The E3 ubiquitin ligase TRIP12 participates in cell cycle progression and chromosome stability

TRIM36 is a member of the tripartite motif (TRIM) family of RING-containing proteins, also known as Haprin, which was first discovered for its abundance in testis and found to be implicated in the spermatozoa acrosome reaction. TRIM36 is a microtubule-associated E3 ubiquitin ligase that plays a role in cytoskeletal organization, and according to data gathered in different species, coordinates growth speed and stability, acting on the microtubules’ plus end, and impacting on cell cycle progression. TRIM36 is also crucial for early developmental processes, in Xenopus, where it is needed for dorso-ventral axis formation, but also in humans as bi-allelic mutations in the TRIM36 gene cause a form of severe neural tube closure defect, called anencephaly. Here, we review TRIM36-related mechanisms implicated in such composite physiological and pathological processes.

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The E3 ubiquitin ligase HECTD1 contributes to cell cycle progression through an effect on mitosis

10.1101/2021.12.17.473173 ◽

2021 ◽

Author(s):

Natalie Vaughan ◽

Nico Scholz ◽

Catherine Lindon ◽

Julien D, F Licchesi

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Time Lapse ◽

Ubiquitin Chain ◽

Cycle Progression ◽

Anaphase Onset ◽

Ubiquitin Ligase Activity ◽

Complex Protein

Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, has provided unique insights which have contributed to the emergence of the Ubiquitin code. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains less well understood. Similarly, the contribution of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains assembled by APC/c during mitosis, also remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell proliferation and we established that this is mediated through loss of its ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase). We confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. To explore the mechanisms involved, we used proteomics to explore the endogenous HECTD1 interactome in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity in mitosis.

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NEK7 is required for G1 progression and procentriole formation

Molecular Biology of the Cell ◽

10.1091/mbc.e16-09-0643 ◽

2017 ◽

Vol 28 (15) ◽

pp. 2123-2134 ◽

Cited By ~ 12

Author(s):

Akshari Gupta ◽

Yuki Tsuchiya ◽

Midori Ohta ◽

Gen Shiratsuchi ◽

Daiju Kitagawa

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Primary Cilia ◽

S Phase ◽

Cycle Progression ◽

Restriction Point ◽

Centriole Duplication ◽

Abnormal Accumulation ◽

Phase Entry

The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin–CDK complexes. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The NIMA-related kinase NEK7 is one of many factors required for proper centriole duplication, as well as for timely cell cycle progression. However, its specific roles in these events are poorly understood. In this study, we find that depletion of NEK7 inhibits progression through the G1 phase in human U2OS cells via down-regulation of various cyclins and CDKs and also inhibits the earliest stages of procentriole formation. Depletion of NEK7 also induces formation of primary cilia in human RPE1 cells, suggesting that NEK7 acts at least before the restriction point during G1. G1-arrested cells in the absence of NEK7 exhibit abnormal accumulation of the APC/C cofactor Cdh1 at the vicinity of centrioles. Furthermore, the ubiquitin ligase APC/CCdh1continuously degrades the centriolar protein STIL in these cells, thus inhibiting centriole assembly. Collectively our results demonstrate that NEK7 is involved in the timely regulation of G1 progression, S-phase entry, and procentriole formation.

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The human GID complex engages two independent modules for substrate recruitment

10.1101/2021.04.07.438752 ◽

2021 ◽

Author(s):

Matthias Peter ◽

Weaam I. Mohamed ◽

Sophia L. Park ◽

Julius Rabl ◽

Alexander Leitner ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Binding Sites ◽

Cell Cycle Progression ◽

Substrate Recognition ◽

E3 Ubiquitin Ligase ◽

Biological Processes ◽

Cycle Progression ◽

Cryo Electron Microscopy ◽

Biochemical Assays

The human GID (hGID) complex is an evolutionary conserved E3 ubiquitin ligase regulating diverse biological processes including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remains poorly understood. While the yeast GID complex recognizes Pro/N-end rule substrates via yeast Gid4, the human GID complex requires a WDR26/Gid7-dependent module to trigger proteasomal degradation of mammalian HBP1. Here, using biochemical assays, crosslinking-mass spectrometry and cryo-electron microscopy, we show that hGID unexpectedly engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 together with RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end rule degron. GID4 substrate binding and ligase activity is regulated by ARMC8 alpha, while the shorter ARMC8 beta; isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring-like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provide insights into the regulation of its ligase activity.

