The tumor suppressor archipelago E3 ligase is required for spermatid differentiation in Drosophila testis.
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ABSTRACT:
SUBMITTER: Vedelek V
PROVIDER: S-EPMC8055871 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The role of cullin E3-ubiquitin ligases for muscle homeostasis is best known during muscle atrophy, as the cullin-1 substrate adaptor atrogin-1 is among the most well-characterized muscle atrogins. We investigated whether cullin activity was also crucial during terminal myoblast differentiation and aggregation of acetylcholine receptors for the establishment of neuromuscular junctions in vitro. The activity of cullin E3-ligases is modulated through post-translational modification with the small ubiquitin-like modifier nedd8. Using either the Nae1 inhibitor MLN4924 (Pevonedistat) or siRNA against nedd8 in early or late stages of differentiation on C2C12 myoblasts, and primary satellite cells from mouse and human, we show that cullin E3-ligase activity is necessary for each step of the muscle cell differentiation program in vitro. We further investigate known transcriptional repressors for terminal muscle differentiation, namely ZBTB38, Bhlhe41, and Id1. Due to their identified roles for terminal muscle differentiation, we hypothesize that the accumulation of these potential cullin E3-ligase substrates may be partially responsible for the observed phenotype. MLN4924 is currently undergoing clinical trials in cancer patients, and our experiments highlight concerns on the homeostasis and regenerative capacity of muscles in these patients who often experience cachexia.
| S-EPMC5395100 | biostudies-literature
Project description:BackgroundUbiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin-proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown.ResultsWe identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133.ConclusionsOur studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.
| S-EPMC9277888 | biostudies-literature
Project description:BACKGROUND:Archipelago (Ago) is a Drosophila homolog of mammalian F-box and WD repeat domain-containing 7 (FBW7, also known as FBXW7). In previous studies, FBW7 has been addressed as a tumor suppressor mediating ubiquitin-dependent proteolysis of several oncogenic proteins. Ubiquitination is a type of protein modification that directs protein for degradation as well as sorting. The level of beta-catenin (?-cat), an intracellular signal transducer in Wnt signaling pathway, is reduced upon overexpression of FBW7 in human cancer cell lines. Loss of function mutations in FBW7 and overactive Wnt signaling have been reported to be responsible for human cancers. RESULTS:We found that Ago is important for the formation of shafts in chemosensory bristles at wing margin. This loss of shaft phenotype by knockdown of ago was rescued by knockdown of wingless (wg) whereas wing notching phenotype by knockdown of wg was rescued by knockdown of ago, establishing an antagonistic relationship between ago and wg. In line with this finding, knockdown of ago increased the level of Armadillo (Arm), a homolog of ?-cat, in Drosophila tissue. Furthermore, knockdown of ago increased the level of Distal-less (Dll) and extracellular Wg in wing discs. In S2 cells, the amount of secreted Wg was increased by knockdown of Ago but decreased by Ago overexpression. Therefore, Ago plays a previously unidentified role in the inhibition of Wg secretion. Ago-overexpressing clones in wing discs exhibited accumulation of Wg in endoplasmic reticulum (ER), suggesting that Ago prevents Wg protein from moving to Golgi from ER. CONCLUSIONS:We concluded that Ago plays dual roles in inhibiting Wg signaling. First, Ago decreases the level of Arm, by which Wg signaling is downregulated in Wg-responding cells. Second, Ago decreases the level of extracellular Wg by inhibiting movement of Wg from ER to Golgi in Wg-producing cells.
| S-EPMC7322864 | biostudies-literature
Project description:The large tumor suppressor 1 (LATS1) is a serine/threonine kinase and tumor suppressor found down-regulated in a broad spectrum of human cancers. LATS1 is a central player of the emerging Hippo-LATS suppressor pathway, which plays important roles in cell proliferation, apoptosis, and stem cell differentiation. Despite the ample data supporting a role for LATS1 in tumor suppression, how LATS1 is regulated at the molecular level remains largely unknown. In this study, we have identified Itch, a HECT class E3 ubiquitin ligase, as a unique binding partner of LATS1. Itch can complex with LATS1 both in vitro and in vivo through the PPxY motifs of LATS1 and the WW domains of Itch. Significantly, we found that overexpression of Itch promoted LATS1 degradation by polyubiquitination through the 26S proteasome pathway. On the other hand, knockdown of endogenous Itch by shRNAs provoked stabilization of endogenous LATS1 proteins. Finally, through several functional assays, we also revealed that change of Itch abundance alone is sufficient for altering LATS1-mediated downstream signaling, negative regulation of cell proliferation, and induction of apoptosis. Taking these data together, our study identifies E3 ubiquitin ligase Itch as a unique negative regulator of LATS1 and presents a possibility of targeting LATS1/Itch interaction as a therapeutic strategy in cancer.
| S-EPMC3064387 | biostudies-literature
Project description:Human Bre1, an E3 ligase for H2B monoubiquitination, binds p53 and enhances activator-dependent transcription. Ebp1, an ErbB3 receptor-binding protein, inhibits cell proliferation and acts as a tumor suppressor. Here, we show that hBre1 acts as an E3 ubiquitin ligase for Ebp1 tumor suppressor and promotes its polyubiquitination and degradation. Ebp1 is polyubiquitinated in cancer cells, which is regulated by its phosphorylation. We identified hBre1 acting as an E3 ligase for Ebp1 and increasing its polyubiquitination. Depletion of hBre1 blocks Ebp1's polyubiquitination and elevates its protein level, preventing cancer proliferation. hBre1 binds Ebp1 and suppresses its repressive effect on E2F-1. Moreover, Ebp1 protein level is substantially diminished in human cancers. It is robustly phosphorylated and localized in the nucleus of primary gliomas, correlating with hBre1 subcellular residency. Thus, hBre1 inhibits Ebp1's tumor suppressive activity through mediating its polyubiquitination and degradation.
