The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting tr...The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting trimer and a membrane-deforming subunit of sorting nexin proteins. Recent studies have shown that the isoforms, sorting nexin 5 (SNX5) and SNX6, have played distinctive regulatory roles in retrograde membrane trafficking. However, the molecular insight determined functional differences within the proteins remains unclear. We reported that SNX5 and SNX6 had distinct binding affinity to the cargo protein vesicular monoamine transporter 2 (VMAT2). SNX5, but not SNX6, specifically interacted with VMAT2 through the Phox domain, which contains an alpha-helix binding motif. Using chimeric mutagenesis, we identified that several key residues within this domain were unique in SNX5, but not SNX6, and played an auxiliary role in its binding to VMAT2. Importantly, we generated a set of mutant SNX6, in which the corresponding key residues were mutated to those in SNX5. In addition to the gain in binding affinity to VMAT2, their overexpression functionally rescued the altered retrograde trafficking of VMAT2 induced by siRNA-mediated depletion of SNX5. These data strongly suggest that SNX5 and SNX6 have different functions in retrograde membrane trafficking, which is determined by the different structural elements within the Phox domain of two proteins. Our work provides a new information on the role of SNX5 and SNX6 in the molecular regulation of retrograde membrane trafficking and vesicular membrane targeting in monoamine neurotransmission and neurological diseases.展开更多
Ricin is a highly toxic type 2 ribosome-inactivating protein(RIP)which is extracted from the seeds of castor beans.Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far.In t...Ricin is a highly toxic type 2 ribosome-inactivating protein(RIP)which is extracted from the seeds of castor beans.Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far.In this study,by structural modification of a retrograde transport blocker Retro-2cyc1,a series of novel compounds were obtained.The primary screen revealed that compound 27 has an improved antiricin activity compare to positive control.In vitro pre-exposure evaluation in Madin-Darby Canine Kidney(MDCK)cells demonstrated that 27 is a powerful anti-ricin compound with an EC50 of 41.05 nmol/L against one LC(lethal concentration,5.56 ng/mL)of ricin.Further studies surprisingly indicated that 27 confers post-exposure activity against ricin intoxication.An in vivo study showed that 1 h post-exposure administration of 27 can improve the survival rate as well as delay the death of ricin-intoxicated mice.A drug combination of 27 with monoclonal antibody mAb4 C13 rescued mice from one LD(lethal dose)ricin challenge and the survival rate of tested animals is 100%.These results represent,for the first time,indication that small molecule retrograde transport blocker confers both in vitro and in vivo post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.展开更多
In this experimental study we coupled nerve growth factor (NGF) with notatin by dicyclohexylcarbodiimide to label NGF and observed the effects of NGF on axonal retrograde transport after axonal injury of motoneurons w...In this experimental study we coupled nerve growth factor (NGF) with notatin by dicyclohexylcarbodiimide to label NGF and observed the effects of NGF on axonal retrograde transport after axonal injury of motoneurons with the aid of notatin chemiluminescence system. The results showed that NGF could be transported through axon not only to sensory neurons in dorsal root ganglia but also to motoneurons in spinal cord ventral horn.展开更多
The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network （TGN）. It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps3...The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network （TGN）. It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins （SNXs）. Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2＇s dimerization partners, SNX6, interacts with the p150Gued subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-pl50Gued interaction. Disruption of the SNX6-pl50Glued interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.展开更多
