Structure of the HECT domain of human WWP2

WWP2 is a HECT-domain ubiquitin ligase of the Nedd4 family, which is involved in various important biological processes, such as protein degradation, membrane-protein sorting and transportation, the immune response, pluripotency of embryonic stem cells, tumourigenesis and metastasis. The HECT domain provides the intrinsic ubiquitin ligase activity of WWP2. Here, the expression, purification, crystallization and crystallographic analysis of the HECT domain of human WWP2 (HECTWWP2) are reported. HECTWWP2 has been crystallized and the crystals diffracted to 2.50 Å resolution. They belonged to space group P41212 and the structure has been solved via molecular replacement. The overall structure of HECTWWP2 has an inverted T-shape. This structure displays a high degree of conservation with previously published structures of Nedd4 subfamily members.

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Regulation of the cell cycle inhibitor p27 and its ubiquitin ligase Skp2 in differentiation of human embryonic stem cells

The FASEB Journal ◽

10.1096/fj.06-7758com ◽

2007 ◽

Vol 21 (11) ◽

pp. 2807-2817 ◽

Cited By ~ 40

Author(s):

Dana Egozi ◽

Maanit Shapira ◽

Galit Paor ◽

Ofer Ben‐Izhak ◽

Karl Skorecki ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Ubiquitin Ligase ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Cell Cycle Inhibitor

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Transplanting stem cells: Potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation

Advanced Drug Delivery Reviews ◽

10.1016/j.addr.2005.08.004 ◽

2005 ◽

Vol 57 (13) ◽

pp. 1944-1969 ◽

Cited By ~ 37

Author(s):

A BOYD ◽

Y HIGASHI ◽

K WOOD

Keyword(s):

Stem Cells ◽

Immune Response ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Embryonic Stem Cell ◽

Cell Transplantation ◽

Embryonic Stem ◽

Immune Attack

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Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens

Stem Cells International ◽

10.1155/2016/8905365 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 7

Author(s):

Gopu Sriram ◽

Vaishali Prakash Natu ◽

Intekhab Islam ◽

Xin Fu ◽

Chaminda Jayampath Seneviratne ◽

...

Keyword(s):

Stem Cells ◽

Immune Response ◽

Mesenchymal Stem Cells ◽

Innate Immune Response ◽

Embryonic Stem ◽

Innate Immune ◽

Microbial Pathogens ◽

Multipotent Stem Cells ◽

Cytokine Expression Profile ◽

The Impact

Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely,Porphyromonas gingivalisandAggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect ofA. actinomycetemcomitanson hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressedTLR-2andTLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib toA. actinomycetemcomitansserotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs.

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KDM2A Targets PFKFB3 for Ubiquitylation to Inhibit the Proliferation and Angiogenesis of Multiple Myeloma Cells

Frontiers in Oncology ◽

10.3389/fonc.2021.653788 ◽

2021 ◽

Vol 11 ◽

Author(s):

Xinling Liu ◽

Jiaqiu Li ◽

Zhanju Wang ◽

Jie Meng ◽

Aihong Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Ubiquitin Ligase ◽

Biological Processes ◽

Potential Mechanism ◽

Histone Demethylation ◽

Ubiquitin Ligase Activity ◽

Lysine Demethylase ◽

Worse Prognosis ◽

Demethylation Activity

The lysine demethylase KDM2A (also known as JHDM1A or FBXL11) demethylates histone H3 at lysine K36 which lead to epigenetic regulation of cell proliferation and tumorigenesis. However, many biological processes are mediated by KDM2A independently by its histone demethylation activity. In the present study, we aimed to characterize the functional significance of KDM2A in multiple myeloma (MM) disease progression. Specifically, we defined that one of the key enzymes of glycolysis PFKFB3 (6-phosphofructo-2-kinase) is ubiquitylated by KDM2A which suppresses MM cell proliferation. Previous study showed that KDM2A and PFKFB3 promoted angiogenesis in various tumor cells. We further reveal that KDM2A targets PFKFB3 for ubiquitination and degradation to inhibit angiogenesis. Several angiogenic cytokines are also downregulated in MM. Clinically, MM patients with low KDM2A and high PFKFB3 levels have shown worse prognosis. These results reveal a novel function of KDM2A through ubiquitin ligase activity by targeting PFKFB3 to induce proliferation, glycolysis and angiogenesis in MM cells. The data provides a new potential mechanism and strategy for MM treatment.

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FANCL Ubiquitinates Beta-Catenin and Enhances Its Nuclear Function

Blood ◽

10.1182/blood.v118.21.1335.1335 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1335-1335

Author(s):

Kim-Hien T. Dao ◽

Michael D. Rotelli ◽

Curtis L. Petersen ◽

Brie R. Brown ◽

Whitney D. Nelson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Beta Catenin ◽

Hematopoietic Stem ◽

Ubiquitin Ligase Activity ◽

Blood Stem Cells ◽

Cord Blood Stem Cells

Abstract Abstract 1335 Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss. A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct). Disclosures: No relevant conflicts of interest to declare.

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miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential

Journal of Experimental Medicine ◽

10.1084/jem.20171312 ◽

2018 ◽

Vol 215 (8) ◽

pp. 2115-2136 ◽

Cited By ~ 8

Author(s):

Sara E. Meyer ◽

David E. Muench ◽

Andrew M. Rogers ◽

Tess J. Newkold ◽

Emily Orr ◽

...

Keyword(s):

Stem Cells ◽

Ubiquitin Ligase ◽

Therapeutic Potential ◽

Embryonic Stem ◽

Leukemia Stem Cells ◽

Monocytic Differentiation ◽

Self Renewal ◽

Ubiquitin Ligase Complex ◽

Pharmacologic Inhibition

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell–like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2–containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.

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