目的:探讨胃癌组织及细胞中Cullin1基因表达水平及其对凋亡的影响及其机制。方法:在TCGA(The Cancer Genome Atlas)数据库下载胃癌数据,分析胃癌组织与正常组织中Cullin1表达差异。应用实时荧光定量PCR(qRT-PCR)技术检测36例胃癌及正常...目的:探讨胃癌组织及细胞中Cullin1基因表达水平及其对凋亡的影响及其机制。方法:在TCGA(The Cancer Genome Atlas)数据库下载胃癌数据,分析胃癌组织与正常组织中Cullin1表达差异。应用实时荧光定量PCR(qRT-PCR)技术检测36例胃癌及正常组织中Cullin1基因表达。合成小干扰RNA(Cullin1-siRNA)转染胃癌细胞系AGS,抑制Cullin1表达;四甲基偶氮唑盐比色法(MTT)检测细胞的增殖活性;流式细胞仪实验检测细胞凋亡率;qRT-PCR和蛋白印迹(Western blot)检测转染各组细胞Cullin1、Bcl2、Bax和Survivin、Livin基因mRNA和蛋白表达。结果:生物信息学结果显示,Cullin1基因在胃癌组织中表达水平明显高于正常组织(P<0.01);不同T分期的胃癌组织中Cullin1基因表达存在差异(P=0.026)。qRT-PCR结果显示验证结果与生物信息学结果符合。Cullin1-siRNA能有效沉默AGS细胞Cullin1基因的表达;有效抑制AGS细胞Cullin1表达后,转染组细胞的活性明显低于NS-siRNA组和空白对照组;凋亡率在Cullin1-siRNA组明显高于NS-siRNA组、空白对照组。Cullin1-siRNA转染后AGS中Cullin1和Bcl2、Survivin、Livin基因和蛋白表达下调,而Bax基因和蛋白表达上调(P<0.05)。结论:Cullin1基因在胃癌中表达增强且与肿瘤浸润深度有关,该基因可能通过抑制胃癌细胞凋亡而发挥作用。展开更多
Protein neddylation is catalyzed by a three-enzyme cascade,namely an E1 NEDD8-activating enzyme(NAE),one of two E2 NEDD8 conjugation enzymes and one of several E3 NEDD8 ligases.The physiological substrates of neddylat...Protein neddylation is catalyzed by a three-enzyme cascade,namely an E1 NEDD8-activating enzyme(NAE),one of two E2 NEDD8 conjugation enzymes and one of several E3 NEDD8 ligases.The physiological substrates of neddylation are the family members of cullin,the scaffold component of cullin RING ligases(CRLs).Currently,a potent E1 inhibitor,MLN4924,also known as pevonedistat,is in several clinical trials for anti-cancer therapy.Here we report the discovery,through virtual screening and structural modifications,of a small molecule compound HA-1141 that directly binds to NAE in both in vitro and in vivo assays and effectively inhibits neddylation of cullins 1 e5.Surprisingly,unlike MLN4924,HA-1141 also triggers non-canonical endoplasmic reticulum(ER)stress and PKR-mediated terminal integrated stress response(ISR)to activate ATF4 at an early stage,and to inhibit protein synthesis and mTORC1 activity at a later stage,eventually leading to autophagy induction.Biologically,HA-1141 suppresses growth and survival of cultured lung cancer cells and tumor growth in in vivo xenograft lung cancer models at a well-tolerated dose.Taken together,our study has identified a small molecule compound with the dual activities of blocking neddylation and triggering ER stress,leading to growth suppression of cancer cells.展开更多
NEDD8 conjugation of Cullin has an important role in ubiquitin-mediated protein degradation. The COP9 signalosome, of which CSN5 is the major catalytic subunit, is a major Cullin deneddylase, Another deneddylase, Dene...NEDD8 conjugation of Cullin has an important role in ubiquitin-mediated protein degradation. The COP9 signalosome, of which CSN5 is the major catalytic subunit, is a major Cullin deneddylase, Another deneddylase, Deneddylase 1, has also been shown to process the Nedd8 precursor. In Drosophila, the DEN1 mutants do not have increased levels of Cullin neddylation, but instead show a significant decrease in neddylated Cullin. This characteristic decrease in neddylated Cullins in the DEN1^null background can be rescued by UAS-dDEN1^WT overexpression but not by overexpression of mature NEDDS, indicating that this phenotype is distinct from the NEDD8-processing function of DENI. We examined the role of DEN 1-CSN interaction in regulating Cullin neddylation. Overexpression of DEN1 in a CSN5^hypo background slightly reduced unneddylated Cullin levels. The CSN5, DEN1 double mutation partially rescues the premature lethality associated with the CSN5 single mutation. These results suggest that DEN1 regulates Cullin neddylation by suppressing CSN deneddylase activity.展开更多
The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism,cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase.However,i...The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism,cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase.However,its upstream regulatory pathways as well as whether it has additional function(s)remain largely unknown.We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1,but not with other Cullin family members.Furthermore,we identified SGK1 as its downstream target.Interestingly,Rictor,but not Raptor or mTOR,promotes SGK1 ubiquitination.As a result,SGK1 expression is elevated in Rictor^(–/–)MEFs.We further defined that as a feedback mechanism,Rictor can be phosphorylated by multiple AGC family kinases including Akt,S6K and SGK1.Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1.On the other hand,it disrupted the interaction between Rictor and Cullin-1.Consequently,T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction.This finding further expands our knowledge of Rictor’s function.Furthermore,our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades,and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.展开更多
