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The COP9 signalosome (CSN) is a conserved protein complex, with CSN1 being one of the largest and most important subunits in the COP9 complex. To investigate the N-terminus function of OsCSN1, we edited the N-terminus of OsCSN1 and found that the mutant of OsCSN1 with 102 amino acids missing at the N-terminus showed insensitivity to red light in terms of the embryonic sheath, stem elongation, and main-root elongation. Moreover, the mutant was able to produce, develop, and bear fruit normally. The research results indicate that OsCSN1 is a negative regulator of stem elongation in rice seedlings regulated by red light. Under red-light treatment, OsCSN1 assembles into CSN, which degrades SLR1 through de NEDDylation, affecting PIL11 activity and ultimately inhibiting stem elongation. OsCSN1 also plays an important regulatory role in the inhibition of rice embryonic sheath elongation under red light. By regulating the degradation of SLR1 and PIL14 through the ubiquitin/26S protease pathway, the elongation of the embryonic sheath is ultimately inhibited. OsCSN1 forms a COP9 complex and is modified with RUB/NEDD8 of the E3 ligase of CUL1 to promote the degradation of SLR1 and PIL14, ultimately affecting the elongation of the embryonic sheath. The regulatory domain is located at the N-terminus of CSN1. [ABSTRACT FROM AUTHOR]

The COP9 signalosome (CSN) is a conserved protein complex, with CSN1 being one of the largest and most important subunits in the COP9 complex. To investigate the N-terminus function of OsCSN1, we edited the N-terminus of OsCSN1 and found that the mutant of OsCSN1 with 102 amino acids missing at the N-terminus showed insensitivity to red light in terms of the embryonic sheath, stem elongation, and main-root elongation. Moreover, the mutant was able to produce, develop, and bear fruit normally. The research results indicate that OsCSN1 is a negative regulator of stem elongation in rice seedlings regulated by red light. Under red-light treatment, OsCSN1 assembles into CSN, which degrades SLR1 through de NEDDylation, affecting PIL11 activity and ultimately inhibiting stem elongation. OsCSN1 also plays an important regulatory role in the inhibition of rice embryonic sheath elongation under red light. By regulating the degradation of SLR1 and PIL14 through the ubiquitin/26S protease pathway, the elongation of the embryonic sheath is ultimately inhibited. OsCSN1 forms a COP9 complex and is modified with RUB/NEDD8 of the E3 ligase of CUL1 to promote the degradation of SLR1 and PIL14, ultimately affecting the elongation of the embryonic sheath. The regulatory domain is located at the N-terminus of CSN1.

Background: Chronic changes caused by a high‐fat diet (HFD) may be associated with weakened lung function in obese patients. However, few studies have focused on the role of senescent cells in HFD‐induced pulmonary fibrosis. This study aimed to determine whether (i) obesity causes the accumulation of aging cells in the lungs, (ii) p16 accumulation in aging epithelial cells or fibroblasts exacerbates long‐term HFD‐induced senescence‐associated pulmonary fibrosis (SAPF) and (iii) p16 deletion or clearance of aging cells ameliorates HFD‐induced SAPF through inactivation of the inflammasome and metabolic remodelling. Methods: Twelve‐month old male mice of p16INK4a (hereafter p16) knockout (p16−/−) and wild‐type (WT), ApoE knockout (ApoE−/−) and ApoE−/−p16−/− were fed a HFD to induce obesity, and the effects of treatment with the senolytic drug ABT263 or the SGK1 specific inhibitor EMD638683 on fibrosis, inflammaging, gene expression, integrin‐inflammasome signalling and metabolism were examined. A549 and IMR‐90 cells were transduced with p16‐overexpressing adenovirus, and treated with palmitic and oleic acids (P&O) to induce steatosis in vitro. Results: We found that long‐term HFD promoted the expression of p16 and the increase of senescent cells in the lung. P16 knockout or ABT263 treatment alleviated pulmonary fibrosis, the increase of senescent cells and senescence‐associated secretory phenotype (SASP) in HFD‐fed mice, as well as in P&O‐treated A549 and IMR‐90 cells. RNA sequencing and bioinformatics analyses revealed that p16 knockout inhibited activation of the integrin‐inflammasome pathway and cellular glycolysis. Mass spectrometry, co‐immunoprecipitation and GST pull‐down assays demonstrated that p16 bound to the N‐terminal of SGK1, thereby interfering with the interaction between the E3 ubiquitin ligase NEDD4L and SGK1, and subsequently inhibiting K48‐polyubiquitin‐dependent degradation of SGK1 mediated by the NEDD4L–Ubch5 complex. EMD638683 was found to alleviate HFD‐induced pulmonary fibrosis and activation of the integrin‐inflammasome pathway. Conclusion: P16 accumulation promoted activation of integrin– inflammasome pathway and cell glycolysis by binding to the N– terminal of SGK1, intefering with the interaction between the E3 ubiquitin ligase NEDD4L and SGK1, thereby inhibiting K48– polyubiquitin– dependent degradation of SGK1 mediated by the NEDD4L–Ubch5 complex. ABT263 or EMD638683 could be used as potential drugs to treat pulmonary fibrosis in obese patients. [ABSTRACT FROM AUTHOR]