| S-EPMC2633391 | biostudies-literature
Project description:Tumor suppressor p53 has a key role in maintaining genomic stability and preventing tumorigenesis through its regulation of cellular stress responses, including apoptosis, cell cycle arrest and senescence. To ensure its proper levels and functions in cells, p53 is tightly regulated mainly through post-translational modifications, such as ubiquitination. Here, we identified E3 ubiquitin ligase TRIM32 as a novel p53 target gene and negative regulator to regulate p53-mediated stress responses. In response to stress, such as DNA damage, p53 binds to the p53 responsive element in the promoter of the TRIM32 gene and transcriptionally induces the expression of TRIM32 in cells. In turn, TRIM32 interacts with p53 and promotes p53 degradation through ubiquitination. Thus, TRIM32 negatively regulates p53-mediated apoptosis, cell cycle arrest and senescence in response to stress. TRIM32 is frequently overexpressed in different types of human tumors. TRIM32 overexpression promotes cell oncogenic transformation and tumorigenesis in mice in a largely p53-dependent manner. Taken together, our results demonstrated that as a novel p53 target and a novel negative regulator for p53, TRIM32 has an important role in regulation of p53 and p53-mediated cellular stress responses. Furthermore, our results also revealed that impairing p53 function is a novel mechanism for TRIM32 in tumorigenesis.
| S-EPMC4211364 | biostudies-literature
Project description:FBXW7 is and E3 ubiquitin ligase and is highly mutated in colorectal cancer. We used human colon organoids with engineered FBXW7 hotspot mutations to investigate novel targets of E3 ligase activity with a combined transcriptomic and proteomic approach uncovering the EGFR-MAPK pathway as highly regulated by the E3 ligase activity.
2024-04-03 | GSE246896 | GEO
Project description:BackgroundThe Drosophila archipelago gene (ago) encodes the specificity component of a ubiquitin ligase that targets the cyclin E and dMyc proteins for degradation. Its human ortholog, Fbw7, is commonly lost in cancers, suggesting that failure to degrade ago/Fbw7 targets drives excess tissue growth.ResultsWe find that ago loss induces hyperplasia of some organs but paradoxically reduces the size of the adult eye. This reflects a requirement for ago to restrict apoptotic activity of the rbf1/de2f1 pathway adjacent to the eye-specific morphogenetic furrow (MF): ago mutant cells display elevated de2f1 activity, express the prodeath dE2f1 targets hid and rpr, and undergo high rates of apoptosis. These phenotypes are dependent on rbf1, de2f1, hid, and the rbf1/de2f1 regulators cyclin E and dacapo but are independent of dp53. A transactivation-deficient de2f1 allele blocks MF-associated apoptosis of ago mutant cells but does not retard their clonal overgrowth, indicating that intact de2f1 function is required for the death but not overproliferation of ago cells. Epidermal growth factor receptor (EGFR) and wingless (wg) alleles also modify the ago apoptotic phenotype, indicating that these pathways may modulate the underlying sensitivity of ago mutant cells to apoptotic signals.ConclusionsThese data show that ago loss requires a collaborating block in cell death to efficiently drive tissue overgrowth and that this conditional phenotype reflects a role for ago in restricting apoptotic output of the rbf1/de2f1 pathway. Moreover, the susceptibility of ago mutant cells to succumb to this apoptotic program appears to depend on local variations in extracellular signaling that could thus determine tissue-specific fates of ago mutant cells.
| S-EPMC2755617 | biostudies-literature
Project description:The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis. Recently, we reported that the ubiquitin E3 ligase ITCH negatively regulates LATS1, thereby increasing YAP activity, which leads to increased cell proliferation and decreased apoptosis. Here, we investigated the role of ITCH in breast tumorigenesis. In particular, we show that ITCH enhances epithelial-to-mesenchymal transition (EMT) through boosting YAP oncogenic function. By contrast, a point mutation in the catalytic domain or WW1 domain of ITCH abolished its EMT-mediated effects. Furthermore, while overexpression of ITCH expression in breast cells is associated with increased incidence of mammary tumor formation and progression, its knockdown inhibited breast cancer cell tumorigenicity and metastasis. Importantly, YAP knockdown was able to attenuate ITCH pro-tumorigenic functions. Lastly, we found that ITCH expression is significantly upregulated in invasive and metastatic breast cancer cases and is associated with worse survival. Together, our results reveal that ITCH pro-tumorigenic functions in breast cancer are mediated, at least in part, through inactivation of the Hippo tumor suppressor pathway.
| S-EPMC4279417 | biostudies-literature
Project description:Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing β-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of β-catenin by β-TRCP, ZNRF3 is ubiquitinated by β-TRCP in both CKI-phosphorylation- and degron-dependent manners. Thus, our findings not only identify a novel substrate for β-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by β-TRCP in a context-dependent manner where β-TRCP negatively regulates Wnt signaling by targeting β-catenin, and positively regulates Wnt signaling by targeting ZNRF3.
| S-EPMC6160385 | biostudies-literature