人类TBC1D7蛋白的表达纯化及其在mTOR通路中的作用机制研究

mTOR信号通路在真核细胞代谢调控中发挥着关键的调控作用,该通路核心元件mTORC1复合物的活性受其上游TSC复合物负调控,TSC复合物包括TSC1、TSC2、TBC1D7三种蛋白.以TBC1D7蛋白为研究对象,通过氨基酸序列比对显示多物种来源的TBC1D7蛋白具有较高的保守性,且蛋白三维结构分析证实其分子表面存在高度保守区域.随后,构建了人类TBC1D7蛋白原核表达载体并在大肠杆菌中表达得到全长TBC1D7蛋白,经过酶切去除标签和凝胶排阻层析得到了高纯度TBC1D7蛋白.另一方面,通过在HCM细胞中对TBC1D7进行敲降和过表达,证实了TBC1D7的表达会直接升高mTORC1的活性;反之,升高TBC1D7的表达则会抑制mTORC1的活性.本研究表达和纯化了人类TBC1D7蛋白,并探讨了在人类心肌细胞中TBC1D7的表达与其下游mTOR信号通路之间的联系,为人类TBC1D7蛋白和mTOR信号通路的进一步研究打下了良好基础.     

Relationships between D1 protein, xanthophyll cycle and photodamage-resistant capacity in rice (Orysa sativa L.)

Relationships between D1 protein, xanthophyll cycle and subspecific difference of photodamage-resistant capacity have been studied inO. japonica rice varieties 02428 and 029 (photoinhibition-tolerance) andO. indica rice varieties 3037 and Palghar (photoinhibition-sensitivity) and their reciprocal cross F₁ hybrids after photoinhibitory treatment. It was shown that PS II photochemical efficiency (F _v /F _m) decreased, and xanthophyll cycle from violaxanthin (V), via anaxanthin (A), to zeaxanthin (Z) was enhanced and non-photochemical quenching (qN) increased accordingly in SM-pretreated leaves of rice when the synthesis of D1 protein was inhibited, and that there was a decrease inqN and, as a result, more loss of D1 protein and a big decrease inF _v/F _m in DTT-pretreated leaves when xanthophyll cycle was inhibited.O. japonica subspecies had a higher maintaining capacity of D1 protein and a decrease ofF _v/F _m in a more narrow range, and exhibited more resistance against photodamage, as compared withO. indica subspecies. The above physiological indexes in reciprocal cross F₁ hybrids, though between the values of their parents, were closer to maternal lines than to paternal lines. Experimental results support the concept that the turnover capacity for D1 protein is an important physiological basis of photoinhibition-tolerance, and will provide the physiological basis for selection of the photoinhibition-tolerant parents and develop a new approach to breed hybrids with high photosynthetic efficiency.     

Relationships between D1 protein, xanthophyll cycle and photodamage-resistant capacity in rice (Orysa sativa L.)

Relationships between D1 protein, xanthophyll cycle and subspecific difference of photodamage-resistant capacity have been studied in O. japonica rice varieties 02428 and 029 (photoinhibition-tolerance) and O. indica rice varieties 3037 and Palghar (photoinhibition-sensitivity) and their reciprocal cross F1 hybrids after photoinhibitory treatment. It was shown that PSⅡ photochemical efficiency (Fv /Fm) decreased, and xanthophyll cycle from violaxanthin (V), via anaxanthin (A), to zeaxanthin (Z) was enhanced and non-photochemical quenching (qN) increased accordingly in SM-pretreated leaves of rice when the synthesis of D1 protein was inhibited, and that there was a decrease in qN and, as a result, more loss of D1 protein and a big decrease in Fv /Fm in DTT-pretreated leaves when xanthophyll cycle was inhibited. O. japonica subspecies had a higher maintaining capacity of D1 protein and a decrease of Fv /Fm in a more narrow range, and exhibited more resistance against photodamage, as compared with O. indica subspecies. The above physiological indexes in reciprocal cross F1 hybrids, though between the values of their parents, were closer to maternal lines than to paternal lines. Experimental results support the concept that the turnover capacity for D1 protein is an important physiological basis of photoinhibition-tolerance, and will provide the physiological basis for selection of the photoinhibition-tolerant parents and develop a new approach to breed hybrids with high photosynthetic efficiency.     

Specific degradation of photosystem II D1 protein by a protease （Air3815） in heterocysts of the cyanobacterium Anabaena sp. PCC7120

Some filamentous cyanobacteria form heterocysts under conditions lacking combined nitrogen for nitrogen fixation.Photosystem II is removed from heterocyst during the process of cell differentiation.Here,we demonstrate that Alr3815 is a protease that is capable of degrading D1 protein of photosystem II.Strain-322,which lacks alr3815,is impaired in nitrogen fixation in air because some oxygen evolving activity is retained in its heterocysts.Our results also suggest that calcium may play a regulatory role in D1 degradation during heterocyst differentiation.     

Involvement of DEG5 and DEG8 proteases in the turnover of the photosystem II reaction center D1 protein under heat stress in Arabidopsis thaliana

Deg5,deg8 and the double mutant,deg5deg8 of Arabidopsis thaliana were used to study the physiological role of the DEG proteases in the repair cycle of photosystem II (PSII) under heat stress. PSII activity in deg mutants showed increased sensitivity to heat stress, and the extent of this effect was greater in the double mutant, deg5deg8, than in the single mutants, deg5 and deg8. Degradation of the D1 protein was slower in the mutants than in the WT plants. Furthermore, the levels of other PSII reaction center proteins tested remained relatively stable in the mutant and WT plants following high-temperature treatment. Thus, our results indicate that DEG5 and DEG8 may have synergistic function in degradation of D1 protein under heat stress.