The Mechanism of Milk Lipid Secretion

乳脂分泌的机制

• 批准号: 7356465
• 负责人: IAN HEYWOOD MATHER
• 金额: $ 26.3万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/7356465
• 关键词: Ablation; Adenovirus Vector; Affinity Chromatography; Apical; Binding; Biochemical Genetics; Biological; Biological Assay; Breast Feeding; Cell membrane; Cells; Chimeric Proteins; Column Chromatography; Complex; Confocal Microscopy; Cytoplasm; Cytoplasmic Tail; Data; Defect; Dominant-Negative Mutation; Epithelial Cells; Fatty acid glycerol esters; Fluorescence Microscopy; Gel Chromatography; Genes; Gland; Glutathione S-Transferase; Green Fluorescent Proteins; Human; Human Milk; In Vitro; Knock-out; Knockout Mice; Label; Lactation; Language; Length; Life; Link; Lipid Bilayers; Lipids; Location; Mammalian Cell; Mammary gland; Membrane; Milk; Milk Proteins; Molecular; Mothers; Mus; Mutant Strains Mice; Oxidases; Phenotype; Proteins; Research Personnel; Role; Rupture; Screening procedure; Surface; Survivors; Testing; Tissues; Transgenes; Triglycerides; Weaning; Weight; Wild Type Mouse; Work; Xanthine Dehydrogenase; Xanthine Oxidase; apical membrane; butyrophilin; cellular imaging; in vitro Assay; in vivo; monomer; neonate; nutrition; programs; promoter; pup; stoichiometry

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.

长期的目标是确定乳腺上皮细胞分泌乳脂的机制 在哺乳期。尽管母乳对哺乳期新生儿的生存和营养很重要， 包括脂质在内的乳汁主要成分分泌的分子和细胞机制 蛋白质仍然未知。大约5%的母乳喂养母亲因不明原因而无法哺乳。在 这项工作提出，牛奶蛋白，亲丁酸蛋白（Btnlat）和黄嘌呤的潜在作用， 脱氢酶/氧化酶（Xdh），在乳脂滴的分泌将进行研究。Btn 1a 1和Xdh是 第一个蛋白质被遗传链接到牛奶脂质分泌，因为消融各自的基因严重 破坏了调节的乳脂质分泌。我们将测试Btn 1a 1被运输到 顶端质膜，并与细胞内脂滴表面上的Xdh结合，形成接头 对细胞出芽和释放脂质必不可少的蛋白质复合物。中的交互式域 Btn 1a 1和Xdh中的胞质尾区将通过测定截短形式的 通过GST pull-down分析和柱层析法对蛋白质进行纯化。Btn 1a 1和Xdh的相互作用域， 将通过表达野生型或截短的功能形式的 这两种蛋白质都来自于由乳腺特异性启动子驱动的转基因。野生型Btn 1a 1或其功能性 Xdh的功能结构域应该恢复Btn 1aT-小鼠中的野生型表型，但是Xdh的功能结构域应该恢复Btn 1aT-小鼠中的野生型表型。 以显性负性方式抑制野生型小鼠的脂质分泌。细胞内分布 Btn 1a 1和Xdh的靶向动力学将通过乳房外植体的共聚焦显微镜来确定 由腺病毒载体转导的腺体制备，腺病毒载体编码荧光标记的融合蛋白， 或者蛋白质。如果Btn 1a 1被转运到顶膜，则支持工作假设 并且当它被掺入到出芽的脂滴中时，在脂双层中变得不那么移动的。Xdh应该绑定 在细胞质中的脂滴和共定位与Btn 1a 1在顶端表面。 外行语言：大约5%的母乳喂养母亲哺乳失败。这个项目将测试这个假设 乳蛋白、亲酪蛋白和黄嘌呤氧化酶是乳脂质分泌所必需的， 提高我们对人类哺乳缺陷的理解和潜在的治疗能力。