Modulation of glycosylation homeostasis by vesicular transport in the Golgi

高尔基体中囊泡运输对糖基化稳态的调节

• 批准号: BB/F006993/1
• 负责人: Daniel Ungar
• 金额: $ 45.4万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF006993%2F1
• 关键词: Modulation; glycosylation; homeostasis; vesicular; transport

Animal cells contain multiple membranous organelles, such as the nucleus, mitochondria, or the various organelles of the secretory pathway. The secretory organelles are responsible for releasing all the proteins from the cell that perform important external functions, such as communication, defence or the physical connectivity between cells. Appropriate sorting of these proteins between organelles is important to ensure that each reaches their correct destination, be it outside the cell, or in one of the organelles themselves to help the organelle's functions. Protein sorting is achieved by relatively small membranous structures, called vesicles, which pinch off one organelle to carry a selected set of its proteins to a second organelle, which is the preferred destination for those proteins. When a vesicle arrives at the target organelle, the two membranes have to be brought into close apposition, or tethered to each other. Subsequently the vesicle and target membrane will merge with each other to deliver the vesicle's cargo. We will study the process of vesicle tethering at the main sorting hub of the secretory pathway, the Golgi apparatus. The tethering of an important vesicle-subset at the Golgi is assisted by a protein complex called COG, which is controlled by GTPases of the Rab family. There are 70 different Rabs known in people, and all are known to shift their shape when hydrolysing a bound GTP molecule. This shape change allows them to control the function of other proteins associated with them, such as COG. We already know, that COG communicates with seven different Rabs. An important unanswered question in vesicle tethering is, how the vesicles find the correct target organelle? We hypothesise that COG functions as a machine to power the tethering of a vesicle, while each Rab may steer COG to use the correct vesicle and tether it to the right target membrane, to allow faithful sorting of the vesicular cargo. The experiments proposed in this application will test this hypothesis by looking at the interplay of COG with three Rabs that we have previously shown to physically contact COG. The physical contact puts the Rabs into the correct position to control COG, but the exact nature of each contact, especially the differences between the various COG-Rab pairs will be important to explain how specific targeting occurs. Vesicle tethering needs other factors besides COG and Rabs as well, and therefore a second question we will address is how those other factors, members of the golgin protein family, will fit into the picture. For the one machine, COG, there are about a handful Rabs, and there could be as many as a dozen golgins, which could also play their part in targeting specificity. As part of this study we will catalogue some of these golgins and Rabs into protein subsets that will form the basis for later more detailed investigations about their specific roles in vesicle tethering and vesicle targeting specificity. The proteins delivered to the outside of the cell by the secretory pathway are often modified in various ways to enhance their functionality, most prominently by sugar chains built of nine different sugar building blocks. Most of the sugar chains, are generated by numerous enzymes that reside in specific sub-compartments of the Golgi, called cisternae. Sugar chains often require a specific sequence of the building blocks added to one another by a specific sequence of the enzymes. This in turn requires the enzymes to be sorted into the correct order. The function of the vesicles using COG is to sort these enzymes into specific cisternae to maintain a given order. This is very important, since defects in COG that cause defects in sugar chains, have been found to cause human diseases. As a final quest of this proposal we will focus our attention on how the different COG-Rab pairs mediate the sorting of enzymes to allow correct sugar chains to be built.

