The host antimicrobial immune response relies on a complex interplay of molecular mechanisms to effectively combat microbial infections.Herein,we investigate the functional role of Cullin-3(Cul3),one critical constitu...The host antimicrobial immune response relies on a complex interplay of molecular mechanisms to effectively combat microbial infections.Herein,we investigate the functional role of Cullin-3(Cul3),one critical constituent of Cullin-RING ubiquitin ligases,in the Drosophila melanogaster(fruit fly)antimicrobial immune defense.Weshow that silencing of Cul3 leads to a decreased induction of antimicrobial peptides and high mortality in adult flies after bacterial infection.Through biochemical approaches,we demonstrate that Cul3 predominantly relies on its BTB-binding domain and neddylation domain to physically associate with death-associated inhibitor of apoptosis 2(Diap2).Importantly,Cul3 ameliorates the Diap2-mediated ubiquitination of death-related ced-3/Nedd2-like caspase(Dredd),a process essential for robust immune deficiency signaling upon bacterial infection.Taken together,our findings highlight a previously unrecognized regulatory axis of Cul3/Diap2/Dredd in the fly antimicrobial immune defense,providing potential insights into therapeutic strategies for combating bacterial infections in humans.展开更多
Ambient temperature affects flowering time in plants,and the MADS-box transcription factor SHORT VEGETATIVE PHASE(SVP)plays a crucial role in the response to changes in ambient temperature.SVP protein stability is reg...Ambient temperature affects flowering time in plants,and the MADS-box transcription factor SHORT VEGETATIVE PHASE(SVP)plays a crucial role in the response to changes in ambient temperature.SVP protein stability is regulated by the 26S proteasome pathway and decreases at high ambient temperature,but the details of SVP degradation are unclear.Here,we show that SVP degradation at high ambient temperature is mediated by the CULLIN3–RING E3 ubiquitin ligase(CRL3)complex in Arabidopsis thaliana.We identified a previously uncharacterized protein that interacts with SVP at high ambient temperature and contains a BTB/POZ domain.We named this protein LATE FLOWERING AT HIGH TEMPERATURE 1(LFH1).Single mutants of LFH1 or CULLIN3A(CUL3A)showed late flowering specifically at 27C.LFH1 protein levels increased at high ambient temperature.We found that LFH1 interacts with CUL3A in the cytoplasm and is important for SVP–CUL3A complex formation.Mutations in CUL3A and/or LFH1 led to increased SVP protein stability at high ambient temperature,suggesting that the CUL3–LFH1 complex functions in SVP degradation.Screening E2 ubiquitin-conjugating enzymes(UBCs)using RING-BOX PROTEIN 1(RBX1),a component of the CRL3 complex,as bait identified UBC15.ubc15 mutants also showed late flowering at high ambient temperature.In vitro and in vivo ubiquitination assays using recombinant CUL3A,LFH1,RBX1,and UBC15 showed that SVP is highly ubiquitinated in an ATP-dependent manner.Collectively,these results indicate that the degradation of SVP at high ambient temperature is mediated by a CRL3 complex comprising CUL3A,LFH1,and UBC15.展开更多
Targeted protein degradation(TPD)technology mainly utilizes the natural degradation systems within cells to specific and efficient degradation of disease-related proteins1.The ubiquitin-proteasome system(UPS)is the mo...Targeted protein degradation(TPD)technology mainly utilizes the natural degradation systems within cells to specific and efficient degradation of disease-related proteins1.The ubiquitin-proteasome system(UPS)is the most important way of protein degradation in eukaryotic cells,which is involved in more than 80%of degradation processes2.Molecular glues are mostly small molecules to stabilize existing interactions between two proteins or induce new protein-protein interactions3.In the case of molecular glues between the target protein and an E3 ligase.展开更多
Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 l...Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 （CUL3）-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ （BPM） proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T （FT）, a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants.展开更多
Hypertension is the largest risk factor for cardiovascular disease,the leading cause of mortality worldwide.As blood pressure regulation is influenced by multiple physiological systems,hypertension cannot be attribute...Hypertension is the largest risk factor for cardiovascular disease,the leading cause of mortality worldwide.As blood pressure regulation is influenced by multiple physiological systems,hypertension cannot be attributed to a single identifiable etiology.Three decades of research into Mendelian forms of hypertension implicated alterations in the renal tubular sodium handling,particularly the distal convoluted tubule(DCT)-native,thiazide-sensitive Na-Cl cotransporter(NCC).Altered functions of the NCC have shown to have profound effects on blood pressure regulation as illustrated by the over activation and inactivation of the NCC in Gordon’s and Gitelman syndromes respectively.Substantial progress has uncovered multiple factors that affect the expression and activity of the NCC.In particular,NCC activity is controlled by phosphorylation/dephosphorylation,and NCC expression is facilitated by glycosylation and negatively regulated by ubiquitination.Studies have even found parvalbumin to be an unexpected regulator of the NCC.In recent years,there have been considerable advances in our understanding of NCC control mechanisms,particularly via the pathway containing the with-no-lysine[K](WNK)and its downstream target kinases,SPS/Ste20-related proline-alanine-rich kinase(SPAK)and oxidative stress responsive 1(OSR1),which has led to the discovery of novel inhibitory molecules.This review summarizes the currently reported regulatory mechanisms of the NCC and discusses their potential as therapeutic targets for treating hypertension.展开更多
Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the un...Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors(TFs) is a dynamic and essential mechanism for plant growth and development. CRL3 BPME3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T(FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays(EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction(ChIP-q PCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3 BPME3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3 BPM E3 ligases in Arabidopsis.展开更多
Folate metabolism plays an essential role in tumor development.Various cancers display therapeutic response to reagents targeting key enzymes of the folate cycle,but obtain chemoresistance later.Therefore,novel target...Folate metabolism plays an essential role in tumor development.Various cancers display therapeutic response to reagents targeting key enzymes of the folate cycle,but obtain chemoresistance later.Therefore,novel targets in folate metabolism are highly demanded.Methylenetetrahydrofolate dehydrogenase/methylenetetrahydrofolate cyclohydrolase 2(MTHFD2)is one of the key enzymes in folate metabolism and its expression is highly increased in mutiple human cancers.However,the underlying mechanism that regulates MTHFD2 expression remains unknown.Here,we elucidate that SIRT4 deacetylates the conserved lysine 50(K50)residue in MTHFD2.K50 deacetylation destabilizes MTHFD2 by elevating cullin 3 E3 ligase-mediated proteasomal degradation in response to stressful stimuli of folate deprivation,leading to suppression of nicotinamide adenine dinucleotide phosphate production in tumor cells and accumulation of intracellular reactive oxygen species,which in turn inhibits the growth of breast cancer cells.Collectively,our study reveals that SIRT4 senses folate availability to control MTHFD2 K50 acetylation and its protein stability,bridging nutrient/folate stress and cellular redox to act on cancer cell growth.展开更多
基金supported by grants from the National Natural Science Foundation of China(32100702).
文摘The host antimicrobial immune response relies on a complex interplay of molecular mechanisms to effectively combat microbial infections.Herein,we investigate the functional role of Cullin-3(Cul3),one critical constituent of Cullin-RING ubiquitin ligases,in the Drosophila melanogaster(fruit fly)antimicrobial immune defense.Weshow that silencing of Cul3 leads to a decreased induction of antimicrobial peptides and high mortality in adult flies after bacterial infection.Through biochemical approaches,we demonstrate that Cul3 predominantly relies on its BTB-binding domain and neddylation domain to physically associate with death-associated inhibitor of apoptosis 2(Diap2).Importantly,Cul3 ameliorates the Diap2-mediated ubiquitination of death-related ced-3/Nedd2-like caspase(Dredd),a process essential for robust immune deficiency signaling upon bacterial infection.Taken together,our findings highlight a previously unrecognized regulatory axis of Cul3/Diap2/Dredd in the fly antimicrobial immune defense,providing potential insights into therapeutic strategies for combating bacterial infections in humans.
基金supported by grants from the National Research Foundation of Korea (NRF-2022R1A3B1078180 and RS-2023-00221182 to J.H.A.and NRF-2022R1A2B5B02001266 to P.J.S.).
文摘Ambient temperature affects flowering time in plants,and the MADS-box transcription factor SHORT VEGETATIVE PHASE(SVP)plays a crucial role in the response to changes in ambient temperature.SVP protein stability is regulated by the 26S proteasome pathway and decreases at high ambient temperature,but the details of SVP degradation are unclear.Here,we show that SVP degradation at high ambient temperature is mediated by the CULLIN3–RING E3 ubiquitin ligase(CRL3)complex in Arabidopsis thaliana.We identified a previously uncharacterized protein that interacts with SVP at high ambient temperature and contains a BTB/POZ domain.We named this protein LATE FLOWERING AT HIGH TEMPERATURE 1(LFH1).Single mutants of LFH1 or CULLIN3A(CUL3A)showed late flowering specifically at 27C.LFH1 protein levels increased at high ambient temperature.We found that LFH1 interacts with CUL3A in the cytoplasm and is important for SVP–CUL3A complex formation.Mutations in CUL3A and/or LFH1 led to increased SVP protein stability at high ambient temperature,suggesting that the CUL3–LFH1 complex functions in SVP degradation.Screening E2 ubiquitin-conjugating enzymes(UBCs)using RING-BOX PROTEIN 1(RBX1),a component of the CRL3 complex,as bait identified UBC15.ubc15 mutants also showed late flowering at high ambient temperature.In vitro and in vivo ubiquitination assays using recombinant CUL3A,LFH1,RBX1,and UBC15 showed that SVP is highly ubiquitinated in an ATP-dependent manner.Collectively,these results indicate that the degradation of SVP at high ambient temperature is mediated by a CRL3 complex comprising CUL3A,LFH1,and UBC15.