Abstract Background Preeclampsia (PE), a placenta-associated pregnancy complication, is the leading cause of maternal and perinatal morbidity and mortality. Met/Erk signaling is inhibited in the placentas of patients with early-onset preeclampsia (E-PE), but the underlying mechanisms remain elusive. In this study, the expression modes of Met and endocytic vesicles in normal and preeclamptic placentas were compared. Biotinylation internalization/recycling assays were used to measure the endocytosis of Met under hypoxia and normoxia in HTR8/SVneo cells. In addition, the expression level of Cbl, a specific E3 ligase of Met, was measured under hypoxia and normoxia, and the endocytosis of Met was studied by using confocal microscopy. Results We found considerable intracellular accumulation of Met, which was colocalized with caveolin-1 (CAV-1), in trophoblasts from E-PE placentas. Prolonged hypoxic stimulation led to the remarkable augmentation of CAV-1-mediated Met endocytosis in HTR8/SVneo cells. In addition, the expression of Cbl was substantially repressed by sustained hypoxia, disrupting ubiquitin degradation and the subsequent intracellular accumulation of Met in HTR8/SVneo cells. The abnormal degradation of Met hampered the ability of hepatocyte growth factor (HGF) to promote trophoblast cell invasion. In E-PE placentas, aberrant upregulation of CAV-1 and downregulation of Cbl were observed in parallel to the intracellular accumulation of Met. Conclusions These findings reveal that prolonged hypoxic stress induces the augmentation of endocytosis and repression of ubiquitin-mediated Met degradation, which leads to the impaired regulation of trophoblast invasion by HGF/Met signaling. These data provide novel evidence for elucidating the pathogenesis of preeclampsia, especially of the early-onset subtype.

The E3 ubiquitin ligase adaptor Speckle-type POZ protein (SPOP) plays an important tumour suppressor role in prostate cancers (PCa), with mutation rate up to 15%. However, how SPOP mutations regulate prostate tumorigenesis remains elusive. Here, we report the identification of cell division cycle associated 5 (CDCA5) as a SPOP substrate. We found that SPOP interacts with CDCA5 and promotes its polyubiquitin degradation in a degron-dependent manner. This effect was greatly impaired by introducing PCa associated SPOP mutations. Importantly, we found that CDCA5 was essential for PCa cells to survive and proliferate. CDCA5 depletion in PCa cells led to cessation of proliferation, G2M arrest, severe sister chromatid aggregation disturbance, and apoptosis. we also found that CDCA5 knockdown decreased the protein expression of p-GSK3β, increased the activity of caspase-3, caspase-9, and the Bax/Bcl-2 ratio. Besides, we confirmed that CDCA5 interrupted cancer cell behavior via the AKT pathway. In contrast, silencing SPOP or overexpressing CDCA5 increased cell proliferation. Consistently, depleting SPOP along with CDCA5, or overexpressing CDCA5 along with SPOP also caused the growth of cells repressed. Consistent with the functional role of CDCA5, the mRNA and protein levels of CDCA5 were significantly increased in PCa, compared to normal tissues, and its high expression was associated with more severe lymph node metastasis, higher Gleason score, and poorer prognosis. Together, our data showed that SPOP plays a crucial role in inhibiting tumorigenesis and partly achieved this by promoting the degradation of oncoprotein CDCA5.