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DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

Cell Death and Disease ◽

10.1038/cddis.2017.222 ◽

2017 ◽

Vol 8 (5) ◽

pp. e2816-e2816 ◽

Cited By ~ 12

Author(s):

Valérie Glorian ◽

Jennifer Allègre ◽

Jean Berthelet ◽

Baptiste Dumetier ◽

Pierre-Marie Boutanquoi ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Ubiquitin Ligase ◽

Transcriptional Activation ◽

Cell Cycle Progression ◽

E3 Ubiquitin Ligase ◽

S Phase ◽

E2f Transcription Factor ◽

Additional Level ◽

Cell Proliferation Control

Abstract The E2F transcription factor 1 is subtly regulated along the cell cycle progression and in response to DNA damage by post-translational modifications. Here, we demonstrated that the E3-ubiquitin ligase cellular inhibitor of apoptosis 1 (cIAP1) increases E2F1 K63-poly-ubiquitination on the lysine residue 161/164 cluster, which is associated with the transcriptional factor stability and activity. Mutation of these lysine residues completely abrogates the binding of E2F1 to CCNE, TP73 and APAF1 promoters, thus inhibiting transcriptional activation of these genes and E2F1-mediated cell proliferation control. Importantly, E2F1 stabilization in response to etoposide-induced DNA damage or during the S phase of cell cycle, as revealed by cyclin A silencing, is associated with K63-poly-ubiquitinylation of E2F1 on lysine 161/164 residues and involves cIAP1. Our results reveal an additional level of regulation of the stability and the activity of E2F1 by a non-degradative K63-poly-ubiquitination and uncover a novel function for the E3-ubiquitin ligase cIAP1.

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Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

Blood ◽

10.1182/blood-2006-11-060350 ◽

2007 ◽

Vol 110 (5) ◽

pp. 1631-1638 ◽

Cited By ~ 71

Author(s):

Tint Lwin ◽

Lori A. Hazlehurst ◽

Sophie Dessureault ◽

Raymond Lai ◽

Wenlong Bai ◽

...

Keyword(s):

Cell Cycle ◽

Cell Adhesion ◽

Tumor Cell ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Response To Therapy ◽

G1 Arrest ◽

Down Regulation ◽

Cycle Progression ◽

Mantle Cell

Abstract Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1 arrest associated with elevated p27Kip1 and p21 (WAF1) proteins. The adhesion-mediated p27Kip1 and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2 ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1 and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1 signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

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Cell cycle oscillators underlying orderly proteolysis of E2F8

10.1101/672964 ◽

2019 ◽

Author(s):

Danit Wasserman ◽

Sapir Nachum ◽

Meital Cohen ◽

Taylor P Enrico ◽

Meirav Noach-Hirsh ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Dynamic Cell ◽

G2 Phase ◽

Differential Stability ◽

Complementary Set ◽

Cyclin F ◽

Transcription Factor E2f1

AbstractE2F8 is a transcriptional repressor that antagonizes the canonical cell cycle transcription factor E2F1. Despite the importance of this atypical E2F family member in cell cycle, apoptosis and cancer, we lack a complete description of the mechanisms that control its dynamics. To address this question, we developed a complementary set of static and dynamic cell-free systems of human origin, which recapitulate inter-mitotic and G1 phases, and a full transition from pro-metaphase to G1. This revealed an interlocking molecular switch controlling E2F8 degradation at mitotic exit, involving dephosphorylation of Cdk1 sites in E2F8 and the activation of APC/CCdh1, but not APC/CCdc20. Further, we revealed a differential stability of E2F8, accounting for its accumulation in late G1 while APC/CCdh1 is still active and suggesting a key role for APC/C in controlling G1-S transcription. Finally, we identified SCF-Cyclin F as the ubiquitin ligase controlling E2F8 in G2-phase. Altogether, our data provide new insights into the regulation of E2F8 throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in promoting orderly cell cycle progression.

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SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function

eLife ◽

10.7554/elife.24325 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 21

Author(s):

Kate E Coleman ◽

Miklós Békés ◽

Jessica R Chapman ◽

Sarah B Crist ◽

Mathew JK Jones ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Cycle Progression ◽

E3 Ligases ◽

Resistant Form ◽

Ligase Function ◽

Conjugating Enzyme

NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to accumulation of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels for CRL-dependent proteostasis.

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