Background： The purpose of this study was to determine the association of three proteins involved in sperm function on the freezability of porcine semen： the heat shock protein 90 alpha（HSP90a）, the Niemann-Pick d...Background： The purpose of this study was to determine the association of three proteins involved in sperm function on the freezability of porcine semen： the heat shock protein 90 alpha（HSP90a）, the Niemann-Pick disease type C2 protein（NPC2）, and lipocalin-type prostaglandin D synthase（L-PGDS）.Six adult boars（each boar was ejaculated three times, 18 in total） were classified by freezability based on the percentage of functionally competent sperm. The male semen with highest freezability（MHF） and the male semen with lowest freezability（MLF） were centrifuged immediately after collection to separate seminal plasma and spermatozoa to make four possible combinations of these two components and to incubate them for 3 h,adjusting the temperature to 17 °C, to freeze them afterwards. The quantification of proteins was performed in two stages： at zero and at 3 h after incubation of the four combinations.Results： The spermatozoa × incubation time（IT） interaction only had effect（P 〈 0.01） on HSP90 a levels; this protein increased in seminal plasma, after 3 h of incubation, in larger quantity（P 〈 0.05） in combinations with MLF spermatozoa. In relation with the NPC2 protein, two isoforms of 16 and 19 kDa were identified. The 19 kDa isoform was affected（P 〈 0.01） only by the seminal plasma × IT interaction, with superior values（P 〈 0.01） both at zero and three hours of incubation, in the combinations with MHF seminal plasma; and 16 kDa isoform was affected（P 〈 0.01） only by the IT with reduction after 3 h of incubation. The levels of L-PGDS was affected（P 〈 0.01）only by the spermatozoa × IT interaction, which reduced（P 〈 0.01） in combinations with MLF spermatozoa after 3 h of incubation.Conclusions： It is possible to consider that the three proteins evaluated were associated with freezability of boar semen due, especially, to the fact that mixtures with MLF spermatozoa showed greater increase levels of the HSP90 a protein and reduction of L-PGDS in plasma. In addition, the seminal plasma of MHF had higher concentration of the NPC2 of 19 kDa protein, which was reduced by incubating with MHF spermatozoa.展开更多
Niemann-Pick type C2(NPC2) is a lysosome luminal protein that functions in concert with NPC1 to mediate egress of lowdensity lipoprotein-derived cholesterol from lysosome. The nuclear factor kappa B subunit 2(NF-κB2)...Niemann-Pick type C2(NPC2) is a lysosome luminal protein that functions in concert with NPC1 to mediate egress of lowdensity lipoprotein-derived cholesterol from lysosome. The nuclear factor kappa B subunit 2(NF-κB2) protein is a component of NF-κB transcription factor complex critically implicated in immune and inflammatory responses. Here, we report that NF-κB2 regulates intracellular cholesterol transport by controlling NPC2 expression. RNAi-mediated disruption of NF-κB2, as well as other signaling members of the non-canonical NF-κB pathway, caused intracellular cholesterol accumulation. Blockage of the non-canonical NF-κB pathway suppressed NPC2 expression, whereas Lymphotoxin β receptor(LTβR) activation or Baff receptor(BaffR) stimulation up-regulated the mRNA abundance and protein level of NPC2. Further, NF-κB2 activated NPC2 transcription through direct binding to its promoter region. We also observed cholesterol accumulation in NF-κB2-deficient zebrafish embryo and NF-κB2 mutant mice. Collectively, these data identify a regulatory role for the non-canonical NF-κB pathway in intracellular cholesterol trafficking and suggest a link between cholesterol transport and immune system.展开更多