AIM:To investigate retrograde tracer transport by gastric enteric neurons in insulin resistant mice with low or high glycosylated hemoglobin(Hb).METHODS:Under anesthesia,the retrograde tracer fluorogold was superficia...AIM:To investigate retrograde tracer transport by gastric enteric neurons in insulin resistant mice with low or high glycosylated hemoglobin(Hb).METHODS:Under anesthesia,the retrograde tracer fluorogold was superficially injected into the fundus or antrum using a microsyringe in KK Cg-Ay/J mice prior to onset of type 2 diabetes mellitus(T2DM;4 wk of age),at onset of T2DM(8 wk of age),and after 8,16,or 24 wk of untreated T2DM and in age-matched KK/HIJ mice.Six days later,mice were sacrificed by CO 2 narcosis followed by pneumothorax.Stomachs were removed and fixed.Sections from fundus,corpus and antrum were excised and mounted on a glass slide.Tracer-labeled neurons were viewed using a microscope and manually counted.Data were expressed as the number of neurons in short and long descending and ascending pathways and in local fundus and antrum pathways,and the number of neurons in all regions labeled after injection of tracer into either the fundus or the antrum.RESULTS:By 8 wk of age,body weights of KKAy mice(n=12,34±1 g) were heavier than KK mice(n=17,29±1 g;F(4,120)=4.414,P=0.002] and glycosylated Hb was higher [KK:(n=7),4.97%±0.04%;KKAy:(n=6),6.57%±0.47%;F(1,26)=24.748,P<0.001].The number of tracer labeled enteric neurons was similar in KK and KKAy mice of all ages in the short descending pathway [F(1,57)=2.374,P=0.129],long descending pathway [F(1,57)=0.922,P=0.341],local fundus pathway [F(1,53)=2.464,P=0.122],local antrum pathway [F(1,57)=0.728,P=0.397],and short ascending pathway [F(1,53)=2.940,P=0.092].In the long ascending pathway,fewer tracer-labeled neurons were present in KKAy as compared to KK mice [KK:(n=34),302±17;KKAy:(n=29),230±15;F(1,53)=8.136,P=0.006].The number of tracer-labeled neurons was decreased in all mice by 16 wk as compared to 8 wk of age in the short descending pathway [8 wk:(n=15),305±26;16 wk:(n=13),210±30;F(4,57)=9.336,P<0.001],local antrum pathway [8 wk:(n=15),349±20;16 wk:(n=13),220±33;F(4,57)=8.920,P<0.001],short ascending pathway [8 wk:(n=14),392±15;16 wk:(n=14),257±33;F(4,53)=17.188,P<0.001],and long ascending pathway [8 wk:(n=14),379±39;16 wk:(n=14),235±26;F(4,53)=24.936,P<0.001].The number of tracer-labeled neurons decreased at 24 wk of age in the local fundus pathway [8 wk:(n=14),33±11;24 wk:(n=12),3±2;F(4,53)=5.195,P=0.001] and 32 wk of age in the long descending pathway [8 wk:(n=15),16±3;32 wk:(n=12),3±2;F(4,57)=2.944,P=0.028].The number of tracer-labeled enteric neurons was correlated to final body weight for local fundus and ascending pathways [KK:(n=34),r =-0.746,P<0.001;KKAy:(n=29),r =-0.842,P<0.001] as well as local antrum and descending pathways [KK(n=36),r =-0.660,P<0.001;KKAy(n=31),r=-0.622,P<0.001].In contrast,glycosylated Hb was not significantly correlated to number of tracer-labeled neurons [KK(n=17),r =-0.164,P=0.528;KKAy(n= 16),r =-0.078,P=0.774].CONCLUSION:Since uncontrolled T2DM did not uniformly impair tracer transport in gastric neurons,long ascending neurons may be more susceptible to persistent hyperglycemia and low effective insulin.展开更多
A number of fluorescent retrograde neuronal tracers have been introduced recently. However, they all have some shortcomings. We have found that a home-made anion fluorescent dye, Titan Yellow (TY; CP; Ting Xiu, Shangh...A number of fluorescent retrograde neuronal tracers have been introduced recently. However, they all have some shortcomings. We have found that a home-made anion fluorescent dye, Titan Yellow (TY; CP; Ting Xiu, Shanghai), was transported retrogradely through axons to their parent cell bodies, with neither migration out of labeled neurons nor tissue damage. Twenty-seven adults albino rats were used in this study.展开更多
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.31371436 and 8157051134 to Y.L.)by the laboratory start-up grant from Nanjing Medical University(to Y.L.).
