POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effec...POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin （Ub）-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu- man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en- dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference （RNAi）, suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.展开更多
Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vanname...Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.展开更多
基金Acknowledgments We are grateful to Dr DA Melton （Harvard University） for shar- ing his human ES cells with us. The study was supported by grants from the National High Technology Research and Development Program of China （2006CB943900）, the National Natural Science Foundation of China （General Program, 30500088）, the Shang- hai Jiao Tong University School of Medicine, and the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The study was also supported by the Shanghai Leading Academic Deciline Project （S30201）.
文摘POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin （Ub）-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu- man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en- dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference （RNAi）, suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.
基金Supported by the National Natural Science Foundation of China(Nos. 31001098 and 30771670)the National High Technology R&D Program (863 Program) (No. 2006BAD01A13)+5 种基金the National Basic Research Program of China (973 Program) (No. 2009CB118702)Shanghai Committee of Science and Technology,China (Nos.08DZ1906401,09ZR1409800,10JC1404100)Shanghai Agriculture Science and Technology Key Grant (No.2-1,2009)the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 200802690012)partially by the E-Institute of Shanghai Municipal Education Commission (No. E03009)the Key and Open Laboratory of Marine and Estuarine Fisheries Resources and Ecology,Ministry of Agriculture
文摘Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.
