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Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases
1Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
Methods in Enzymology
|January 10, 2017
Summary
Intramembrane proteases, crucial for cellular processes and implicated in diseases like Alzheimer's, are integral membrane enzymes. This chapter details their structural determination and enzymatic assays, advancing mechanistic understanding.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Intramembrane proteases are integral membrane enzymes catalyzing peptide bond hydrolysis within the lipid bilayer.
- They are classified into four main types: S2P, rhomboid, Rce1-type, and aspartyl proteases (presenilin, SPP).
- Dysfunction of these proteases is linked to neurodegenerative diseases like Parkinson's and Alzheimer's.
Purpose of the Study:
- To discuss the structural elucidation of key intramembrane proteases.
- To highlight advancements in expressing and purifying human γ-secretase for structural studies.
- To describe enzymatic activity assays for intramembrane proteases.
Main Methods:
- Determination of crystal structures for bacterial S2P, E. coli rhomboid (GlpG), and archaeal presenilin/SPP (PSH).
- Development of a novel expression method for human γ-secretase.
- Cryoelectron microscopy (cryo-EM) for human γ-secretase structure determination.
- Enzymatic activity assays for assessing protease function.
Main Results:
- Crystal structures of several intramembrane proteases have been determined.
- A method for expressing human γ-secretase enabling its structure determination by cryo-EM has been established.
- Enzymatic assays provide a means to study protease activity.
Conclusions:
- Structural insights into intramembrane proteases are crucial for understanding their cellular roles and disease associations.
- Advances in expression and structural biology techniques facilitate the study of these challenging membrane proteins.
- Enzymatic assays are essential for functional characterization of intramembrane proteases.
