The p53 tumor suppressor protein coordinates the cellular responses to a broad range of cellular stresses, leading to DNA repair, cell cycle arrest or apoptosis. The stability of p53 is essential for its tumor suppres...The p53 tumor suppressor protein coordinates the cellular responses to a broad range of cellular stresses, leading to DNA repair, cell cycle arrest or apoptosis. The stability of p53 is essential for its tumor suppressor function, which is tightly controlled by ubiquitin-dependent degradation primarily through its negative regulator mudne double minute 2 （Mdm2）. To better understand the regulation of p53, we tested the interaction between p53 and USP11 using co-immunoprecipitation. The results show that USP11, an ubiquitin-specific protease, forms specific complexes with p53 and stabilizes p53 by deubiquitinating it. Moreover, down-regulation of USP11 dramatically attenuated p53 in- duction in response to DNA damage stress. These findings reveal that USP11 is a novel regulator of p53, which is required for p53 activation in response to DNA damage.展开更多
Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)...Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)plays an instrumental role in aerobic glycolysis and tumor progression.However,whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer(SCLC)is largely unclear.In this study,we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC.Methods:Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts.CSC-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model.Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133.The expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting,immunoprecipitation,immunofluorescence,and immunohistochemistry assay.RNA expression levels of Nanog,POU5F1,Lin28,HK2,Prominin-1 were analyzed through quantitative reverse transcription PCR.Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay.CD133+cells were sorted by flow cytometry using an anti-CD133 antibody.Results:We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts.HK2 depletion inhibited CSC stemness and promoted CSC differentiation.Mechanistically,nonmitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels.The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11(USP11)to CD133,thereby inhibiting CD133 polyubiquitylation and degradation.HK2-mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators,SCLC cell stemness,and tumor growth in mice.In addition,HK2 expression was positively correlated with CD133 expression in human SCLC specimens,and their expression levels were associated with poor prognosis of SCLC patients.Conclusions:These results revealed a critical non-metabolic function of HK2 in promotion of cancer cell stemness.Our findings provided new insights into the multifaceted roles of HK2 in tumor development.展开更多
Phosphorylation affects ubiquitination, stability, and activity of transcriptional factors, thus regulating various cellular functions. E2F transcriptional factor I （E2F1） regulates paternally expressed imprinted ge...Phosphorylation affects ubiquitination, stability, and activity of transcriptional factors, thus regulating various cellular functions. E2F transcriptional factor I （E2F1） regulates paternally expressed imprinted gene 10 （Peg10） expression, thereby promoting cell proliferation. However, the effect of E2FZ stability on PeglO expression and the molecular regulation of E2FZ stability by its phos- phorylation have not been well demonstrated. Here, we describe a new pathway in which phosphorylation of E2F1 by GSK3p increases E2FZ association with the deubiquitinating enzyme, ubiquitin-specific protease 11 （USPll）, which removes K63-1inked ubiquitin chains thereby preventing E2FZ degradation in the nuclei. Downregulation of USPlZ increases E2FZ ubiquitination and reduces E2F1 stability and protein levels, thereby decreasing PeglO mRNA levels. Physiologically, USPll depletion suppresses cell proliferation and wound healing in lung epithelial cells, and these effects are reversed by E2F1 and PEGIO overexpression. Thus, our study reveals a new molecular model that phosphorylation promotes substrate stability through increasing its associ- ation with a deubiquitinating enzyme. The data suggest that GSK3p and USPll act in concert to modulate E2FZ abundance and PEGIO expression in lung epithelial celts to affect cell wound healing. This study provides new therapeutic targets to lessen lung injury by improving lung epithelial cell repair and remodeling after injury.展开更多
基金Project supported by the Key Project of Colleges and Universities in Fujian Province Serving the Construction of the West-strait(No.A101)the Foundation for the 2013 Research Plan of University Key Teacher Domestic Visitor of the Ministry of Educationthe Foundation for the Second Batch of Key Teacher of Quanzhou Normal College(the personnel department of QNC[2012]No.1),China
文摘The p53 tumor suppressor protein coordinates the cellular responses to a broad range of cellular stresses, leading to DNA repair, cell cycle arrest or apoptosis. The stability of p53 is essential for its tumor suppressor function, which is tightly controlled by ubiquitin-dependent degradation primarily through its negative regulator mudne double minute 2 （Mdm2）. To better understand the regulation of p53, we tested the interaction between p53 and USP11 using co-immunoprecipitation. The results show that USP11, an ubiquitin-specific protease, forms specific complexes with p53 and stabilizes p53 by deubiquitinating it. Moreover, down-regulation of USP11 dramatically attenuated p53 in- duction in response to DNA damage stress. These findings reveal that USP11 is a novel regulator of p53, which is required for p53 activation in response to DNA damage.
