Decoding intramembrane aspartyl protease substrate preferences and activity

细胞通过化学信号进行交流。细胞内信号的保真度对维持整体健康至关重要，并由多种酶产生的信号分子控制。这里感兴趣的是通过蛋白质或多肽的分解而产生的一类信号分子，称为蛋白分解，由膜内蛋白水解酶(IP)执行，膜内蛋白水解酶是在细胞缺水的脂膜中发现的酶。虽然蛋白水解酶是众所周知的，但IP在缺水的细胞膜上进行这种依赖于水的化学作用。IP的作用是细胞分裂、发育和新陈代谢的关键，但这种独特的化学环境使该系统难以研究，因此该过程的关键细节仍不清楚。佐治亚理工学院的凝聚力研究/教育使命涉及高中、本科生和研究生，让他们在分子水平上理解特定知识产权类型的化学和结构。这项研究将回答特定的IP如何在独特的膜环境中对其许多底物进行蛋白质分解化学，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，学生们将使用跨学科、尖端和高分辨率的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，将为K-12学生开发、实施和传播教育模块。从长远来看，对IP的化学和结构的了解将导致未来的研究，以更好地了解IP衍生的信号是如何在细胞、组织和生物水平上调节的，以获得特定的生物结果。天门冬氨酸IP亚类(IAP)将被研究，作为模型，使用来自甲烷甲烷菌JR1的IAP同源基因。学生将探索化学和位置考虑之间的相互作用，评估切割过程的程度，并使用量身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射线结晶学和小角中子散射来研究活化底物/IAP络合物的结构，这些结果将由分子动力学模拟来补充。这些研究将为使用定义明确的模型系统对膜内蛋白分解过程提供关键的新的机制洞察。这些研究的结果将挑战基于较低分辨率或间接方法的该领域的原则，使IAP的识别底物在生物学中得到广泛的应用，并将导致未来的研究，以探索膜内蛋白水解酶的调节机制。新的基于结构的假说将用于进一步研究，包括用于催化的大量水进入的来源。量身定做的定量方法也有望在需要类似知识的IAP直系物、实验条件和IP类中广泛采用。任何前所未有的用于结构确定的策略都将适用于其他IAP、其他IP类和膜蛋白的研究。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的交叉学科中广泛传播。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.