动物细胞含有多个膜性细胞器，如细胞核、线粒体或分泌途径的各种细胞器。分泌细胞器负责从细胞中释放所有蛋白质，这些蛋白质执行重要的外部功能，例如通信，防御或细胞之间的物理连接。这些蛋白质在细胞器之间的适当分类对于确保每个蛋白质到达其正确的目的地是重要的，无论是在细胞外，还是在细胞器本身中，以帮助细胞器的功能。蛋白质分选是通过相对较小的膜结构（称为囊泡）实现的，囊泡夹住一个细胞器，将其选定的一组蛋白质携带到第二个细胞器，这是这些蛋白质的首选目的地。当囊泡到达目标细胞器时，两个膜必须紧密贴合，或彼此束缚。随后，囊泡和靶膜将彼此合并以递送囊泡的货物。我们将研究囊泡拴系在分泌途径的主要分类枢纽，高尔基体的过程。一个重要的囊泡子集在高尔基体的束缚是由称为COG的蛋白质复合物辅助的，该复合物由Rab家族的GTP酶控制。在人类中已知有70种不同的Rabs，并且已知所有Rabs在水解结合的GTP分子时都会改变它们的形状。这种形状的变化使它们能够控制与它们相关的其他蛋白质的功能，例如COG。我们已经知道，COG与七个不同的Rabs通信。囊泡束缚中一个重要的未解问题是，囊泡如何找到正确的靶细胞器？我们假设，COG作为一个机器的功能，为一个囊泡的拴系提供动力，而每个Rab可以引导COG使用正确的囊泡，并将其拴系到正确的靶膜，以允许忠实地分选囊泡货物。本申请中提出的实验将通过观察COG与我们先前已经示出的与COG物理接触的三个Rabs的相互作用来测试该假设。物理接触使Rabs进入正确的位置以控制COG，但每次接触的确切性质，特别是各种COG-Rab对之间的差异对于解释特定目标是如何发生的非常重要。除了COG和Rabs外，囊泡束缚还需要其他因素，因此我们要解决的第二个问题是，这些其他因素，golgin蛋白家族的成员，将如何融入这幅图景。对于一台机器，COG，大约有几个Rabs，可能有十几个Golgins，它们也可以在靶向特异性方面发挥作用。作为本研究的一部分，我们将这些golgins和Rabs中的一些归类为蛋白质子集，这些蛋白质子集将为以后更详细地研究它们在囊泡束缚和囊泡靶向特异性中的特定作用奠定基础。通过分泌途径传递到细胞外的蛋白质通常以各种方式进行修饰以增强其功能，最突出的是由九种不同的糖结构单元构建的糖链。大多数糖链是由位于高尔基体特定亚区室（称为池）中的许多酶产生的。糖链通常需要特定序列的构建单元通过特定序列的酶彼此添加。这反过来又需要将酶分类为正确的顺序。使用COG的囊泡的功能是将这些酶分类到特定的池中以维持给定的顺序。这是非常重要的，因为已经发现引起糖链缺陷的COG缺陷会引起人类疾病。作为这个提议的最后探索，我们将把注意力集中在不同的COG-Rab对如何介导酶的分选，以允许正确的糖链被建立。

项目成果

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions.

• DOI: 10.1007/s00418-013-1117-6
• 发表时间: 2013-09
• 期刊: HISTOCHEMISTRY AND CELL BIOLOGY
• 影响因子: 2.3
• 作者: Willett, Rose;Ungar, Daniel;Lupashin, Vladimir
• 通讯作者: Lupashin, Vladimir

Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay.

无细胞荧光高尔基体内逆行囊泡运输测定。

• DOI: 10.21769/bioprotoc.2616
• 发表时间: 2017
• 期刊: Bio-protocol
• 影响因子: 0.8
• 作者: Cottam NP

Golgi linked protein glycosylation and associated diseases.

• DOI: 10.1016/j.semcdb.2009.03.004
• 发表时间: 2009-09
• 期刊: Seminars in cell & developmental biology
• 影响因子: 7.3
• 作者: D. Ungar

Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling.

• DOI: 10.1021/pr401043r
• 发表时间: 2014-03-07
• 期刊: JOURNAL OF PROTEOME RESEARCH
• 影响因子: 4.4
• 作者: Rahman, Salina Abdul;Bergstroem, Ed;Watson, Christopher J.;Wilson, Katherine M.;Ashford, David A.;Thomas, Jerry R.;Ungar, Daniel;Thomas-Oates, Jane E.
• 通讯作者: Thomas-Oates, Jane E.