The Mechanism of Milk Lipid Secretion

乳脂分泌的机制

• 批准号: 7227903
• 负责人: IAN HEYWOOD MATHER
• 金额: $ 27.32万
• 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/7227903
• 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

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（由申请方提供）：长期目标是确定哺乳期间乳腺上皮细胞的乳脂质分泌机制。尽管母乳对于哺乳新生儿的存活和营养具有重要意义，但母乳主要成分（包括脂质和蛋白质）分泌的分子和细胞机制仍不清楚。大约5%的母乳喂养母亲因不明原因而无法哺乳。在这项拟议的工作中，乳蛋白，亲丁酸蛋白（Btn 1a 1）和黄嘌呤脱氢酶/氧化酶（Xdh），在乳脂滴的分泌的潜在作用将被研究。Btn 1a 1和Xdh是第一个与乳脂分泌有遗传联系的蛋白质，因为切除相应的基因会严重破坏乳脂的调节分泌。我们将测试的假设，Btn 1a 1被运送到顶端质膜，并结合到细胞内脂滴的表面上的XDH形成的连接蛋白复合物的萌芽和释放的脂质从细胞中必不可少的。通过GST下拉试验和柱色谱法测定截短形式蛋白质之间的结合，鉴定Btn 1a 1胞质尾区和Xdh中的相互作用结构域。在体外鉴定的Btn 1a 1和Xdh的相互作用结构域将通过表达来自由乳腺特异性启动子驱动的转基因的任一蛋白质的野生型或截短功能形式来筛选体内功能。野生型Btn 1a 1或其功能结构域应恢复Btn 1a 1-/-小鼠中的野生型表型，但Xdh的功能结构域应通过以显性负性方式起作用来抑制野生型小鼠中的脂质分泌。Btn 1a 1和Xdh的细胞内分布和靶向动力学将通过乳腺外植体的共聚焦显微镜来确定，所述乳腺外植体从用编码荧光标记的任一蛋白质的融合蛋白的腺病毒载体转导的腺体制备。如果Btn 1a 1被转运到顶膜，并在脂质双层中变得不那么移动的，因为它被掺入到出芽的脂滴中，则工作假设将得到支持。Xdh应该结合到细胞质中的脂滴，并与Btn 1a 1在顶端表面共定位。 外行语言：大约5%的母乳喂养母亲哺乳失败。本项目将检验乳蛋白、亲酪蛋白和黄嘌呤氧化酶对乳脂质分泌至关重要的假设，从而提高我们对人类泌乳不足的理解和治疗的潜在能力。