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中乳脂分泌的机理 在哺乳期间。尽管母乳对于哺乳新生儿的生存和营养很重要，但 牛奶主要成分（包括脂质）的主要成分的分子和细胞机制 蛋白质仍然未知。出于未知原因，大约5％的母乳喂养母亲的泌乳失败。在 这项提出的工作，牛奶蛋白，丁烷蛋白（BTNLAT）和黄嘌呤的潜在作用 将研究脱氢酶/氧化酶（XDH），在分泌乳脂液滴中。 BTN1A1和XDH是 首先要与牛奶脂质分泌有基因相关的蛋白质，因为各个基因的消融严重消融 破坏牛奶脂质的调节分泌。我们将检验以下假设，即BTN1A1被运输到 顶端质膜并与XDH结合在形成接头的细胞内脂质液滴表面 蛋白质复合物对于从细胞中萌芽和释放脂质必不可少。交互式域中 通过分析截短形式的结合，将确定BTN1A1和XDH中的细胞质尾巴 GST下拉测定法和色谱柱色谱法的蛋白质。 BTN1A1和XDH的交互式域， 在体外确定，将通过表达野生型或截短的功能形式来筛选在体内的功能 由乳腺特异性启动子驱动的转基因的蛋白质。野生型BTN1A1或其功能 域应恢复btn1at'〜小鼠中的野生型表型，但XDH的功能域应 通过以显性阴性方式起作用，抑制野生型小鼠的脂质分泌。细胞内分布 BTN1A1和XDH的靶向动力学将通过乳腺爆炸剂的共聚焦显微镜确定 由用腺病毒载体转导的腺体制备，编码荧光标记的融合蛋白 任何一种蛋白质。如果将BTN1A1运输到顶部膜，将支持工作假设 并在脂质双层中变成较少的移动性，因为它被掺入了萌芽的脂质液滴中。 XDH应该绑定 要在细胞质中的脂质液滴，并与顶部表面的BTN1A1共定位。 外行语言：哺乳期约5％的母乳喂养母亲失败。该项目将检验假设 牛奶蛋白，丁醇和黄嘌呤氧化酶对于牛奶脂质的分泌至关重要 增强我们对治疗人类泌乳缺陷的潜在能力的理解。