such as signal transduction, but its function in the context of heterogeneous ubiquitin chains remains unclear. Here, we report that K63 ubiquitylation plays a critical role in proteasome-mediated substrate degradation by serving as a "seed" for K48/K63 branched ubiquitin chains. Quantitative analysis revealed that K48/K63 branched linkages preferentially associate with proteasomes in cells. We found that ITCH-dependent K63 ubiquitylation of the proapoptotic regulator TXNIP triggered subsequent assembly of K48/K63 branched chains by recruiting ubiquitin-interacting ligases such as UBR5, leading to TXNIP degradation. These results reveal a role for K63 chains as a substrate-specific mark for proteasomal degradation involved in regulating cell fate. Our findings provide insight into how cellular interpretation of the ubiquitin code is altered by combinations of ubiquitin linkages.
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http://dx.doi.org/10.1073/pnas.1716673115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816176PMC
February 2018

Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

Mol Cell 2017 Oct;68(2):350-360.e7

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Electronic address:

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.
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http://dx.doi.org/10.1016/j.molcel.2017.09.037DOI Listing
October 2017

Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30.

Nat Struct Mol Biol 2017 Nov 25;24(11):911-919. Epub 2017 Sep 25.

Structural Life Science Division, Synchrotron Radiation Research Organization, The University of Tokyo, Tokyo, Japan.

Parkin ubiquitin (Ub) ligase (also known as PARK2) ubiquitinates damaged mitochondria for their clearance and quality control. USP30 deubiquitinase opposes parkin-mediated Ub-chain formation on mitochondria by preferentially cleaving Lys6-linked Ub chains. Here, we report the crystal structure of zebrafish USP30 in complex with a Lys6-linked diubiquitin (diUb or Ub) at 1.87-Å resolution. The distal Ub-recognition mechanism of USP30 is similar to those of other USP family members, whereas Phe4 and Thr12 of the proximal Ub are recognized by a USP30-specific surface. Structure-based mutagenesis showed that the interface with the proximal Ub is critical for the specific cleavage of Lys6-linked Ub chains, together with the noncanonical catalytic triad composed of Cys-His-Ser. The structural findings presented here reveal a mechanism for Lys6-linkage-specific deubiquitination.
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http://dx.doi.org/10.1038/nsmb.3469DOI Listing
November 2017

Ubiquitination of stalled ribosome triggers ribosome-associated quality control.

Nat Commun 2017 07 31;8(1):159. Epub 2017 Jul 31.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan.

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.
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http://dx.doi.org/10.1038/s41467-017-00188-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5534433PMC
July 2017

Ubiquitination of exposed glycoproteins by SCF directs damaged lysosomes for autophagy.

Proc Natl Acad Sci U S A 2017 08 25;114(32):8574-8579. Epub 2017 Jul 25.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan;

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCF ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCF ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy.
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http://dx.doi.org/10.1073/pnas.1702615114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559013PMC
August 2017

Inhibitory effects of local anesthetics on the proteasome and their biological actions.

Sci Rep 2017 07 11;7(1):5079. Epub 2017 Jul 11.

Research Reactor Institute, Kyoto University, Kumatori, Osaka, Japan.

Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the β5 subunit of the 20S proteasome, and not to the other active subunits, β1 and β2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells.
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http://dx.doi.org/10.1038/s41598-017-04652-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506043PMC
July 2017

In Vivo Ubiquitin Linkage-type Analysis Reveals that the Cdc48-Rad23/Dsk2 Axis Contributes to K48-Linked Chain Specificity of the Proteasome.

Mol Cell 2017 May;66(4):488-502.e7

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo 156-8506, Japan. Electronic address:

Ubiquitin-binding domain (UBD) proteins regulate numerous cellular processes, but their specificities toward ubiquitin chain types in cells remain obscure. Here, we perform a quantitative proteomic analysis of ubiquitin linkage-type selectivity of 14 UBD proteins and the proteasome in yeast. We find that K48-linked chains are directed to proteasomal degradation through selectivity of the Cdc48 cofactor Npl4. Mutating Cdc48 results in decreased selectivity, and lacking Rad23/Dsk2 abolishes interactions between ubiquitylated substrates and the proteasome. Among them, only Npl4 has K48 chain specificity in vitro. Thus, the Cdc48 complex functions as a K48 linkage-specifying factor upstream of Rad23/Dsk2 for proteasomal degradation. On the other hand, K63 chains are utilized in endocytic pathways, whereas both K48 and K63 chains are found in the MVB and autophagic pathways. Collectively, our results provide an overall picture of the ubiquitin network via UBD proteins and identify the Cdc48-Rad23/Dsk2 axis as a major route to the proteasome.
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http://dx.doi.org/10.1016/j.molcel.2017.04.024DOI Listing
May 2017

HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains.

PLoS Pathog 2017 01 19;13(1):e1006162. Epub 2017 Jan 19.

Division of Cellular and Molecular Biology, Department of Cancer Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation.
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http://dx.doi.org/10.1371/journal.ppat.1006162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283754PMC
January 2017

Ubiquitin recognition by the proteasome.

Authors:
Yasushi Saeki

J Biochem 2017 Feb;161(2):113-124

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.

The 26S proteasome is a 2.5-MDa complex responsible for the selective, ATP-dependent degradation of ubiquitylated proteins in eukaryotic cells. Substrates in hundreds cellular pathways are timely ubiquitylated and converged to the proteasome by direct recognition or by multiple shuttle factors. Engagement of substrate protein triggers conformational changes of the proteasome, which drive substrate unfolding, deubiquitylation and translocation of substrates to proteolytic sites. Recent studies have challenged the previous paradigm that Lys48-linked tetraubiquitin is a minimal degradation signal: in addition, monoubiquitylation or multiple short ubiquitylations can serve as the targeting signal for proteasomal degradation. In this review, I highlight recent advances in our understanding of the proteasome structure, the ubiquitin topology in proteasome targeting, and the cellular factors that regulate proteasomal degradation.
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http://dx.doi.org/10.1093/jb/mvw091DOI Listing
February 2017