文摘目的:探讨胃癌组织及细胞中Cullin1基因表达水平及其对凋亡的影响及其机制。方法:在TCGA(The Cancer Genome Atlas)数据库下载胃癌数据,分析胃癌组织与正常组织中Cullin1表达差异。应用实时荧光定量PCR(qRT-PCR)技术检测36例胃癌及正常组织中Cullin1基因表达。合成小干扰RNA(Cullin1-siRNA)转染胃癌细胞系AGS,抑制Cullin1表达;四甲基偶氮唑盐比色法(MTT)检测细胞的增殖活性;流式细胞仪实验检测细胞凋亡率;qRT-PCR和蛋白印迹(Western blot)检测转染各组细胞Cullin1、Bcl2、Bax和Survivin、Livin基因mRNA和蛋白表达。结果:生物信息学结果显示,Cullin1基因在胃癌组织中表达水平明显高于正常组织(P<0.01);不同T分期的胃癌组织中Cullin1基因表达存在差异(P=0.026)。qRT-PCR结果显示验证结果与生物信息学结果符合。Cullin1-siRNA能有效沉默AGS细胞Cullin1基因的表达;有效抑制AGS细胞Cullin1表达后,转染组细胞的活性明显低于NS-siRNA组和空白对照组;凋亡率在Cullin1-siRNA组明显高于NS-siRNA组、空白对照组。Cullin1-siRNA转染后AGS中Cullin1和Bcl2、Survivin、Livin基因和蛋白表达下调,而Bax基因和蛋白表达上调(P<0.05)。结论:Cullin1基因在胃癌中表达增强且与肿瘤浸润深度有关,该基因可能通过抑制胃癌细胞凋亡而发挥作用。
基金National Key R&D Program of China(2016YFA0501800 to Yi Sun)for financial support。
文摘Protein neddylation is catalyzed by a three-enzyme cascade,namely an E1 NEDD8-activating enzyme(NAE),one of two E2 NEDD8 conjugation enzymes and one of several E3 NEDD8 ligases.The physiological substrates of neddylation are the family members of cullin,the scaffold component of cullin RING ligases(CRLs).Currently,a potent E1 inhibitor,MLN4924,also known as pevonedistat,is in several clinical trials for anti-cancer therapy.Here we report the discovery,through virtual screening and structural modifications,of a small molecule compound HA-1141 that directly binds to NAE in both in vitro and in vivo assays and effectively inhibits neddylation of cullins 1 e5.Surprisingly,unlike MLN4924,HA-1141 also triggers non-canonical endoplasmic reticulum(ER)stress and PKR-mediated terminal integrated stress response(ISR)to activate ATF4 at an early stage,and to inhibit protein synthesis and mTORC1 activity at a later stage,eventually leading to autophagy induction.Biologically,HA-1141 suppresses growth and survival of cultured lung cancer cells and tumor growth in in vivo xenograft lung cancer models at a well-tolerated dose.Taken together,our study has identified a small molecule compound with the dual activities of blocking neddylation and triggering ER stress,leading to growth suppression of cancer cells.
文摘NEDD8 conjugation of Cullin has an important role in ubiquitin-mediated protein degradation. The COP9 signalosome, of which CSN5 is the major catalytic subunit, is a major Cullin deneddylase, Another deneddylase, Deneddylase 1, has also been shown to process the Nedd8 precursor. In Drosophila, the DEN1 mutants do not have increased levels of Cullin neddylation, but instead show a significant decrease in neddylated Cullin. This characteristic decrease in neddylated Cullins in the DEN1^null background can be rescued by UAS-dDEN1^WT overexpression but not by overexpression of mature NEDDS, indicating that this phenotype is distinct from the NEDD8-processing function of DENI. We examined the role of DEN 1-CSN interaction in regulating Cullin neddylation. Overexpression of DEN1 in a CSN5^hypo background slightly reduced unneddylated Cullin levels. The CSN5, DEN1 double mutation partially rescues the premature lethality associated with the CSN5 single mutation. These results suggest that DEN1 regulates Cullin neddylation by suppressing CSN deneddylase activity.
基金supported in part by the DOD Prostate New Investigator award to W.W.NIH grant GM089763 to W.W.
文摘The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism,cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase.However,its upstream regulatory pathways as well as whether it has additional function(s)remain largely unknown.We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1,but not with other Cullin family members.Furthermore,we identified SGK1 as its downstream target.Interestingly,Rictor,but not Raptor or mTOR,promotes SGK1 ubiquitination.As a result,SGK1 expression is elevated in Rictor^(–/–)MEFs.We further defined that as a feedback mechanism,Rictor can be phosphorylated by multiple AGC family kinases including Akt,S6K and SGK1.Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1.On the other hand,it disrupted the interaction between Rictor and Cullin-1.Consequently,T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction.This finding further expands our knowledge of Rictor’s function.Furthermore,our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades,and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.
