Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade.The bestknown substrates of ...Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade.The bestknown substrates of neddylation are cullin family proteins,which are the core component of Cullin-RING E3 ubiquitin ligases(CRLs).Given that cullin neddylation is required for CRL activity,and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers,targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics.In the past decade,we have witnessed significant progress in the field of protein neddylation from preclinical target validation,to drug screening,then to the clinical trials of neddylation inhibitors.In this review,we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade,followed by a summary of all reported chemical inhibitors of neddylation enzymes.We then discussed the structure-based targeting of protein-protein interaction in neddylation cascade,and finally the available approaches for the discovery of new neddylation inhibitors.This review will provide a focused,up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.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
NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 （NEDD8） and the roles of NEDDylation involved in the DNA dam...NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 （NEDD8） and the roles of NEDDylation involved in the DNA damage response （DDR） are largely unknown. Translesion synthesis （TLS） is a damage-tolerance mechanism, in which RAD181RAD6-mediated monoubiq- uitinated proliferating cell nuclear antigen （PCNA） pro- motes recruitment of polymerase q （polq） to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to H202 stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to H2O2-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.展开更多
The primary cilium is a microtubule-based sensory organelle.The molecular mechanism that regulates ciliary dynamics remains elusive.Here,we report an unexpected finding that MLN4924,a small molecule inhibitor of NEDD8...The primary cilium is a microtubule-based sensory organelle.The molecular mechanism that regulates ciliary dynamics remains elusive.Here,we report an unexpected finding that MLN4924,a small molecule inhibitor of NEDD8-activating enzyme(NAE),blocks primary ciliary formation by inhibiting synthesis/assembly and promoting disassembly.This is mainly mediated by MLN4924-induced phosphorylation of AKT1 at Ser473 under serum-starved,ciliary-promoting conditions.Indeed,pharmaceutical inhibition(by MK2206)or genetic depletion(via siRNA)of AKT1 rescues MLN4924 effect,indicating its causai role.Interestingly,pAKT 1-Ser473 activity regulates both ciliary synthesis/assembly and disassembly in a MLN4924 dependent manner,whereas pAKT-Thr308 determines the ciliary length in MLN4924-independent but VHL-dependent manner.Finally,MLN4924 inhibits mouse hair regrowth,a process requires ciliogenesis?Collectively,our study dem on strates an unexpected role of a neddylation inhibitor in regulation of ciliogenesis via AKT1,and pro?vides a proof-of-concept for potential utility of MLN4924 in the treatment of human diseases associated with abnormal ciliogenesis.展开更多
Background:The metabolic syndrome is a consequence of modern lifestyle that causes synaptic insulin resistance and cognitive deficits and that in interaction with a high amyloid load is an important risk factor for Al...Background:The metabolic syndrome is a consequence of modern lifestyle that causes synaptic insulin resistance and cognitive deficits and that in interaction with a high amyloid load is an important risk factor for Alzheimer’s dis-ease.It has been proposed that neuroinflammation might be an intervening variable,but the underlying mechanisms are currently unknown.Methods:We utilized primary neurons to induce synaptic insulin resistance as well as a mouse model of high-risk aging that includes a high amyloid load,neuroinflammation,and diet-induced obesity to test hypotheses on underly-ing mechanisms.Results:We found that neddylation and subsequent activation of cullin-RING ligase complexes induced synaptic insulin resistance through ubiquitylation and degradation of the insulin-receptor substrate IRS1 that organizes synap-tic insulin signaling.Accordingly,inhibition of neddylation preserved synaptic insulin signaling and rescued memory deficits in mice with a high amyloid load,which were fed with a’western diet’.Conclusions:Collectively,the data suggest that neddylation and degradation of the insulin-receptor substrate is a nodal point that links high amyloid load,neuroinflammation,and synaptic insulin resistance to cognitive decline and impaired synaptic plasticity in high-risk aging.展开更多
基金the financial support by the National Key R&D Program of China(2016YFA0501800 to YS)
文摘Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade.The bestknown substrates of neddylation are cullin family proteins,which are the core component of Cullin-RING E3 ubiquitin ligases(CRLs).Given that cullin neddylation is required for CRL activity,and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers,targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics.In the past decade,we have witnessed significant progress in the field of protein neddylation from preclinical target validation,to drug screening,then to the clinical trials of neddylation inhibitors.In this review,we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade,followed by a summary of all reported chemical inhibitors of neddylation enzymes.We then discussed the structure-based targeting of protein-protein interaction in neddylation cascade,and finally the available approaches for the discovery of new neddylation inhibitors.This review will provide a focused,up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.
