Allopolyploid Brassica juncea is particularly enriched in sinigrin,a kind of 3C aliphatic glucosinolates(GSLs),giving rise to characteristic taste after picking.However,the molecular mechanism underlying 3C aliphatic ...Allopolyploid Brassica juncea is particularly enriched in sinigrin,a kind of 3C aliphatic glucosinolates(GSLs),giving rise to characteristic taste after picking.However,the molecular mechanism underlying 3C aliphatic GSLs biosynthesis in this species remains unknown.In this study,we genome-widely identified GSLs metabolic genes,indicating different evolutionary rate of GSLs metabolic genes between subgenomes of B.juncea.Eight methythioalkylmalate synthase(MAMs)homologs were identified from B.juncea,in which six MAM1s were located in chloroplast and the other two were not detected with any expression.Furthermore,BjMAM1-4,BjMAM1-5,and BjMAM1-6 displayed higher expression levels in leaves than other tissues.Silenced expression analysis revealed that BjMAM1-4 and BjMAM1-6 function in 3C and 4C aliphatic GSLs accumulation.The specificity of the substrate selection for the second cycle reaction is much lower than that of the first cycle,suggesting these genes may preferentially catalyze 3C aliphatic GSLs biosynthesis.Our study provides insights into the molecular mechanism underlying the accumulation of 3C aliphatic GSLs,thereby facilitating the manipulation of aliphatic GSLs content in B.juncea.展开更多
We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins （i.e.,...We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins （i.e., Tac-β3, Tac-β3△741, Tac-β3△747, Tac-β3△754, Tac-β3△759, and Tac-β3ANITY） consisting of the extracellular and transmembrane domains of human IL-2 receptor （Tac） and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin uIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3△NITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3△759, Tac-β3ANITY, and Tac-β3△754, but not Tac-β3△747 or Tac-β3△741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3△ and Tac-β3ANITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3△759 and 123/Tac-β3△754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3△747 and 123/Tac-β3△741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.展开更多
基金The authors thank Prof.Q.Wang for GSLs analysis.This work was supported by grants from the National Natural Science Foundation of Zhejiang Province(Grant no.LZ20C150002)the National Natural Science Foundation of China(Grant no.31872095).
文摘Allopolyploid Brassica juncea is particularly enriched in sinigrin,a kind of 3C aliphatic glucosinolates(GSLs),giving rise to characteristic taste after picking.However,the molecular mechanism underlying 3C aliphatic GSLs biosynthesis in this species remains unknown.In this study,we genome-widely identified GSLs metabolic genes,indicating different evolutionary rate of GSLs metabolic genes between subgenomes of B.juncea.Eight methythioalkylmalate synthase(MAMs)homologs were identified from B.juncea,in which six MAM1s were located in chloroplast and the other two were not detected with any expression.Furthermore,BjMAM1-4,BjMAM1-5,and BjMAM1-6 displayed higher expression levels in leaves than other tissues.Silenced expression analysis revealed that BjMAM1-4 and BjMAM1-6 function in 3C and 4C aliphatic GSLs accumulation.The specificity of the substrate selection for the second cycle reaction is much lower than that of the first cycle,suggesting these genes may preferentially catalyze 3C aliphatic GSLs biosynthesis.Our study provides insights into the molecular mechanism underlying the accumulation of 3C aliphatic GSLs,thereby facilitating the manipulation of aliphatic GSLs content in B.juncea.
基金We thank all the laboratory members for helpful discussion. This work was supported by grants from National Natural Science Foundation of China （No. 81270594）, National Basic Research Program of China （No. 2012CB518000, No. 2013CB966800） and National Natural Science Foundation of China （No. 81070414）. Jiansong Huang is a recipient of a fellowship from The China Postdoctoral Science Foundation （No. 2013M531185）.
文摘We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins （i.e., Tac-β3, Tac-β3△741, Tac-β3△747, Tac-β3△754, Tac-β3△759, and Tac-β3ANITY） consisting of the extracellular and transmembrane domains of human IL-2 receptor （Tac） and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin uIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3△NITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3△759, Tac-β3ANITY, and Tac-β3△754, but not Tac-β3△747 or Tac-β3△741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3△ and Tac-β3ANITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3△759 and 123/Tac-β3△754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3△747 and 123/Tac-β3△741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.
