Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a gua...Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a guanine nucleotide exchange factor(GEF)for the RAB GTPase RAB7A and is indispensable for directing RAB7A recruitment to autophagosome or lysosomal membranes.Despite its critical role,the precise molecular mechanism underlying the assembly of the human MON1A-CCZ1(HsMC1)complex and its specific GEF activity towards RAB7A has remained unclear.In this study,we report the high-resolution cryo-electron microscopy(cryo-EM)structure of the HsMC1 GEF domain in a complex with the nucleotide-free RAB7A^(N125I)at 2.85 A resolution.Our structural data demonstrate that engagement with the HsMC1 complex induces marked conformational shifts in the phosphate-binding loop(P-loop)and SwitchⅠ/Ⅱregions of RAB7A.A striking feature of this complex is the direct interaction between the P-loop of RAB7A and CCZ1,a structural detail not previously observed.Furthermore,biochemical assays targeting residues within InterfaceⅠorⅡof the HsMC1-RAB7A complex highlight their critical role in mediating the interaction and suggest a unique mechanism for nucleotide exchange facilitated by the HsMC1 complex.These findings provide novel molecular insights into the functional mechanisms of the HsMC1-RAB7A complex,offering a robust structural framework to inform future investigations into disease-related targets and therapeutic development.展开更多
目的:本实验阐述了Rab7对Raw264.7巨噬细胞中R848激活TLR7(Toll like receptor-7)所产生的细胞因子的影响,并探讨Rab7对MAPK信号转导的影响。方法:使用Rab7刺激silence-Rab7-Raw264.7细胞,检测R848激活TLR7后产生的TNF-α、IL-6、IFN-α...目的:本实验阐述了Rab7对Raw264.7巨噬细胞中R848激活TLR7(Toll like receptor-7)所产生的细胞因子的影响,并探讨Rab7对MAPK信号转导的影响。方法:使用Rab7刺激silence-Rab7-Raw264.7细胞,检测R848激活TLR7后产生的TNF-α、IL-6、IFN-α、IFN-β与IP-10,通过Western blot检测MAPK的磷酸化水平,分析Rab7对MAPK的信号转导的作用。结果:Rab7对TLR7激活后细胞因子的产生具有抑制作用,Rab7对MAPK信号通路具有抑制作用。结论:本实验结果进一步说明Rab7是TLR7信号转导通路的负向调控因子,并且参与到MAPK信号通路中。展开更多
基金supported by the grants from the National Natural Science Foundation of China(32201025 to D.T.,32071214 to S.Q.,32470738 to S.Q.,and 32471311 to D.T.)。
文摘Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a guanine nucleotide exchange factor(GEF)for the RAB GTPase RAB7A and is indispensable for directing RAB7A recruitment to autophagosome or lysosomal membranes.Despite its critical role,the precise molecular mechanism underlying the assembly of the human MON1A-CCZ1(HsMC1)complex and its specific GEF activity towards RAB7A has remained unclear.In this study,we report the high-resolution cryo-electron microscopy(cryo-EM)structure of the HsMC1 GEF domain in a complex with the nucleotide-free RAB7A^(N125I)at 2.85 A resolution.Our structural data demonstrate that engagement with the HsMC1 complex induces marked conformational shifts in the phosphate-binding loop(P-loop)and SwitchⅠ/Ⅱregions of RAB7A.A striking feature of this complex is the direct interaction between the P-loop of RAB7A and CCZ1,a structural detail not previously observed.Furthermore,biochemical assays targeting residues within InterfaceⅠorⅡof the HsMC1-RAB7A complex highlight their critical role in mediating the interaction and suggest a unique mechanism for nucleotide exchange facilitated by the HsMC1 complex.These findings provide novel molecular insights into the functional mechanisms of the HsMC1-RAB7A complex,offering a robust structural framework to inform future investigations into disease-related targets and therapeutic development.
文摘目的:本实验阐述了Rab7对Raw264.7巨噬细胞中R848激活TLR7(Toll like receptor-7)所产生的细胞因子的影响,并探讨Rab7对MAPK信号转导的影响。方法:使用Rab7刺激silence-Rab7-Raw264.7细胞,检测R848激活TLR7后产生的TNF-α、IL-6、IFN-α、IFN-β与IP-10,通过Western blot检测MAPK的磷酸化水平,分析Rab7对MAPK的信号转导的作用。结果:Rab7对TLR7激活后细胞因子的产生具有抑制作用,Rab7对MAPK信号通路具有抑制作用。结论:本实验结果进一步说明Rab7是TLR7信号转导通路的负向调控因子,并且参与到MAPK信号通路中。