Background: Preeclampsia (PE), a placenta-associated pregnancy complication, is the leading cause of maternal and perinatal morbidity and mortality. Met/Erk signaling is inhibited in the placentas of patients with early-onset preeclampsia (E-PE), but the underlying mechanisms remain elusive. In this study, the expression modes of Met and endocytic vesicles in normal and preeclamptic placentas were compared. Biotinylation internalization/recycling assays were used to measure the endocytosis of Met under hypoxia and normoxia in HTR8/SVneo cells. In addition, the expression level of Cbl, a specific E3 ligase of Met, was measured under hypoxia and normoxia, and the endocytosis of Met was studied by using confocal microscopy. Results: We found considerable intracellular accumulation of Met, which was colocalized with caveolin-1 (CAV-1), in trophoblasts from E-PE placentas. Prolonged hypoxic stimulation led to the remarkable augmentation of CAV-1-mediated Met endocytosis in HTR8/SVneo cells. In addition, the expression of Cbl was substantially repressed by sustained hypoxia, disrupting ubiquitin degradation and the subsequent intracellular accumulation of Met in HTR8/SVneo cells. The abnormal degradation of Met hampered the ability of hepatocyte growth factor (HGF) to promote trophoblast cell invasion. In E-PE placentas, aberrant upregulation of CAV-1 and downregulation of Cbl were observed in parallel to the intracellular accumulation of Met. Conclusions: These findings reveal that prolonged hypoxic stress induces the augmentation of endocytosis and repression of ubiquitin-mediated Met degradation, which leads to the impaired regulation of trophoblast invasion by HGF/Met signaling. These data provide novel evidence for elucidating the pathogenesis of preeclampsia, especially of the early-onset subtype. [ABSTRACT FROM AUTHOR]

A previous study on hepatoblastoma revealed novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex, including the tumor suppressor speckle-type BTB/POZ (SPOP). Moreover, the SPOP gene affected cell growth, and its S119N mutation was identified as a loss-of-function mutation in hepatoblastoma. This study aimed to explore more functions and the potential mechanism of SPOP and its S119N mutation. The in vitro effects of SPOP on cell proliferation, invasion, apoptosis, and in vivo tumor growth were investigated by western blot analysis, Cell Counting Kit-8, colony formation assay, flow cytometry, and xenograft animal experiments. The substrate of SPOP was discovered by a protein quantification assay and quantitative ubiquitination modification assay. The present study further proved that SPOP functioned as an anti-oncogene through the phosphatidylinositol 3-kinase/Akt signaling pathway to affect various malignant biological behaviors of hepatoblastoma both in vitro and in vivo. Furthermore, experimental results also suggested that solute carrier family 7 member 1 (SLC7A1) might be a substrate of SPOP and influence cell phenotype by regulating arginine metabolism. In conclusion, these findings demonstrated the function of SPOP and revealed a potential substrate related to hepatoblastoma tumorigenesis, which might thus provide a novel therapeutic target for hepatoblastoma.

