The Mechanism of Milk Lipid Secretion

乳脂分泌的机制

• 批准号: 7576149
• 负责人: IAN HEYWOOD MATHER
• 金额: $ 26.44万
• 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 2010-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7576149
• 关键词: Ablation; Adenovirus Vector; Affinity Chromatography; Apical; Binding; Biochemical Genetics; Biological; Biological Assay; Breast Feeding; Cell membrane; Cells; Chimeric Proteins; Column Chromatography; Confocal Microscopy; Cytoplasm; Cytoplasmic Tail; Data; Defect; Dominant-Negative Mutation; Epithelial Cells; Fatty acid glycerol esters; Filtration; Fluorescence Microscopy; Gel; Genes; Gland; 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; protein complex; 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。 在哺乳期间。尽管母乳对哺乳新生儿的生存和营养很重要， 牛奶中包括脂肪在内的主要成分分泌的分子和细胞机制 和蛋白质仍然未知。大约5%的哺乳母亲因不明原因而哺乳失败。在……里面 这项拟议的工作，乳蛋白，丁咯菲林(Btnlat)和黄嘌呤的潜在作用 将对乳脂滴分泌中的脱氢酶/氧化酶(XDH)进行研究。Btn1a1和Xdh是 第一个与乳脂分泌有遗传联系的蛋白质，因为各自的基因严重消融 扰乱乳脂的调节分泌。我们将检验Btn1a1被传输到 顶端质膜，并与细胞内脂滴表面的XDH键结合形成接头 蛋白质复合体，是细胞萌发和释放脂质所必需的。互动域(S)在 Btn1a1和XdH的细胞质尾巴将通过分析截断形式的 经GST下拉分析和柱层析鉴定。Btn1a1和XdH的相互作用结构域， 在体外鉴定的，将通过表达野生型或截短的功能形式在体内进行功能筛选 或者是由乳房特异性启动子驱动的转基因蛋白质。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 通过以显性的负面方式抑制野生型小鼠的脂肪分泌。胞内分布 Btn1a1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 用编码荧光标记融合蛋白的腺病毒载体转导的腺体制备 两种蛋白质都不是。如果Btn1a1被运输到根尖膜，工作假说将得到支持 并且当它被结合到萌芽中的脂滴中时，在脂双层中的流动性变得较小。Xdh应该绑定 到细胞质中的脂滴，并与Btn1a1在根尖表面共定位。 外行语言：大约5%的哺乳母亲哺乳失败。这个项目将检验这一假说 牛奶中的蛋白质--丁咯菲林和黄嘌呤氧化酶--对乳脂的分泌是必不可少的，因此 增强我们对人类哺乳缺陷的理解和潜在的治疗能力。

项目成果

Kinetics of milk lipid droplet transport, growth, and secretion revealed by intravital imaging: lipid droplet release is intermittently stimulated by oxytocin.

• DOI: 10.1091/mbc.e16-11-0776
• 发表时间: 2017-04-01
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Masedunskas A;Chen Y;Stussman R;Weigert R;Mather IH
• 通讯作者: Mather IH

A test of current models for the mechanism of milk-lipid droplet secretion.

• DOI: 10.1111/tra.12087
• 发表时间: 2013-09
• 期刊: Traffic (Copenhagen, Denmark)
• 作者: Jeong J;Lisinski I;Kadegowda AK;Shin H;Wooding FB;Daniels BR;Schaack J;Mather IH
• 通讯作者: Mather IH