The Mechanism of Milk Lipid Secretion

乳脂分泌的机制

• 批准号: 7034219
• 负责人: IAN HEYWOOD MATHER
• 金额: $ 28.46万
• 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/7034219
• 关键词: Adenoviridae; affinity chromatography; confocal scanning microscopy; gel filtration chromatography; gene expression; genetic promoter element; genetically modified animals; glycoproteins; green fluorescent proteins; lactation; lipid transport; mammary epithelium; milk; molecular /cellular imaging; protein binding; protein localization; protein protein interaction; protein transport; tissue /cell culture; transfection /expression vector; xanthine oxidase

DESCRIPTION (provided by applicant): 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 (Btn1a1) 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 Btn1a1-/- 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 mammary explants 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.

描述（由申请人提供）：长期目标是确定哺乳期乳腺上皮细胞的乳脂分泌机制。尽管母乳对于哺乳新生儿的生存和营养很重要，但包括脂质和蛋白质在内的主要成分（包括脂质和蛋白质）的主要成分的分子和细胞机制仍然未知。出于未知原因，大约5％的母乳喂养母亲的泌乳失败。在这项提议的工作中，将研究牛奶蛋白，丁烷蛋白（BTN1A1）和黄嘌呤脱氢酶/氧化酶（XDH）的潜在作用，研究将研究牛奶脂液滴的分泌。 BTN1A1和XDH是第一个与牛奶脂分泌有关的蛋白质，因为各个基因的消融严重破坏了牛奶脂质的调节分泌。我们将检验以下假设：BTN1A1被转运到顶端质膜，并与XDH结合在细胞内脂质液滴的表面上，形成了接头蛋白蛋白复合物，对于从细胞中萌芽和释放脂质必不可少。通过通过GST下拉测定法和色谱柱色谱法测定在蛋白质截短形式之间结合，可以鉴定出BTN1A1和XDH中的胞质尾部的交互结构域。在体外鉴定的BTN1A1和XDH的交互式结构域将通过表达由乳腺特异性启动子驱动的转基因的野生型或截短的功能形式来在体内筛选。野生型BTN1A1或其功能结构域应在BTN1A1 - / - 小鼠中恢复野生型表型，但是XDH的功能结构域应抑制野生型小鼠中的脂质分泌，通过以优势负方式作用。 BTN1A1和XDH的细胞内分布和靶向动力学将由由用腺病毒载体转导的腺体载体制备的乳腺外植体的共聚焦显微镜确定，这些腺病毒载体编码了两种蛋白质的荧光标记的融合蛋白。如果将BTN1A1运输到顶端膜，并且在脂质双层中将其移动较低，则应支持该假设的假设。 XDH应与细胞质中的脂质液滴结合，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。该项目将检验以下假设：牛奶蛋白，丁烷蛋白和黄嘌呤氧化酶对于分泌牛奶脂质，从而增强我们对人类泌乳缺陷的潜在能力至关重要。