The E3 ubiquitin ligase adaptor Speckle-type POZ protein (SPOP) plays an important tumour suppressor role in prostate cancers (PCa), with mutation rate up to 15%. However, how SPOP mutations regulate prostate tumorigenesis remains elusive. Here, we report the identification of cell division cycle associated 5 (CDCA5) as a SPOP substrate. We found that SPOP interacts with CDCA5 and promotes its polyubiquitin degradation in a degron-dependent manner. This effect was greatly impaired by introducing PCa associated SPOP mutations. Importantly, we found that CDCA5 was essential for PCa cells to survive and proliferate. CDCA5 depletion in PCa cells led to cessation of proliferation, G2M arrest, severe sister chromatid aggregation disturbance, and apoptosis. we also found that CDCA5 knockdown decreased the protein expression of p-GSK3β, increased the activity of caspase-3, caspase-9, and the Bax/Bcl-2 ratio. Besides, we confirmed that CDCA5 interrupted cancer cell behavior via the AKT pathway. In contrast, silencing SPOP or overexpressing CDCA5 increased cell proliferation. Consistently, depleting SPOP along with CDCA5, or overexpressing CDCA5 along with SPOP also caused the growth of cells repressed. Consistent with the functional role of CDCA5, the mRNA and protein levels of CDCA5 were significantly increased in PCa, compared to normal tissues, and its high expression was associated with more severe lymph node metastasis, higher Gleason score, and poorer prognosis. Together, our data showed that SPOP plays a crucial role in inhibiting tumorigenesis and partly achieved this by promoting the degradation of oncoprotein CDCA5. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an invasive human fungal pathogen that causes more than 181,000 deaths each year. Studies have demonstrated that pulmonary C. neoformans infection induces innate immune responses involving copper, and copper detoxification in C. neoformans improves its fitness and pathogenicity during pulmonary C. neoformans infection. However, the molecular mechanism by which copper inhibits C. neoformans proliferation is unclear. We used a metallothionein double-knockout C. neoformans mutant that was highly sensitive to copper to demonstrate that exogenous copper ions inhibit fungal cell growth by inducing reactive oxygen species generation. Using liquid chromatography-tandem mass spectrometry, we found that copper down-regulated factors involved in protein translation, but up-regulated proteins involved in ubiquitin-mediated protein degradation. We propose that the down-regulation of protein synthesis and the up-regulation of protein degradation are the main effects of copper toxicity. The ubiquitin modification of total protein and proteasome activity were promoted under copper stress, and inhibition of the proteasome pathway alleviated copper toxicity. Our proteomic analysis sheds new light on the antifungal mechanisms of copper. [ABSTRACT FROM AUTHOR]

Zygotic genomic activation (ZGA) is a landmark event in the maternal-to-zygotic transition (MZT), and the regulation of ZGA by maternal factors remains to be elucidated. In this study, the depletion of maternal ring finger protein 114 (RNF114), a ubiquitin E3 ligase, led to developmental arrest of two-cell mouse embryos. Using immunofluorescence and transcriptome analysis, RNF114 was proven to play a crucial role in major ZGA. To study the underlying mechanism, we performed protein profiling in mature oocytes and found a potential substrate for RNF114, chromobox 5 (CBX5), ubiquitylation and degradation of which was regulated by RNF114. The overexpression of CBX5 prevented embryonic development and impeded major ZGA. Furthermore, TAB1 was abnormally accumulated in mutant two-cell embryos, which was consistent with the result of in vitro knockdown of Rnf114. Knockdown of Cbx5 or Tab1 in maternal RNF114-depleted embryos partially rescued developmental arrest and the defect of major ZGA. In summary, our study reveals that maternal RNF114 plays a precise role in degrading some important substrates during the MZT, the misregulation of which may impede the appropriate activation of major ZGA in mouse embryos. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an invasive human fungal pathogen that causes more than 181,000 deaths each year. Studies have demonstrated that pulmonary C. neoformans infection induces innate immune responses involving copper, and copper detoxification in C. neoformans improves its fitness and pathogenicity during pulmonary C. neoformans infection. However, the molecular mechanism by which copper inhibits C. neoformans proliferation is unclear. We used a metallothionein double-knockout C. neoformans mutant that was highly sensitive to copper to demonstrate that exogenous copper ions inhibit fungal cell growth by inducing reactive oxygen species generation. Using liquid chromatography-tandem mass spectrometry, we found that copper down-regulated factors involved in protein translation, but up-regulated proteins involved in ubiquitin-mediated protein degradation. We propose that the down-regulation of protein synthesis and the up-regulation of protein degradation are the main effects of copper toxicity. The ubiquitin modification of total protein and proteasome activity were promoted under copper stress, and inhibition of the proteasome pathway alleviated copper toxicity. Our proteomic analysis sheds new light on the antifungal mechanisms of copper.