文摘The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting trimer and a membrane-deforming subunit of sorting nexin proteins. Recent studies have shown that the isoforms, sorting nexin 5 (SNX5) and SNX6, have played distinctive regulatory roles in retrograde membrane trafficking. However, the molecular insight determined functional differences within the proteins remains unclear. We reported that SNX5 and SNX6 had distinct binding affinity to the cargo protein vesicular monoamine transporter 2 (VMAT2). SNX5, but not SNX6, specifically interacted with VMAT2 through the Phox domain, which contains an alpha-helix binding motif. Using chimeric mutagenesis, we identified that several key residues within this domain were unique in SNX5, but not SNX6, and played an auxiliary role in its binding to VMAT2. Importantly, we generated a set of mutant SNX6, in which the corresponding key residues were mutated to those in SNX5. In addition to the gain in binding affinity to VMAT2, their overexpression functionally rescued the altered retrograde trafficking of VMAT2 induced by siRNA-mediated depletion of SNX5. These data strongly suggest that SNX5 and SNX6 have different functions in retrograde membrane trafficking, which is determined by the different structural elements within the Phox domain of two proteins. Our work provides a new information on the role of SNX5 and SNX6 in the molecular regulation of retrograde membrane trafficking and vesicular membrane targeting in monoamine neurotransmission and neurological diseases.
基金the financial supports of the National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2018ZX09711003-001-001)of China.
文摘Ricin is a highly toxic type 2 ribosome-inactivating protein(RIP)which is extracted from the seeds of castor beans.Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far.In this study,by structural modification of a retrograde transport blocker Retro-2cyc1,a series of novel compounds were obtained.The primary screen revealed that compound 27 has an improved antiricin activity compare to positive control.In vitro pre-exposure evaluation in Madin-Darby Canine Kidney(MDCK)cells demonstrated that 27 is a powerful anti-ricin compound with an EC50 of 41.05 nmol/L against one LC(lethal concentration,5.56 ng/mL)of ricin.Further studies surprisingly indicated that 27 confers post-exposure activity against ricin intoxication.An in vivo study showed that 1 h post-exposure administration of 27 can improve the survival rate as well as delay the death of ricin-intoxicated mice.A drug combination of 27 with monoclonal antibody mAb4 C13 rescued mice from one LD(lethal dose)ricin challenge and the survival rate of tested animals is 100%.These results represent,for the first time,indication that small molecule retrograde transport blocker confers both in vitro and in vivo post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.
文摘In this experimental study we coupled nerve growth factor (NGF) with notatin by dicyclohexylcarbodiimide to label NGF and observed the effects of NGF on axonal retrograde transport after axonal injury of motoneurons with the aid of notatin chemiluminescence system. The results showed that NGF could be transported through axon not only to sensory neurons in dorsal root ganglia but also to motoneurons in spinal cord ventral horn.
基金We thank Yingfang Liu （Institute of Biophysics, Chinese Acad- emy of Sciences） for advice on PX domain structure and SNX6 mutations. We are particularly grateful to Yanmin Yang （Stanford University, USA） for insightful discussions and the Flag-MAP1B LC construct. We also thank Juan S Bonifacino （NIH, USA） for the rabbit anti-CI-MPR antibody, Hiroyoshi Ariga （Hokkaido University, Japan） for Flag- and HA-tagged human SNX6 overexpression constructs, and Li Yu （Tsinghua University, China） for the YFP-EEA1 expression construct. We thank Chonglin Yang （Institute of Genetics and Developmental Biology, Chinese Academy of Sciences）, Dahua Chen （Institute of Zoology, Chinese Academy of Sciences） and Li Yu for critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China （30770675） and Chinese Academy of Sciences （KSCX1-YW-R-37）. J-J Liu is supported by the CAS 100-Tal- ents Program.
文摘The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network （TGN）. It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins （SNXs）. Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2＇s dimerization partners, SNX6, interacts with the p150Gued subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-pl50Gued interaction. Disruption of the SNX6-pl50Glued interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.
