Calcium-binding EF-hand proteins and protein domains; an NMR and biophysical approach

钙结合 EF-hand 蛋白和蛋白结构域；

• 批准号: RGPIN-2020-04210
• 负责人: Vogel, Hans
• 金额: $ 3.64万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739745
• 关键词: Calcium; binding; EF; hand; proteins

Eukaryotic cells contain a remarkable variety of proteins that have to work together in a coordinated manner to achieve a specific physiological outcome. In order to attain such control, the activities of many proteins are regulated by various chemical signals, that can turn specific groups of proteins on and off. There are a number of distinct chemical signals that can orchestrate these cellular responses, such as cyclic nucleotides and calcium. The research program described here, is focussed on a group of regulatory calcium-binding proteins, called the EF-hand proteins. This name refers to a specific helix-loop-helix structural motif, that was found when the 3D structure of the first protein of this class was determined in the 1970's. By now we know that human and plant genomes code for over 250 distinct EF-hand proteins and that most of these have multiple EF-hand motifs. For example, the most widely distributed protein calmodulin, has four EF-hand motifs that are organized in a pairwise manner into two connected lobes. Calmodulin is present in all eukaryotes, ranging from humans, birds, fish, amoeba, plants and yeast, indicating that this is an almost universal signalling system. While calcium is the signal, the EF-hand proteins are the on- and off-switches: after binding calcium they can bind to and alter the activity of their target proteins. Since the 1980's the structures of many EF-hand proteins, or EF-hand domains that are part of larger proteins, have been solved, and their functions have been elucidated through elegant cell biology studies. It has recently become clear, however, that plants and yeasts contain a number of EF-hand proteins, that carry specific mutations, and these can no longer respond to calcium in the same manner. Nonetheless, these 'inactivated switches' are completely conserved, suggesting that they must have another important biological function; we will investigate these poorly understood functions for several plant and yeast actin-bundling fimbrin/plastin proteins, and in ATP-magnesium mitochondrial carrier proteins. We will also study another multi-domain EF-hand protein called nucleobindin2; this protein is unique as it is secreted into the Endoplasmic Reticulum, where the calcium regulation is very different from that in the cytoplasm. Nucleobindin2 provides the precursor for the peptide hormone nesfatin, and we plan to demonstrate that its EF-hand domain can play a decisive role in the proteolytic release of this hormone. Finally, we will investigate in detail the interactions between calmodulin and the aquaporin 0 and 4 transmembrane water channels, as this leads to intriguingly different physiological outcomes, channel inhibition in the former and channel translocation in the latter case. Taken together, these studies will provide new molecular level insights into the biological roles of these unique EF-hand protein interactions and further expand our understanding of their evolutionary relationships.

真核细胞含有多种蛋白质，它们必须以协调的方式共同作用以实现特定的生理结果。为了实现这种控制，许多蛋白质的活性受到各种化学信号的调节，这些信号可以打开和关闭特定的蛋白质组。有许多不同的化学信号可以协调这些细胞反应，如环核苷酸和钙。这里描述的研究计划集中在一组调节钙结合蛋白，称为EF-手蛋白。这个名字是指一个特定的螺旋-环-螺旋结构基序，这是在20世纪70年代确定这类蛋白质的第一个3D结构时发现的。到目前为止，我们知道人类和植物基因组编码超过250种不同的EF-手蛋白，其中大多数具有多个EF-手基序。例如，分布最广泛的蛋白质钙调素，有四个EF-手基序，以成对的方式组织成两个连接的叶。钙调素存在于所有真核生物中，包括人类、鸟类、鱼类、变形虫、植物和酵母，表明这是一种几乎通用的信号系统。虽然钙是信号，但EF-手蛋白是开关：在结合钙后，它们可以结合并改变其靶蛋白的活性。自20世纪80年代以来，许多EF-手蛋白或作为较大蛋白质的一部分的EF-手结构域的结构已经被解决，并且它们的功能已经通过优雅的细胞生物学研究被阐明。然而，最近已经清楚的是，植物和酵母含有许多携带特定突变的EF手蛋白，并且这些EF手蛋白不再以相同的方式对钙做出反应。尽管如此，这些“失活开关”是完全保守的，这表明他们必须有另一个重要的生物学功能，我们将调查这些知之甚少的功能，为几个植物和酵母肌动蛋白捆绑fimplant/plastin蛋白，并在ATP-镁线粒体载体蛋白。我们还将研究另一种称为核结合蛋白2的多结构域EF手蛋白;这种蛋白质是独特的，因为它分泌到内质网中，其中钙调节与细胞质中的钙调节非常不同。Nucleobindin 2提供了肽激素nesfatin的前体，我们计划证明其EF-手结构域可以在这种激素的蛋白水解释放中发挥决定性作用。最后，我们将详细研究钙调蛋白和水通道蛋白0和4跨膜水通道之间的相互作用，因为这会导致有趣的不同生理结果，前者的通道抑制和后者的通道易位。总之，这些研究将为这些独特的EF-手蛋白相互作用的生物学作用提供新的分子水平的见解，并进一步扩大我们对其进化关系的理解。