Familial hyperkalemic hypertension (FHHt; also called pseudohypoaldosteronism type II) is a hereditary hypertensive disease which can be caused by mutations in four genes: WNK1 [with no lysine (K) 1], WNK4, Kelch-like3 (KLHL3), and cullin3 (CUL3). Decreased KLHL3 expression was identified as being involved in the pathogenesis of FHHt caused by cullin 3 disease mutations. Recent studies have revealed an increased WNK4 and hence Na-Cl cotransporter (NCC) activity in the db/db mice, resulting from PKC-mediated KLHL3 phosphorylation, which impairs the degradation of its substrate, WNK4. However, whether WNK4 and NCC were activated in type 1 diabetes still remains unclear. We created streptozotocin-induced type 1 diabetic mice and revealed that renal WNK-oxidative stress response kinase-1/STE20/SPS1-related proline alanine–rich kinase (OSR1/SPAK)-NCC cascade was activated, whereas KLHL3 expression was markedly decreased and CUL3 was heavily neddylated. Moreover, decreased KLHL3 was reversed and WNK1 and WNK4 abundance increased by MLN4924, a neddylation inhibitor. In vitro, our study also showed decreased KLHL3 abundance without any significant change in phosphorylated KLHL3 under high glucose exposure. These results indicate that decreased KLHL3 likely plays a role in the pathogenesis of renal sodium reabsorption in hyperglycemic conditions. [ABSTRACT FROM AUTHOR]

Pituitary adenoma (PA) is one of the most common intracranial tumors, and approximately 40% of all PAs are prolactinomas. Dopamine agonists (DAs), such as cabergoline (CAB), have been successfully used in the treatment of prolactinomas. The expression of dopamine type 2 receptor (DRD2) determines the therapeutic effect of DAs, but the molecular mechanisms of DRD2 regulation are not fully understood. In this study, we first demonstrated that DRD2 underwent proteasome-mediated degradation. We further employed the yeast two-hybrid system and identified kelch repeat and BTB (POZ) domain containing 7 (KBTBD7), a substrate adaptor for the CUL3-RING ubiquitin (Ub) ligase complex, as a DRD2-interacting protein. KBTBD6/7 directly interacted with, and ubiquitinated DRD2 at five ubiquitination sites (K221, K226, K241, K251, and K258). CAB, a high-affinity DRD2 agonist, induced DRD2 internalization, and cytoplasmic DRD2 was degraded via ubiquitination under the control of KBTBD6/7, the activity of which attenuated CAB-mediated inhibition of the AKT/mTOR pathway. KBTBD7 knockout (KO) mice were generated using the CRISPR-Cas9 technique, in which the static level of DRD2 protein was elevated in the pituitary gland, thalamus, and heart, compared to that of WT mice. Consistently, the expression of KBTBD6/7 was negatively correlated with that of DRD2 in human pituitary tumors. Moreover, KBTBD7 was highly expressed in dopamine-resistant prolactinomas, but at low levels in dopamine-sensitive prolactinomas. Knockdown of KBTBD6/7 sensitized MMQ cells and primary pituitary tumor cells to CAB treatment. Conversely, KBTBD7 overexpression increased CAB resistance of estrogen-induced in situ rat prolactinoma model. Together, our findings have uncovered the novel mechanism of DRD2 protein degradation and shown that the KBTBD6/7-DRD2 axis regulates PA sensitivity to DA treatment. KBTBD6/7 may thus become a promising therapeutic target for pituitary tumors. [ABSTRACT FROM AUTHOR]

F-box proteins are critical components of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases and involved in the ubiquitin-dependent proteolytic pathway. Dysregulation of F-box protein-mediated proteolysis often leads to human malignancies. F-box only protein 8 (FBX8), a novel component of F-box proteins, is down-regulated in several tumors and closely correlates with tumor progression. However, little is known about its function, regulatory mechanisms and substrates in the progression of colorectal carcinoma (CRC). Combining microRNA (miRNA) assay, functional characterization, mechanistic studies with clinical validation, we identify FBX8 as a CRC metastasis suppressor downstream of miR-223, a metastasis promoting miRNA that is transcriptionally regulated by Myocyte enhancer factor (MEF2A). mTOR is a substrate of FBX8 for ubiquitin-mediated degradation and is required for FBX8 induced cell proliferation and invasion in CRC cells. FBX8 is down-regulated in human CRC tissues and correlates with MEF2A, miR-223 and mTOR expression levels. Notably, low FBX8 expression status in CRC tissues was a significant prognostic factor for poor overall survival of patients. These findings illustrate FBX8 as a metastasis suppressor that functions through mTOR signaling pathway and has significant prognostic power. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is clinically challenging due to the development of distant metastasis following initial therapy. Therefore, it is necessary to elucidate the mechanisms underlying metastases to develop novel therapeutic strategies. Nucleophosmin 1 (NPM1) has been directly linked to the development of human tumors and may have both tumor-suppressing and oncogenic properties. Although NPM1 is often overexpressed in solid tumors of various histopathological origins, its specific function in mediating the development of NPC is still unknown. Here, we investigated the role of NPM1 in NPC and discovered that NPM1 was elevated in clinical NPC samples and served as a predictor of the worst prognosis in NPC patients. Furthermore, the upregulation of NPM1 promoted the migration and the cancer stemness of NPC both in vitro and in vivo. Mechanistic analyses revealed that the E3 ubiquitin ligase Mdm2 was recruited by NPM1 to induce the ubiquitination-mediated proteasomal degradation of p53. Ultimately, knockdown of NPM1 suppressed the stemness and EMT signals. In summation, this study demonstrated the role and the underlying molecular mechanism of NPM1 in NPC, providing the evidence for the clinical application of NPM1 as a therapeutic target for the treatment of patients with NPC., Competing Interests: None., (AJCR Copyright © 2023.)