ML(MD-2 related lipid recognition,ML)蛋白超家族是一类能特异性结合脂类物质的蛋白,广泛分布于植物、动物以及真菌中。ML蛋白在哺乳动物中有明确的分类,其中功能研究比较清楚的有MD-2(myeloid differentiation protein-2)和NPC2(Niem...ML(MD-2 related lipid recognition,ML)蛋白超家族是一类能特异性结合脂类物质的蛋白,广泛分布于植物、动物以及真菌中。ML蛋白在哺乳动物中有明确的分类,其中功能研究比较清楚的有MD-2(myeloid differentiation protein-2)和NPC2(Nieman-Pick type C2 protein,NPC2)家族,它们在胆固醇代谢和先天免疫信号传导途径中起重要作用。而ML蛋白家族在昆虫中成员多且复杂,其功能更多元化。本文总结了ML蛋白家族在昆虫中的分类以及其在胆固醇代谢、先天免疫通路、甘油三酯的积累及化学信息素传递中的功能研究进展。脂类物质作为昆虫生长过程中的一类重要物质,对其结合载体的研究,将帮助我们进一步阐明昆虫脂类物质的识别及代谢过程,同时也为害虫防治和益虫利用提供理论指导。展开更多
【目的】蜜蜂触角作为嗅觉器官在蜜蜂的采集、繁衍等方面发挥着极其重要的作用。本研究以中华蜜蜂Apis cerana cerana(简称中蜂)采集蜂触角为材料,通过蛋白质全谱分析鉴定中蜂采集蜂触角所含蛋白质,为研究蜜蜂嗅觉感受机制奠定数据基础...【目的】蜜蜂触角作为嗅觉器官在蜜蜂的采集、繁衍等方面发挥着极其重要的作用。本研究以中华蜜蜂Apis cerana cerana(简称中蜂)采集蜂触角为材料,通过蛋白质全谱分析鉴定中蜂采集蜂触角所含蛋白质,为研究蜜蜂嗅觉感受机制奠定数据基础。【方法】使用LC-MS/MS对中蜂采集蜂触角总蛋白进行鉴定;利用OmicsBean软件对中蜂采集蜂触角中鉴定的所有表达蛋白质进行GO分析和KEGG分析;采用q RT-PCR方法对不同级型中蜂触角中的NPC2(Niemann-Pick proteins of type C2)基因的转录情况进行检测。【结果】LC-MS/MS鉴定结果显示,中蜂采集蜂触角中共鉴定到1231个蛋白,其中包括14种与嗅觉相关的蛋白;GO分析与KEGG分析显示,分子功能分类中,绑定分子功能类别和催化活性功能类别所占比例最多。参与碳代谢、氨基酸、三羧酸循环、脂代谢等代谢通路的蛋白有297个,参与到环境信息加工和发育的有43个,参与到遗传信息加工的有67个,参与到小分子结合的约12%;qRT-PCR检测结果显示,NPC2在雄蜂的触角内表达量最高。【结论】本研究所鉴定到的中蜂采集蜂触角蛋白质为触角的发育及其功能的发挥提供了物质基础与能量基础。此外,嗅觉相关蛋白的分析也为后续深入研究中蜂对气味物质的识别和转运机制提供了分子数据支撑。展开更多
Niemann-Pick type C2 （NPC2） is a type of small soluble protein involved in lipid metabolism and triglyceride accumulation in vertebrates and arthropods. Recent stud- ies have determined that NPC2 also participates i...Niemann-Pick type C2 （NPC2） is a type of small soluble protein involved in lipid metabolism and triglyceride accumulation in vertebrates and arthropods. Recent stud- ies have determined that NPC2 also participates in chemical communication of arthropods. In this work, two novel NPC2 proteins （MmedNPC2a and MmedNPC2b） in Microplitis mediator were identified. Real-time quantitative PCR （qPCR） analysis revealed that Mmed- NPC2a was expressed higher in the antennae than in other tissues of adult wasps compared with MmedNPC2b. Subsequent immunolocalization results demonstrated that NPC2a was located in the lymph cavities of sensilla placodea. To further explore the binding charac- terization of recombinant MmedNPC2a to 54 candidate odor molecules, a fluorescence binding assay was performed. It was found MmedNPC2a could not bind with selected fatty acids, such as linoleic acid, palmitic acid, stearic acid and octadecenoic acid. How- ever, seven cotton volatiles, 4-ethylbenzaldehyde, 3,4-dimethylbenzaldehyde, fl-ionone, linalool, m-xylene, benzaldehyde and trans-2-hexen-l-al showed certain binding abilities with MmedNPC2a. Moreover, the predicted 3D model of MmedNPC2a was composed of seven r-sheets and three pairs of disulfide bridges. In this model, the key binding residues for oleic acid in CjapNPC2 of Camponotus japonicus, Lue68, Lys69, Lys70, Phe97, Thr103 and Phe127, are replaced with Phe85, Ser86, His87, Leu113, Tyr119 and Ile143 in MmedNPC2a, respectively. We proposed that MmedNPC2a in M. mediator may play roles in perception of plant volatiles.展开更多
Endosomal compartments sort and deliver exogenous lipoprotein-derived cholesterol to the endoplasmic reticulum for regulating cellular cholesterol homeostasis.A large number of studies have focused on the removal of e...Endosomal compartments sort and deliver exogenous lipoprotein-derived cholesterol to the endoplasmic reticulum for regulating cellular cholesterol homeostasis.A large number of studies have focused on the removal of endosomal cholesterol,since its accumulation leads to devastating human diseases.Recent studies suggest that cytoplasmic sterol-binding proteins may be involved in endosomal cholesterol transport.In particular,endosome/lysosome-localized or-associated cholesterol-binding proteins may serve as key mediators of cholesterol removal in a non-vesicular manner.Further characterization of these cholesterol-binding proteins will shed light on the molecular mechanisms that regulate endosomal cholesterol sorting.展开更多
