The Mechanism of Milk Lipid Secretion
乳脂分泌的机制
基本信息
- 批准号:7356465
- 负责人:
- 金额:$ 26.3万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2006
- 资助国家:美国
- 起止时间:2006-05-01 至 2010-02-28
- 项目状态:已结题
- 来源:
- 关键词:AblationAdenovirus VectorAffinity ChromatographyApicalBindingBiochemical GeneticsBiologicalBiological AssayBreast FeedingCell membraneCellsChimeric ProteinsColumn ChromatographyComplexConfocal MicroscopyCytoplasmCytoplasmic TailDataDefectDominant-Negative MutationEpithelial CellsFatty acid glycerol estersFluorescence MicroscopyGel ChromatographyGenesGlandGlutathione S-TransferaseGreen Fluorescent ProteinsHumanHuman MilkIn VitroKnock-outKnockout MiceLabelLactationLanguageLengthLifeLinkLipid BilayersLipidsLocationMammalian CellMammary glandMembraneMilkMilk ProteinsMolecularMothersMusMutant Strains MiceOxidasesPhenotypeProteinsResearch PersonnelRoleRuptureScreening procedureSurfaceSurvivorsTestingTissuesTransgenesTriglyceridesWeaningWeightWild Type MouseWorkXanthine DehydrogenaseXanthine Oxidaseapical membranebutyrophilincellular imagingin vitro Assayin vivomonomerneonatenutritionprogramspromoterpupstoichiometry
项目摘要
The long-term objective is to determine the mechanism of milk-lipid secretion from mammary epithelial cells
during lactation. Despite the importance of breast milk for the survival and nutrition of the suckling neonate,
molecular and cellular mechanisms underlying secretion of the principal components of milk including lipid
and protein remain unknown. Lactation fails in about 5% of breast-feeding mothers for unknown reasons. In
this proposed work, the potential role of the milk proteins, butyrophilin (Btnlat) and xanthine
dehydrogenase/ oxidase (Xdh), in the secretion of milk-lipid droplets will be studied. Btn1a1 and Xdh are the
first proteins to be genetically linked to milk-lipid secretion because ablation of the respective genes severely
disrupts the regulated secretion of milk lipid. We will test the hypothesis that Btn1a1 is transported to the
apical plasma membrane and binds to Xdh on the surface of intracellular lipid droplets forming a linker
protein complex essential for the budding and release of lipid from the cell. Interactive domain(s) in the
cytoplasmic tail of Btn1a1 and in Xdh will be identified by assaying for binding between truncated forms of
the proteins by GST pull-down assays and column chromatography. Interactive domains of Btn1a1 and Xdh,
identified in vitro, will be screened for function in vivo by expressing wild-type or truncated functional forms of
either protein from transgenes driven by a mammary-specific promoter. Wild-type Btn1a1 or its functional
domain should restore the wild-type phenotype in Btn1aT'~ mice but the functional domain of Xdh should
inhibit lipid secretion in wild-type mice by acting in a dominant negative manner. The intracellular distribution
and targeting dynamics of Btn1a1 and Xdh will be determined by confocal microscopy of mammaryexplants
prepared from glands transduced with adenoviral vectors encoding fluorescently labeled fusion proteins of
either protein. The working hypothesis will be supported if Btn1a1 is transported to the apical membrane
and becomes less mobile in the lipid bilayer as it is incorporated into budding lipid droplets. Xdh should bind
to lipid droplets in the cytoplasm and colocalize with Btn1a1 at the apical surface.
Lay language: Lactation fails in about 5% of breast-feeding mothers. This project will test the hypothesis
that the milk proteins, butyrophilin and xanthine oxidase, are essential for the secretion of milk lipid and thus
enhance our understanding of, and potential ability to treat lactation deficiencies in humans.
长期目标是确定乳腺上皮细胞分泌乳脂的机制
哺乳期间。尽管母乳对于哺乳新生儿的生存和营养非常重要,
乳汁主要成分(包括脂质)分泌的分子和细胞机制
和蛋白质仍然未知。大约 5% 的母乳喂养母亲由于不明原因无法哺乳。在
这项拟议的工作,乳蛋白、嗜丁蛋白 (Btnlat) 和黄嘌呤的潜在作用
将研究乳脂滴分泌中的脱氢酶/氧化酶(Xdh)。 Btn1a1 和 Xdh 是
第一个与乳脂分泌在遗传上相关的蛋白质,因为各个基因的严重消融
扰乱乳脂的调节分泌。我们将测试 Btn1a1 被转运到
顶端质膜并与细胞内脂滴表面的 Xdh 结合形成连接子
蛋白质复合物对于细胞中脂质的出芽和释放至关重要。交互域
Btn1a1 和 Xdh 的细胞质尾部将通过测定截短形式之间的结合来鉴定
通过 GST Pull-down 分析和柱色谱分析蛋白质。 Btn1a1 和 Xdh 的交互域,
体外鉴定的,将通过表达野生型或截短的功能形式来筛选体内功能
来自由乳腺特异性启动子驱动的转基因的任一蛋白质。野生型 Btn1a1 或其功能
结构域应该恢复 Btn1aT'~ 小鼠的野生型表型,但 Xdh 的功能结构域应该
通过显性失活方式抑制野生型小鼠的脂质分泌。细胞内分布
Btn1a1 和 Xdh 的靶向动态将通过乳腺外植体的共聚焦显微镜确定
由用编码荧光标记融合蛋白的腺病毒载体转导的腺体制备
任一蛋白质。如果 Btn1a1 被转运到顶膜,则工作假设将得到支持
当它并入出芽的脂滴时,它在脂质双层中的移动性变得较低。 Xdh 应该绑定
细胞质中的脂滴并与顶端表面的 Btn1a1 共定位。
通俗语言:大约 5% 的母乳喂养母亲会出现泌乳失败。该项目将测试假设
乳蛋白、嗜丁酸蛋白和黄嘌呤氧化酶对于乳脂的分泌至关重要,因此
增强我们对治疗人类泌乳缺陷的理解和潜在能力。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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IAN HEYWOOD MATHER其他文献
IAN HEYWOOD MATHER的其他文献
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{{ truncateString('IAN HEYWOOD MATHER', 18)}}的其他基金
SORTING OF PLASMA MEMBRANE PROTEINS IN EPITHELIAL CELLS
上皮细胞中血浆膜蛋白的分类
- 批准号:
3023196 - 财政年份:1990
- 资助金额:
$ 26.3万 - 项目类别:
GORDON RESEARCH CONFERENCE ON MAMMARY GLAND BIOLOGY
戈登乳腺生物学研究会议
- 批准号:
3433961 - 财政年份:1987
- 资助金额:
$ 26.3万 - 项目类别:
GORDON RESEARCH CONFERENCE ON MAMMARY GLAND BIOLOGY
戈登乳腺生物学研究会议
- 批准号:
3433838 - 财政年份:1985
- 资助金额:
$ 26.3万 - 项目类别:
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