Gastric cancer is a type of serious malignant tumors all around the world. TCGA data showed that the expression of TRIM65 (E3 ubiquitin ligase) was enhanced in the gastric cancer tissues. The role of TRIM65 in the tumorigenesis of gastric cancer remains unclear. In this study, we successfully established TRIM65-knockdown gastric cancer cells. Next, CCK-8, colony formation assays and transwell assays were performed to detect the cell proliferation and invasion. The results showed that suppression of TRIM65 inhibited the proliferation and invasion of gastric cancer cells. Interestingly, the Western blot assay confirmed that downregulation of TRIM65 increased the level of PPM1A and decreased the level of p -TBK1 in gastric cancer cells. Mechanistically, immunoprecipitation assay revealed that knockdown of TRIM65 inhibited the ubiquitin degradation of PPM1A. In rescue experiments, suppression of PPM1A promoted the proliferation and invasion of gastric cancer cells transfected with sh-TRIM65. Therefore, our results suggested that knockdown of TRIM65 inhibited the proliferation and invasion of gastric cancer cells by suppressing the ubiquitin degradation of PPM1A and phosphorylation of TBK1. [ABSTRACT FROM AUTHOR]

CCDC65 is a member of the coiled-coil domain-containing protein family and was only reported in gastric cancer by our group. We first observed that it is downregulated in lung adenocarcinoma based on the TCGA database. Reduced CCDC65 protein was shown as an unfavorable factor promoting the clinical progression in lung adenocarcinoma. Subsequently, CCDC65 -/- mice were found possibly dead of hydrocephalus. Compared with the CCDC65 +/+ mice, the downregulation of CCDC65 in CCDC65 +/- mice significantly increased the formation ability of lung cancer induced by urethane. In the subsequent investigation, we observed that CCDC65 functions as a tumor suppressor repressing cell proliferation in vitro and in vivo . Molecular mechanism showed that CCDC65 recruited E3 ubiquitin ligase FBXW7 to induce the ubiquitination degradation of c-Myc, an oncogenic transcription factor in tumors, and reduced c-Myc binding to ENO1 promoter, which suppressed the transcription of ENO1. In addition, CCDC65 also recruited FBXW7 to degrade ENO1 protein by ubiquitinated modulation. The downregulated ENO1 further reduced the phosphorylation activation of AKT1, which thus inactivated the cell cycle signal. Our data demonstrated that CCDC65 is a potential tumor suppressor by recruiting FBWX7 to suppress c-Myc/ENO1-induced cell cycle signal in lung adenocarcinoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Zygotic genomic activation (ZGA) is a landmark event in the maternal-to-zygotic transition (MZT), and the regulation of ZGA by maternal factors remains to be elucidated. In this study, the depletion of maternal ring finger protein 114 (RNF114), a ubiquitin E3 ligase, led to developmental arrest of two-cell mouse embryos. Using immunofluorescence and transcriptome analysis, RNF114 was proven to play a crucial role in major ZGA. To study the underlying mechanism, we performed protein profiling in mature oocytes and found a potential substrate for RNF114, chromobox 5 (CBX5), ubiquitylation and degradation of which was regulated by RNF114. The overexpression of CBX5 prevented embryonic development and impeded major ZGA. Furthermore, TAB1 was abnormally accumulated in mutant two-cell embryos, which was consistent with the result of in vitro knockdown of Rnf114. Knockdown of Cbx5 or Tab1 in maternal RNF114-depleted embryos partially rescued developmental arrest and the defect of major ZGA. In summary, our study reveals that maternal RNF114 plays a precise role in degrading some important substrates during the MZT, the misregulation of which may impede the appropriate activation of major ZGA in mouse embryos., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background: Caffeic acid 3,4-dihydroxyphenethyl ester (CADPE) is a natural polyphenolic ester isolated as a minor component from a water extract of the Chinese medicine Zhongjiefeng [Sarcandra glabra (Thunb.) Nakai (Chloranthaceae)] and has previously shown to have activity against solid tumors through the modulation of multiple targets or signal pathways. However, the activity and potential mechanism of CADPE against leukemia cells have not yet been characterized.Purpose: To investigate whether and how CADPE kills leukemia cells.Method: (1) The activity of CADPE inhibiting the growth of different leukemia cell lines was evaluated by MTT assay; (2) Cell cycle arrest and apoptosis induced by CADPE were determined by flow cytometry with FlowJo software for quantification; (3) The protein levels were analyzed by Western blot and ubiquitin-binding c-Myc was acquired by co-immunoprecipitation.Results: CADPE exerted potent activity against different leukemia cell lines with low toxicity in normal cells. In terms of mechanism of action, CADPE promoted ubiquitin-proteasome-dependent degradation of c-Myc through activating glycogen synthase kinase-3β (GSK3β) and downregulating deubiquitinating enzyme USP28 to trigger the interaction of c-Myc with ubiquitin ligase Fbw7, resulting in the downregulation of cell cycle regulators and anti-apoptotic proteins and consequently, cell cycle arrest and cell apoptosis.Conclusion: CADPE is a novel c-Myc inhibitor with high activity and a unique mechanism for killing leukemia cells. [ABSTRACT FROM AUTHOR]

