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。