RUI:MCB: Glutathionylation of beta-amylase3 in Arabidopsis thaliana

该项目最终将提高预测某些蛋白质在环境压力条件下如何被修饰的能力。这种修饰可能导致蛋白质功能的变化，帮助生物体对改变的条件做出反应，包括温度变化。这项研究的重点是一种植物淀粉酶，一种分解叶淀粉的酶，似乎在寒冷胁迫下的生存中发挥作用。这种酶受到一种机制的抑制，该机制涉及在冷应激期间可能发生的某些氨基酸的可逆修饰。这种类型的修饰知之甚少，但被认为是广泛存在于所有对环境压力作出反应的生物体中。淀粉酶中的修饰氨基酸已经被鉴定，本项目将表征这些修饰如何影响酶活性，以及是什么使这些氨基酸对这种特定修饰敏感。这项研究将在一个以本科为主的机构进行，涉及至少10名本科生，帮助他们为研究生院，专业学校或其他科学职业做好准备。学生将参与工作的各个方面，并能够在区域和国家会议上展示他们的成果。此外，该项目将支持一名博士后学者，因为她获得了教授和指导本科生研究的经验，这些技能对于她在本科院校工作的最终目标至关重要。蛋白质在特定半胱氨酸残基处的谷胱甘肽化是一种重要的翻译后修饰，但对于是什么使得一些半胱氨酸对谷胱甘肽化敏感而另一些半胱氨酸对谷胱甘肽化不敏感知之甚少。此外，在植物中还没有了解蛋白质谷胱甘肽化的生理作用的例子。在这个项目中，充分研究的模式植物拟南芥将被用来研究#946;-淀粉酶（BAM）基因家族的两个成员，其中一个，BAM 3，被谷胱甘肽化抑制，另一个，BAM 1，没有。该项目的两个目标是：1）使用定点诱变、质谱和同源建模来表征BAM 3的谷胱甘肽化以及这种修饰对催化活性的影响，以及2）鉴定影响半胱氨酸对谷胱甘肽化敏感性的关键因素。 一个保守的半胱氨酸残基被确定为是敏感的BAM 3中的谷胱甘肽化，但在BAM 1不敏感。跨植物的BAM 3和BAM 1序列的序列比对和同源性建模揭示了两种BAM之间不同的许多残基，但在每种类型内是保守的，并且可能影响对谷胱甘肽化的敏感性。计算建模和蛋白质诱变的组合将用于表征影响谷胱甘肽化事件的因素。从这些实验的结果将导致一个更准确的模型预测半胱氨酸残基的敏感性谷胱甘肽在其他蛋白质的发展，并更好地了解参与应激驯化的分子机制。

This project will ultimately lead to an improved ability to predict how certain proteins can be modified under conditions of environmental stress. Such modifications may lead to changes in the protein's function that help organisms respond to altered conditions, including temperature change. The research is focused on a plant amylase, an enzyme that breaks down leaf starch and appears to play a role in cold stress survival. This enzyme is inhibited by a mechanism involving the reversible modification of certain amino acids that may occur during cold stress. This type of modification is poorly understood but is thought to be widespread in all organisms responding to environmental stress. The modified amino acids in the amylase have been identified and this project will characterize how these modifications affect enzyme activity and what it is that makes these amino acids sensitive to this specific modification. The research will be carried out at a predominantly undergraduate institution and involve at least 10 undergraduates, helping prepare them for graduate school, professional schools or other careers in science. The students will be involved in all aspects of the work and be able to present their results at regional and national conferences. In addition, the project will support a postdoctoral scholar as she gains experience in teaching and mentoring undergraduates in research, skills that are essential to her eventual goal of working at an undergraduate institution. Glutathionylation of proteins at specific cysteine residues is an important post-translational modification but little is known about what makes some cysteines sensitive to glutathionylation while others are insensitive. In addition, there are no examples in plants where the physiological effects of protein glutathionylation are understood. In this project the well-studied model plant Arabidopsis thaliana will be used to investigate two members of the β-amylase (BAM) gene family, one of which, BAM3, is inhibited by glutathionylation and the other, BAM1, is not. The two objectives of the project are: 1) to characterize the glutathionylation of BAM3 and the effects of this modification on catalytic activity using site directed mutagenesis, mass spectrometry and homology modeling, and 2) to identify key factors influencing the susceptibility of cysteines to glutathionylation. A conserved Cys residue was identified that is sensitive to glutathionylation in BAM3 but is insensitive in BAM1. Sequence alignments and homology modeling of BAM3 and BAM1 sequences across plants revealed a number of residues that differ between the two BAMs, but are conserved within each type and may influence the sensitivity to glutathionylation. A combination of computational modeling and protein mutagenesis will be used to characterize the factors affecting this glutathionylation event. Results from these experiments will lead to the development of a more accurate model for predicting the sensitivity of cysteine residues to glutathionylation in other proteins, and to a better understanding of the molecular mechanisms involved in stress acclimation.