基金funded by the Vice-Rector’s Office at Universidad de Caldas
文摘Background： The purpose of this study was to determine the association of three proteins involved in sperm function on the freezability of porcine semen： the heat shock protein 90 alpha（HSP90a）, the Niemann-Pick disease type C2 protein（NPC2）, and lipocalin-type prostaglandin D synthase（L-PGDS）.Six adult boars（each boar was ejaculated three times, 18 in total） were classified by freezability based on the percentage of functionally competent sperm. The male semen with highest freezability（MHF） and the male semen with lowest freezability（MLF） were centrifuged immediately after collection to separate seminal plasma and spermatozoa to make four possible combinations of these two components and to incubate them for 3 h,adjusting the temperature to 17 °C, to freeze them afterwards. The quantification of proteins was performed in two stages： at zero and at 3 h after incubation of the four combinations.Results： The spermatozoa × incubation time（IT） interaction only had effect（P 〈 0.01） on HSP90 a levels; this protein increased in seminal plasma, after 3 h of incubation, in larger quantity（P 〈 0.05） in combinations with MLF spermatozoa. In relation with the NPC2 protein, two isoforms of 16 and 19 kDa were identified. The 19 kDa isoform was affected（P 〈 0.01） only by the seminal plasma × IT interaction, with superior values（P 〈 0.01） both at zero and three hours of incubation, in the combinations with MHF seminal plasma; and 16 kDa isoform was affected（P 〈 0.01） only by the IT with reduction after 3 h of incubation. The levels of L-PGDS was affected（P 〈 0.01）only by the spermatozoa × IT interaction, which reduced（P 〈 0.01） in combinations with MLF spermatozoa after 3 h of incubation.Conclusions： It is possible to consider that the three proteins evaluated were associated with freezability of boar semen due, especially, to the fact that mixtures with MLF spermatozoa showed greater increase levels of the HSP90 a protein and reduction of L-PGDS in plasma. In addition, the seminal plasma of MHF had higher concentration of the NPC2 of 19 kDa protein, which was reduced by incubating with MHF spermatozoa.
基金supported by National Natural Science Foundation of China (91754102, 31771568, 31701030, 31601147, 31600651)111 Project of Ministry of Education of China (B16036)Natural Science Foundation of Hubei Province (2016CFA012)
文摘Niemann-Pick type C2(NPC2) is a lysosome luminal protein that functions in concert with NPC1 to mediate egress of lowdensity lipoprotein-derived cholesterol from lysosome. The nuclear factor kappa B subunit 2(NF-κB2) protein is a component of NF-κB transcription factor complex critically implicated in immune and inflammatory responses. Here, we report that NF-κB2 regulates intracellular cholesterol transport by controlling NPC2 expression. RNAi-mediated disruption of NF-κB2, as well as other signaling members of the non-canonical NF-κB pathway, caused intracellular cholesterol accumulation. Blockage of the non-canonical NF-κB pathway suppressed NPC2 expression, whereas Lymphotoxin β receptor(LTβR) activation or Baff receptor(BaffR) stimulation up-regulated the mRNA abundance and protein level of NPC2. Further, NF-κB2 activated NPC2 transcription through direct binding to its promoter region. We also observed cholesterol accumulation in NF-κB2-deficient zebrafish embryo and NF-κB2 mutant mice. Collectively, these data identify a regulatory role for the non-canonical NF-κB pathway in intracellular cholesterol trafficking and suggest a link between cholesterol transport and immune system.