基金Ministry of Science and Technology of the People’s Republic of China,Grant/Award Number:2020YFA0803300National Natural Science Foundation of China,Grant/Award Numbers:82188102,82030074,82122053,32100574+10 种基金Beijing Municipal Science&Technology Commission,Grant/Award Number:Z191100006619115R&D Program of Beijing Municipal Education commission,Grant/Award Number:KJZD20191002302CAMS Innovation Fund for Medical Science,Grant/Award Numbers:2021-1-I2M-012,2021-I2M-1-067Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences,Grant/Award Number:2021-PT310-001Key-Area Research and Development Program of Guangdong Province,Grant/Award Number:2021B0101420005Sanming Project of Medicine in Shenzhen,Grant/Award Numbers:SZSM201612097,SZSM201812062Aiyou Foundation,Grant/Award Number:KY201701Natural Science Foundation of Shandong Province,Grant/Award Number:ZR2020QH191Zhejiang Natural Science Foundation-Key Project,Grant/Award Number:LD21H160003Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang,Grant/Award Number:2019R01001Zhimin Lu is the Kuancheng Wang Distinguished Chair。
文摘Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)plays an instrumental role in aerobic glycolysis and tumor progression.However,whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer(SCLC)is largely unclear.In this study,we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC.Methods:Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts.CSC-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model.Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133.The expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting,immunoprecipitation,immunofluorescence,and immunohistochemistry assay.RNA expression levels of Nanog,POU5F1,Lin28,HK2,Prominin-1 were analyzed through quantitative reverse transcription PCR.Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay.CD133+cells were sorted by flow cytometry using an anti-CD133 antibody.Results:We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts.HK2 depletion inhibited CSC stemness and promoted CSC differentiation.Mechanistically,nonmitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels.The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11(USP11)to CD133,thereby inhibiting CD133 polyubiquitylation and degradation.HK2-mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators,SCLC cell stemness,and tumor growth in mice.In addition,HK2 expression was positively correlated with CD133 expression in human SCLC specimens,and their expression levels were associated with poor prognosis of SCLC patients.Conclusions:These results revealed a critical non-metabolic function of HK2 in promotion of cancer cell stemness.Our findings provided new insights into the multifaceted roles of HK2 in tumor development.
文摘Phosphorylation affects ubiquitination, stability, and activity of transcriptional factors, thus regulating various cellular functions. E2F transcriptional factor I （E2F1） regulates paternally expressed imprinted gene 10 （Peg10） expression, thereby promoting cell proliferation. However, the effect of E2FZ stability on PeglO expression and the molecular regulation of E2FZ stability by its phos- phorylation have not been well demonstrated. Here, we describe a new pathway in which phosphorylation of E2F1 by GSK3p increases E2FZ association with the deubiquitinating enzyme, ubiquitin-specific protease 11 （USPll）, which removes K63-1inked ubiquitin chains thereby preventing E2FZ degradation in the nuclei. Downregulation of USPlZ increases E2FZ ubiquitination and reduces E2F1 stability and protein levels, thereby decreasing PeglO mRNA levels. Physiologically, USPll depletion suppresses cell proliferation and wound healing in lung epithelial cells, and these effects are reversed by E2F1 and PEGIO overexpression. Thus, our study reveals a new molecular model that phosphorylation promotes substrate stability through increasing its associ- ation with a deubiquitinating enzyme. The data suggest that GSK3p and USPll act in concert to modulate E2FZ abundance and PEGIO expression in lung epithelial celts to affect cell wound healing. This study provides new therapeutic targets to lessen lung injury by improving lung epithelial cell repair and remodeling after injury.