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.
基金We sincerely thank Profs. Jun Huang, Wensheng Wei, and Caixia Guo for providing the plasmids used in this study. We thank Millennium Pharmaceuticals for providing the MLN4924 used in this study. This work was supported by the National Natural Science Foundation of China （Grant Nos. 31470754, 81730080 and 31670786）, the National Key Research and Development Program of China （2016YFC1302401）.
文摘NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 （NEDD8） and the roles of NEDDylation involved in the DNA damage response （DDR） are largely unknown. Translesion synthesis （TLS） is a damage-tolerance mechanism, in which RAD181RAD6-mediated monoubiq- uitinated proliferating cell nuclear antigen （PCNA） pro- motes recruitment of polymerase q （polq） to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to H202 stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to H2O2-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.
文摘The primary cilium is a microtubule-based sensory organelle.The molecular mechanism that regulates ciliary dynamics remains elusive.Here,we report an unexpected finding that MLN4924,a small molecule inhibitor of NEDD8-activating enzyme(NAE),blocks primary ciliary formation by inhibiting synthesis/assembly and promoting disassembly.This is mainly mediated by MLN4924-induced phosphorylation of AKT1 at Ser473 under serum-starved,ciliary-promoting conditions.Indeed,pharmaceutical inhibition(by MK2206)or genetic depletion(via siRNA)of AKT1 rescues MLN4924 effect,indicating its causai role.Interestingly,pAKT 1-Ser473 activity regulates both ciliary synthesis/assembly and disassembly in a MLN4924 dependent manner,whereas pAKT-Thr308 determines the ciliary length in MLN4924-independent but VHL-dependent manner.Finally,MLN4924 inhibits mouse hair regrowth,a process requires ciliogenesis?Collectively,our study dem on strates an unexpected role of a neddylation inhibitor in regulation of ciliogenesis via AKT1,and pro?vides a proof-of-concept for potential utility of MLN4924 in the treatment of human diseases associated with abnormal ciliogenesis.
基金the Deutsche Forschungsgemeinschaft(DFG)(Kr 1879/9-1/FOR 2419,Kr1879/5-1/6-1/10-1CRC1436 A02 Project-ID 425899996+2 种基金Research Training Group 2413 SynAGE,TP4),BMBF‘Energi’FKZ:01GQ1421B,The EU Joint Programme-Neurodegenerative Disease Research(JPND)project STAD(01ED1613)and Leibniz Foundation SAW‘ISAS2’,’SynMetAge’,’Neurotranslation’and’SynErca’to MRK.CRC1436 A02 Project-ID 425899996 to AKCRC1436 A05 Project-ID 425899996,Research Training Group 2413 SynAGE TP5 and BMBF‘Energi’FKZ:01GQ1421A to AD.G.M.G.was supported by the Alexander-von-Humboldt Foundation/CAPES post-doctoral research fellowship(99999.001756/2014-01).
文摘Background:The metabolic syndrome is a consequence of modern lifestyle that causes synaptic insulin resistance and cognitive deficits and that in interaction with a high amyloid load is an important risk factor for Alzheimer’s dis-ease.It has been proposed that neuroinflammation might be an intervening variable,but the underlying mechanisms are currently unknown.Methods:We utilized primary neurons to induce synaptic insulin resistance as well as a mouse model of high-risk aging that includes a high amyloid load,neuroinflammation,and diet-induced obesity to test hypotheses on underly-ing mechanisms.Results:We found that neddylation and subsequent activation of cullin-RING ligase complexes induced synaptic insulin resistance through ubiquitylation and degradation of the insulin-receptor substrate IRS1 that organizes synap-tic insulin signaling.Accordingly,inhibition of neddylation preserved synaptic insulin signaling and rescued memory deficits in mice with a high amyloid load,which were fed with a’western diet’.Conclusions:Collectively,the data suggest that neddylation and degradation of the insulin-receptor substrate is a nodal point that links high amyloid load,neuroinflammation,and synaptic insulin resistance to cognitive decline and impaired synaptic plasticity in high-risk aging.