文摘ML(MD-2 related lipid recognition,ML)蛋白超家族是一类能特异性结合脂类物质的蛋白,广泛分布于植物、动物以及真菌中。ML蛋白在哺乳动物中有明确的分类,其中功能研究比较清楚的有MD-2(myeloid differentiation protein-2)和NPC2(Nieman-Pick type C2 protein,NPC2)家族,它们在胆固醇代谢和先天免疫信号传导途径中起重要作用。而ML蛋白家族在昆虫中成员多且复杂,其功能更多元化。本文总结了ML蛋白家族在昆虫中的分类以及其在胆固醇代谢、先天免疫通路、甘油三酯的积累及化学信息素传递中的功能研究进展。脂类物质作为昆虫生长过程中的一类重要物质,对其结合载体的研究,将帮助我们进一步阐明昆虫脂类物质的识别及代谢过程,同时也为害虫防治和益虫利用提供理论指导。
文摘【目的】蜜蜂触角作为嗅觉器官在蜜蜂的采集、繁衍等方面发挥着极其重要的作用。本研究以中华蜜蜂Apis cerana cerana(简称中蜂)采集蜂触角为材料,通过蛋白质全谱分析鉴定中蜂采集蜂触角所含蛋白质,为研究蜜蜂嗅觉感受机制奠定数据基础。【方法】使用LC-MS/MS对中蜂采集蜂触角总蛋白进行鉴定;利用OmicsBean软件对中蜂采集蜂触角中鉴定的所有表达蛋白质进行GO分析和KEGG分析;采用q RT-PCR方法对不同级型中蜂触角中的NPC2(Niemann-Pick proteins of type C2)基因的转录情况进行检测。【结果】LC-MS/MS鉴定结果显示,中蜂采集蜂触角中共鉴定到1231个蛋白,其中包括14种与嗅觉相关的蛋白;GO分析与KEGG分析显示,分子功能分类中,绑定分子功能类别和催化活性功能类别所占比例最多。参与碳代谢、氨基酸、三羧酸循环、脂代谢等代谢通路的蛋白有297个,参与到环境信息加工和发育的有43个,参与到遗传信息加工的有67个,参与到小分子结合的约12%;qRT-PCR检测结果显示,NPC2在雄蜂的触角内表达量最高。【结论】本研究所鉴定到的中蜂采集蜂触角蛋白质为触角的发育及其功能的发挥提供了物质基础与能量基础。此外,嗅觉相关蛋白的分析也为后续深入研究中蜂对气味物质的识别和转运机制提供了分子数据支撑。
基金This work was supported by the China National ‘973＇ Basic Research Program （2012CBl14104） and the Na- tional Natural Science Foundation of China （31272048, 31471778, and 31672038）.
文摘Niemann-Pick type C2 （NPC2） is a type of small soluble protein involved in lipid metabolism and triglyceride accumulation in vertebrates and arthropods. Recent stud- ies have determined that NPC2 also participates in chemical communication of arthropods. In this work, two novel NPC2 proteins （MmedNPC2a and MmedNPC2b） in Microplitis mediator were identified. Real-time quantitative PCR （qPCR） analysis revealed that Mmed- NPC2a was expressed higher in the antennae than in other tissues of adult wasps compared with MmedNPC2b. Subsequent immunolocalization results demonstrated that NPC2a was located in the lymph cavities of sensilla placodea. To further explore the binding charac- terization of recombinant MmedNPC2a to 54 candidate odor molecules, a fluorescence binding assay was performed. It was found MmedNPC2a could not bind with selected fatty acids, such as linoleic acid, palmitic acid, stearic acid and octadecenoic acid. How- ever, seven cotton volatiles, 4-ethylbenzaldehyde, 3,4-dimethylbenzaldehyde, fl-ionone, linalool, m-xylene, benzaldehyde and trans-2-hexen-l-al showed certain binding abilities with MmedNPC2a. Moreover, the predicted 3D model of MmedNPC2a was composed of seven r-sheets and three pairs of disulfide bridges. In this model, the key binding residues for oleic acid in CjapNPC2 of Camponotus japonicus, Lue68, Lys69, Lys70, Phe97, Thr103 and Phe127, are replaced with Phe85, Ser86, His87, Leu113, Tyr119 and Ile143 in MmedNPC2a, respectively. We proposed that MmedNPC2a in M. mediator may play roles in perception of plant volatiles.
基金supported by research grants from the Ara Parseghian Medical Research Foundationthe National Health and Medical Research Council of Australia(#510271).
文摘Endosomal compartments sort and deliver exogenous lipoprotein-derived cholesterol to the endoplasmic reticulum for regulating cellular cholesterol homeostasis.A large number of studies have focused on the removal of endosomal cholesterol,since its accumulation leads to devastating human diseases.Recent studies suggest that cytoplasmic sterol-binding proteins may be involved in endosomal cholesterol transport.In particular,endosome/lysosome-localized or-associated cholesterol-binding proteins may serve as key mediators of cholesterol removal in a non-vesicular manner.Further characterization of these cholesterol-binding proteins will shed light on the molecular mechanisms that regulate endosomal cholesterol sorting.