文摘Targeted protein degradation(TPD)technology mainly utilizes the natural degradation systems within cells to specific and efficient degradation of disease-related proteins1.The ubiquitin-proteasome system(UPS)is the most important way of protein degradation in eukaryotic cells,which is involved in more than 80%of degradation processes2.Molecular glues are mostly small molecules to stabilize existing interactions between two proteins or induce new protein-protein interactions3.In the case of molecular glues between the target protein and an E3 ligase.
文摘Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 （CUL3）-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ （BPM） proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T （FT）, a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants.
基金supported by the University of Exeter Medical School(UK)NIH Grants R01 NS109358(USA)。
文摘Hypertension is the largest risk factor for cardiovascular disease,the leading cause of mortality worldwide.As blood pressure regulation is influenced by multiple physiological systems,hypertension cannot be attributed to a single identifiable etiology.Three decades of research into Mendelian forms of hypertension implicated alterations in the renal tubular sodium handling,particularly the distal convoluted tubule(DCT)-native,thiazide-sensitive Na-Cl cotransporter(NCC).Altered functions of the NCC have shown to have profound effects on blood pressure regulation as illustrated by the over activation and inactivation of the NCC in Gordon’s and Gitelman syndromes respectively.Substantial progress has uncovered multiple factors that affect the expression and activity of the NCC.In particular,NCC activity is controlled by phosphorylation/dephosphorylation,and NCC expression is facilitated by glycosylation and negatively regulated by ubiquitination.Studies have even found parvalbumin to be an unexpected regulator of the NCC.In recent years,there have been considerable advances in our understanding of NCC control mechanisms,particularly via the pathway containing the with-no-lysine[K](WNK)and its downstream target kinases,SPS/Ste20-related proline-alanine-rich kinase(SPAK)and oxidative stress responsive 1(OSR1),which has led to the discovery of novel inhibitory molecules.This review summarizes the currently reported regulatory mechanisms of the NCC and discusses their potential as therapeutic targets for treating hypertension.
基金supported by National Natural Science Foundation of China(31670179,and 91854201)the Research Grants Council of Hong Kong(Ao E/M-05/12,CUHK14104716,and C4002-17G)to L.J.+1 种基金RGC(CUHK14104716)CUHK Direct Grants(4053143,4053174,4053243)to L.C.
文摘Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors(TFs) is a dynamic and essential mechanism for plant growth and development. CRL3 BPME3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T(FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays(EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction(ChIP-q PCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3 BPME3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3 BPM E3 ligases in Arabidopsis.
基金supported by the National Key R&D Program of China(2020YFA0803400/2020YFA0803402 and 2019YFA0801703 to Q.-Y.L.)the National Natural Science Foundation of China(81872240 to M.Y.,82002951 to J.L,and 81790250/81790253,91959202,and 82121004 to Q.-Y.L.)the Innovation Program of Shanghai Municipal Education Commission(N173606 to Q.-Y.L.).
文摘Folate metabolism plays an essential role in tumor development.Various cancers display therapeutic response to reagents targeting key enzymes of the folate cycle,but obtain chemoresistance later.Therefore,novel targets in folate metabolism are highly demanded.Methylenetetrahydrofolate dehydrogenase/methylenetetrahydrofolate cyclohydrolase 2(MTHFD2)is one of the key enzymes in folate metabolism and its expression is highly increased in mutiple human cancers.However,the underlying mechanism that regulates MTHFD2 expression remains unknown.Here,we elucidate that SIRT4 deacetylates the conserved lysine 50(K50)residue in MTHFD2.K50 deacetylation destabilizes MTHFD2 by elevating cullin 3 E3 ligase-mediated proteasomal degradation in response to stressful stimuli of folate deprivation,leading to suppression of nicotinamide adenine dinucleotide phosphate production in tumor cells and accumulation of intracellular reactive oxygen species,which in turn inhibits the growth of breast cancer cells.Collectively,our study reveals that SIRT4 senses folate availability to control MTHFD2 K50 acetylation and its protein stability,bridging nutrient/folate stress and cellular redox to act on cancer cell growth.