基金Supported by Office of Research and Sponsored Programs,Midwestern University, Downers Grove, IL, United States
文摘AIM:To investigate retrograde tracer transport by gastric enteric neurons in insulin resistant mice with low or high glycosylated hemoglobin(Hb).METHODS:Under anesthesia,the retrograde tracer fluorogold was superficially injected into the fundus or antrum using a microsyringe in KK Cg-Ay/J mice prior to onset of type 2 diabetes mellitus(T2DM;4 wk of age),at onset of T2DM(8 wk of age),and after 8,16,or 24 wk of untreated T2DM and in age-matched KK/HIJ mice.Six days later,mice were sacrificed by CO 2 narcosis followed by pneumothorax.Stomachs were removed and fixed.Sections from fundus,corpus and antrum were excised and mounted on a glass slide.Tracer-labeled neurons were viewed using a microscope and manually counted.Data were expressed as the number of neurons in short and long descending and ascending pathways and in local fundus and antrum pathways,and the number of neurons in all regions labeled after injection of tracer into either the fundus or the antrum.RESULTS:By 8 wk of age,body weights of KKAy mice(n=12,34±1 g) were heavier than KK mice(n=17,29±1 g;F(4,120)=4.414,P=0.002] and glycosylated Hb was higher [KK:(n=7),4.97%±0.04%;KKAy:(n=6),6.57%±0.47%;F(1,26)=24.748,P<0.001].The number of tracer labeled enteric neurons was similar in KK and KKAy mice of all ages in the short descending pathway [F(1,57)=2.374,P=0.129],long descending pathway [F(1,57)=0.922,P=0.341],local fundus pathway [F(1,53)=2.464,P=0.122],local antrum pathway [F(1,57)=0.728,P=0.397],and short ascending pathway [F(1,53)=2.940,P=0.092].In the long ascending pathway,fewer tracer-labeled neurons were present in KKAy as compared to KK mice [KK:(n=34),302±17;KKAy:(n=29),230±15;F(1,53)=8.136,P=0.006].The number of tracer-labeled neurons was decreased in all mice by 16 wk as compared to 8 wk of age in the short descending pathway [8 wk:(n=15),305±26;16 wk:(n=13),210±30;F(4,57)=9.336,P<0.001],local antrum pathway [8 wk:(n=15),349±20;16 wk:(n=13),220±33;F(4,57)=8.920,P<0.001],short ascending pathway [8 wk:(n=14),392±15;16 wk:(n=14),257±33;F(4,53)=17.188,P<0.001],and long ascending pathway [8 wk:(n=14),379±39;16 wk:(n=14),235±26;F(4,53)=24.936,P<0.001].The number of tracer-labeled neurons decreased at 24 wk of age in the local fundus pathway [8 wk:(n=14),33±11;24 wk:(n=12),3±2;F(4,53)=5.195,P=0.001] and 32 wk of age in the long descending pathway [8 wk:(n=15),16±3;32 wk:(n=12),3±2;F(4,57)=2.944,P=0.028].The number of tracer-labeled enteric neurons was correlated to final body weight for local fundus and ascending pathways [KK:(n=34),r =-0.746,P<0.001;KKAy:(n=29),r =-0.842,P<0.001] as well as local antrum and descending pathways [KK(n=36),r =-0.660,P<0.001;KKAy(n=31),r=-0.622,P<0.001].In contrast,glycosylated Hb was not significantly correlated to number of tracer-labeled neurons [KK(n=17),r =-0.164,P=0.528;KKAy(n= 16),r =-0.078,P=0.774].CONCLUSION:Since uncontrolled T2DM did not uniformly impair tracer transport in gastric neurons,long ascending neurons may be more susceptible to persistent hyperglycemia and low effective insulin.
文摘A number of fluorescent retrograde neuronal tracers have been introduced recently. However, they all have some shortcomings. We have found that a home-made anion fluorescent dye, Titan Yellow (TY; CP; Ting Xiu, Shanghai), was transported retrogradely through axons to their parent cell bodies, with neither migration out of labeled neurons nor tissue damage. Twenty-seven adults albino rats were used in this study.
