Development of Secretory Mechanisms During Lactogenesis

该项目将重点关注乳腺上皮细胞从妊娠到哺乳的第二阶段过渡期间观察到的胞吐和脂质分泌途径的分子变化。我们的主要假设是，居民高尔基体和细胞质脂滴（CLD）蛋白的一个子集的差异表达和/或翻译后修饰允许高尔基体的扩增和CLD的变化，以准备广泛的牛奶蛋白和脂质分泌。将在泌乳过程中的多个时间点进行无偏倚的整体蛋白质组学分析（2D-凝胶电泳/质谱法），以鉴定内源性和新型蛋白质差异表达/修饰导致高尔基体扩增和脂质分泌的序列。这些蛋白质的一个选择组将在从妊娠到哺乳的过渡期间进行功能表征。RLP 30是一种新的高尔基体基质样蛋白，对它的初步研究使我们提出了目前的假设，即高尔基体基质的重塑在细胞器扩张中起着至关重要的作用。脂质分泌的机制涉及黄嘌呤氧化还原酶（XOR）、亲丁酸蛋白（Btn）、脂肪细胞分化相关蛋白（ADRP）和膜结合巯基氧化酶之间的相互作用。这些相互作用将在分子和功能水平上进行研究，蛋白质组学将用于识别脂质分泌过程的其他组分。这些方法将为今后的乳汁分泌研究建立基本框架。

This project will focus on the molecular changes in exocytic and lipid secretory pathways observed to be functionally up-regulated during the Stage II transition from pregnancy from pregnancy to lactation in mammary epithelial cells. Our major hypothesis is that differential expression and/or post-translational modifications of a subset of resident of resident Golgi and cytoplasmic lipid droplet (CLD) proteins allow for the amplification of the Golgi and changes in the CLDs in preparation for extensive milk protein and lipid secretion. An unbiased global proteomics analysis (2D-gel electrophoresis/mass spectrometry) will be carried out a multiple time points during lactogenesis to identify the sequence in which endogenous and novel proteins are differentially expressed/modified resulting in Golgi expansion and lipid secretion. A select group of these proteins will be functionally characterized during the transition from pregnancy to lactation. Preliminary studies on RLP30, a novel Golgi matrix-like protein have led us to our current hypothesis that remodeling of the Golgi matrix plays a crucial role in organellar expansion. The mechanism of lipid secretion prosed involves interactions among xanthine oxidoreductase (XOR), butyrophilin (Btn), adipocyte differentiation related protein (ADRP), and a membrane associated thiol-oxidase. These interactions will be investigated at molecular and functional levels and proteomics will be used to identify additional components of the lipid secretion process. These approaches will establish the fundamental framework for future studies of milk secretion.