Decoding intramembrane aspartyl protease substrate preferences and activity

细胞通过化学信号进行交流。细胞中信号的保真度对维持整体健康至关重要，并由各种酶产生的信号分子控制。这里感兴趣的是通过蛋白质或肽的分解作用产生的信号分子的类别，称为蛋白质水解，由膜内蛋白酶（IPs）执行，这是在细胞的水耗尽脂质膜中发现的酶。虽然蛋白酶进行的蛋白质水解是众所周知的，但IPs在缺水的细胞膜上进行这种依赖水的化学反应。IPs的作用是细胞分裂、发育和代谢的关键，但这种独特的化学环境使得该系统难以研究，因此该过程的关键细节仍然未知。佐治亚理工学院的凝聚力研究/教育任务涉及高中，本科和研究生水平的学生，以在分子水平上理解特定IP类型的化学和结构。该研究将回答特定的IP如何在独特的膜环境中对其许多底物进行蛋白质水解化学以产生信号分子产物，作为更大的IP家族的模型。为了回答这些问题，学生将使用针对膜的跨学科，尖端和高分辨率生化和生物物理方法，为他们未来的STEM职业生涯进行培训。反过来，K-12学生的教育模块将被开发、实施和传播。从长远来看，对ip的化学和结构的理解将有助于未来的研究，以更好地了解ip衍生的信号是如何在细胞、组织和有机体水平上调控的，从而引发特定的生物学结果。以古菌Methanoculleus marisnigri JR1的IAP同源物为模型，研究天冬氨酸IP亚类（IAP）。学生将探索化学因素和位置因素之间的相互作用，评估裂解能力的程度，并使用定制的高分辨率和定量实验方法，在分子细节上阐明底物- iap相互作用。然后，学生将使用化学交联、x射线晶体学和小角度中子散射来研究活化底物/IAP复合物的结构，这些结果将通过分子动力学模拟来补充。这些研究将提供关键的新的机制洞察到膜内蛋白水解过程使用一个定义良好的模型系统。这些研究的结果将挑战基于低分辨率或间接方法的领域原则，使IAPs的底物鉴定在整个生物学中广泛存在，并将导致未来的研究探索膜内蛋白酶的调节机制。将出现新的基于结构的进一步研究假设，包括催化的大量水进入的起源。量身定制的定量方法也有望在IAP同源物、实验条件和需要类似知识的知识产权类别中得到广泛采用。任何用于结构测定的前所未有的策略都将适用于其他iap，其他IP类别和膜蛋白的研究。研究结果将在生物化学、蛋白质组学、膜生物物理学、结构生物学和中子科学等跨学科领域广泛传播。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Cells communicate through chemical signals. The fidelity of signaling in the cell is critical to maintaining overall health and is controlled by signaling molecules that are generated by a variety of enzymes. Of interest here is the category of signaling molecules generated via the action of the breakdown of proteins or peptides, known as proteolysis, performed by intramembrane proteases (IPs), which are enzymes found in the water-depleted lipid membrane of the cell. Although proteolysis performed by proteases is well-known, IPs conduct this water-dependent chemistry in the water-depleted cell membrane. The action of IPs is key to cell division, development, and metabolism, but this unique chemical environment renders the system difficult to study and thus key details of the process remain unknown. The cohesive research/educational mission at the Georgia Institute of Technology involves students at high school, undergraduate, and graduate levels to comprehend, at the molecular level, the chemistry and structure of a particular IP type. The research will answer how the specific IP performs proteolysis chemistry on its many substrates in the distinctive membrane environment to generate the signaling molecule products, as a model for the larger IP family. To answer these questions, students will use cross-disciplinary, cutting edge, and high-resolution biochemical and biophysical methods tailored to membranes, training them for future STEM careers. In turn, educational modules for K-12 students will be developed, implemented, and disseminated. In the long term, an understanding of the chemistry and structure of IPs will lead to future research to better understand how IP-derived signaling is regulated at the cellular, tissue, and organismal level to elicit specific biological outcome.The aspartyl IP sub-class (IAP) will be studied, using as a model an IAP ortholog from the archaeon Methanoculleus marisnigri JR1. Students will probe the interplay between chemical and positional considerations, evaluate the extent of cleavage processivity, and elucidate substrate-IAP interactions in molecular detail, using tailored high-resolution and quantitative experimental methods. Students will then study the structure of activated substrate/IAP complexes using chemical crosslinking, X-ray crystallography, and small angle neutron scattering, results of which will be complemented by molecular dynamics simulations. These studies will provide critical new mechanistic insight into the process of intramembrane proteolysis using a well-defined model system. Results of these studies will challenge tenets in the field based on lower resolution or indirect methods, enable the identification substrates for IAPs broadly throughout biology, and will lead to future studies to probe the mechanisms by which intramembrane proteases are regulated. New structure-based hypotheses for further study, including the origin of bulk water entry for catalysis, will emerge. The tailored quantitative methods also hold promise for broad adoption across IAP orthologs, experimental conditions, and IP classes where similar knowledge is desirable. Any unprecedented strategies used for structure determination will be applicable to studies of other IAPs, other IP classes, and membrane proteins. Results will be broadly disseminated in the cross-disciplines of biochemistry, proteomics, membrane biophysics, structural biology, and neutron sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.