A previous study on hepatoblastoma revealed novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex, including the tumor suppressor speckle-type BTB/POZ (SPOP). Moreover, the SPOP gene affected cell growth, and its S119N mutation was identified as a loss-of-function mutation in hepatoblastoma. This study aimed to explore more functions and the potential mechanism of SPOP and its S119N mutation. The in vitro effects of SPOP on cell proliferation, invasion, apoptosis, and in vivo tumor growth were investigated by western blot analysis, Cell Counting Kit-8, colony formation assay, flow cytometry, and xenograft animal experiments. The substrate of SPOP was discovered by a protein quantification assay and quantitative ubiquitination modification assay. The present study further proved that SPOP functioned as an anti-oncogene through the phosphatidylinositol 3-kinase/Akt signaling pathway to affect various malignant biological behaviors of hepatoblastoma both in vitro and in vivo . Furthermore, experimental results also suggested that solute carrier family 7 member 1 (SLC7A1) might be a substrate of SPOP and influence cell phenotype by regulating arginine metabolism. In conclusion, these findings demonstrated the function of SPOP and revealed a potential substrate related to hepatoblastoma tumorigenesis, which might thus provide a novel therapeutic target for hepatoblastoma., (© 2020 The Authors.)

F-box proteins are critical components of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases and involved in the ubiquitin-dependent proteolytic pathway. Dysregulation of F-box protein-mediated proteolysis often leads to human malignancies. F-box only protein 8 (FBX8), a novel component of F-box proteins, is down-regulated in several tumors and closely correlates with tumor progression. However, little is known about its function, regulatory mechanisms and substrates in the progression of colorectal carcinoma (CRC). Combining microRNA (miRNA) assay, functional characterization, mechanistic studies with clinical validation, we identify FBX8 as a CRC metastasis suppressor downstream of miR-223, a metastasis promoting miRNA that is transcriptionally regulated by Myocyte enhancer factor (MEF2A). mTOR is a substrate of FBX8 for ubiquitin-mediated degradation and is required for FBX8 induced cell proliferation and invasion in CRC cells. FBX8 is down-regulated in human CRC tissues and correlates with MEF2A, miR-223 and mTOR expression levels. Notably, low FBX8 expression status in CRC tissues was a significant prognostic factor for poor overall survival of patients. These findings illustrate FBX8 as a metastasis suppressor that functions through mTOR signaling pathway and has significant prognostic power. [ABSTRACT FROM AUTHOR]