Post-Reductionist Protein Folding: Determining the In-cell Folding Energy Landsca

后还原论蛋白质折叠：确定细胞内折叠能量景观

基本信息

批准号：
8492500
负责人：
ANNE GERSHENSON
金额：
$ 5.9万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8492500
关键词：
Address Aging Alzheimer&apos s Disease Amino Acid Sequence Amino Acids Bacillus amyloliquefaciens ribonuclease Beginning of Life Biogenesis Biological Birth Blood coagulation Case Study Cells Chemicals Complex Crowding Cystic Fibrosis Data Defect Disease Disease Attributes Distal Employee Strikes Endoplasmic Reticulum Environment Enzymes Event Evolution Explosion Failure Family Fluorescence Goals Grant Immunoglobulins In Vitro Inflammation Knowledge Label Lead Length Light Liver Cirrhosis Maps Measures Methods Molecular Chaperones Muramidase Mutation Nature Pathway interactions Pattern Peptide Hydrolases Physiological Processes Plasma Play Process Protein Secretion Proteins Psyche structure Pulmonary Emphysema Quality Control Reaction Research Ribonucleases Ribosomes Role Scientist Serine Proteinase Inhibitors Serpins Structure Suicide Techniques Testing Therapeutic Therapeutic Intervention Tube Work biological research disease-causing mutation improved in vivo insight interest polypeptide prevent protein folding protein misfolding public health relevance research study secretory protein single molecule

项目摘要

The endoplasmic reticulum (ER) is a folding factory for the cell, able to churn out thousands of secretory proteins per second. Misfolding of a number of these proteins results in diseases ranging from cystic fibrosis to liver cirrhosis. While full-length proteins can be studied in vitro using a myriad of biophysical techniques, thus allowing biophysicists to characterize in detail their folding energy landscapes, studying protein folding in the more physiologically relevant ER environment presents daunting technical obstacles. Using recently developed, powerful single molecule fluorescence techniques on nascent polypeptide chains that contain fluorescently-labeled amino acids, we will determine the conformational evolution as a nascent polypeptide chain elongates in the ER and how conformational space is modulated by interactions with the chaperones and modifying enzymes resident in the ER. The goal is to obtain detailed information on the folding landscape of the growing nascent chain, rivaling the data available in test-tube experiments. Co- and post-translational interactions between the ER-resident proteins and nascent chains likely smooth the folding energy landscape, biasing against misfolded and aggregation-prone intermediates. This remodeling of the folding landscape is particularly important for proteins with high contact order, i.e. many contacts between distal parts of the chain. We will focus on an important family of secreted proteins, the inhibitory serpins, which fold into native structures with high contact order. Intriguingly serpin native states are energetically metastable, poised to change to a different structure. Serpins play crucial biological roles by regulating proteases involved in key physiological processes including blood coagulation and inflammation. A number of serpin mutations, associated with diseases such as liver cirrhosis and emphysema (together called 'serpinopathies'), lead to serpin aggregation in the ER. By studying serpin folding in the ER and comparing it to in vitro folding, we will determine how the folding landscape is altered by interactions with ER-resident proteins and by disease-associated mutations. The results of this research will provide unprecedented insight into how serpins misfold and how their energy landscapes differ in vitro and in vivo. This work will also provide a readily accessible experimental toolbox for scientists studying protein folding in the ER as well as a platform for potential therapeutic strategies to treat serpinopathies and other ER folding diseases.

内质网（ER）是细胞的折叠工厂，每秒能够产生数千个分泌蛋白。许多这些蛋白质的错误折叠会导致从囊性纤维化到肝硬化等疾病。虽然可以使用多种生物物理技术在体外研究全长蛋白质，从而使生物物理学家能够详细描述其折叠能量景观，但在更生理相关的内质网环境中研究蛋白质折叠却面临着令人畏惧的技术障碍。使用最近开发的强大的单分子荧光技术对包含荧光标记氨基酸的新生多肽链进行分析，我们将确定新生多肽链在内质网中伸长时的构象演化，以及如何通过与分子伴侣和修饰酶的相互作用来调节构象空间住在急诊室。目标是获得有关不断增长的新生链折叠景观的详细信息，与试管实验中可用的数据相媲美。内质网驻留蛋白和新生链之间的共翻译和翻译后相互作用可能会平滑折叠能量景观，从而偏向于错误折叠和易于聚集的中间体。这种折叠景观的重塑对于具有高接触顺序的蛋白质尤其重要，即链的远端部分之间有许多接触。我们将重点关注一个重要的分泌蛋白家族，即抑制性丝氨酸蛋白酶抑制剂，它以高接触顺序折叠成天然结构。有趣的是，丝氨酸蛋白酶抑制剂的原生状态在能量上是亚稳态的，准备转变为不同的结构。丝氨酸蛋白酶抑制剂通过调节参与关键生理过程（包括凝血和炎症）的蛋白酶来发挥重要的生物学作用。许多与肝硬化和肺气肿（统称为“丝氨酸蛋白酶抑制剂病”）等疾病相关的丝氨酸蛋白酶抑制剂突变会导致丝氨酸蛋白酶抑制剂在内质网中聚集。通过研究内质网中的丝氨酸蛋白酶抑制剂折叠并将其与体外折叠进行比较，我们将确定折叠景观如何通过与内质网驻留蛋白的相互作用以及疾病相关突变而改变。这项研究的结果将为了解丝氨酸蛋白酶抑制剂如何错误折叠以及它们在体外和体内的能量景观如何不同提供前所未有的见解。这项工作还将为研究内质网蛋白质折叠的科学家提供一个易于使用的实验工具箱，以及治疗丝氨酸病和其他内质网折叠疾病的潜在治疗策略的平台。