Enzymology at the Membrane Interface: Intramembrane Proteases

Chapter One: Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease

• Abstract
• 1 Introduction
• 2 The Use of MBP-Tagged Model Substrates for Analysis of Proteolytic Activity of RseP
• 3 In Vitro Analysis of Proteolysis of SPs by RseP
• 4 Structural Analysis of RseP Using Thiol-Specific Modification Reagents
• 5 In Vivo Cross-Linking Analysis to Probe the Interaction of RseP With Its Substrate in the Membrane
• Acknowledgments

Chapter Two: Signal Peptidase Enzymology and Substrate Specificity Profiling

• Abstract
• 1 Introduction
• 2 Purification of Δ2-75 SP1
• 3 Purification of SP1 Substrate pONA
• 4 In Vitro Signal Peptide Cleavage Assay
• 5 In Vivo Assay of Signal Peptidase Activity
• 6 Substrate Specificity Profiling
• 7 Conclusions
• Acknowledgment

Chapter Three: Screening and Characterization Strategies for Nanobodies Targeting Membrane Proteins

• Abstract
• 1 Introduction
• 2 Phage Display
• 3 Critical Aspects of Biopannings
• 4 Screening Strategies
• 5 Characterization of Nanobodies
• 6 Potential Applications
• Acknowledgments

Chapter Four: Probing the Activity of Eukaryotic Rhomboid Proteases In Vitro

• Abstract
• 1 Introduction
• 2 Expression and Solubilization of Rhomboid Proteases From Bacteria
• 3 In Vitro Probing of Rhomboid Protease Activity
• Acknowledgments

Chapter Five: Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases

• Abstract
• 1 Introduction to Intramembrane Proteases
• 2 Expression and Purification of GlpG, S2P, and PSH
• 3 Enzymatic Activity Assays for Intramembrane Proteases
• 4 Expression and Purification of Human γ-Secretase
• 5 Enzymatic Activity Assay for Human γ-Secretase
• 6 Structures of I-CLiPs
• 7 Concluding Remarks
• Acknowledgments

Chapter Six: Analyzing Amyloid-β Peptide Modulation Profiles and Binding Sites of γ-Secretase Modulators

• Abstract
• 1 Introduction
• 2 Methods
• Acknowledgments

Chapter Seven: Probing the Structure and Function Relationships of Presenilin by Substituted-Cysteine Accessibility Method

• Abstract
• 1 Introduction
• 2 Principle of Cysteine-Based Structural Analyses of Presenilin
• 3 Protocol
• 4 Summary and Perspectives
• Acknowledgments

Chapter Eight: A New Method to Determine the Transmembrane Conformation of Substrates in Intramembrane Proteolysis by Deep-UV Resonance Raman Spectroscopy

• Abstract
• 1 Introduction
• 2 In Vitro Intramembrane Proteolysis Assay
• 3 Intramembrane Proteolysis of the Substrate Monitored by dUVRR Spectroscopy
• 4 Conclusions
• Acknowledgment

Chapter Nine: An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane

• Abstract
• 1 Introduction
• 2 Enzymatic Considerations for Kinetic Analysis of Proteolysis in Detergent Micelle Systems
• 3 A New Strategy: Quantitative Analysis in Proteoliposomes
• 4 Preparation of Liposomes
• 5 Preparation of Fluorophore-Labeled Substrate
• 6 Establishing an Inducible and Fluorogenic Intramembrane Protease Assay
• 7 Real-Time Kinetic Analysis of Membrane-Immersed Proteolysis
• 8 Summary
• Acknowledgments

Chapter Ten: Production of Recombinant Rhomboid Proteases

• Abstract
• 1 Introduction
• 2 Overview
• 3 Example of an Expression and Purification Protocol—The pBAD System
• Acknowledgments

Chapter Eleven: Mechanism and Inhibition of Rhomboid Proteases

• Abstract
• 1 Introduction
• 2 Rhomboid Protease Mechanism
• 3 Substrate Specificity of Rhomboid Proteases
• 4 Rhomboid Protease Inhibitors
• 5 Concluding Remarks and Perspectives
• Acknowledgments

Chapter Twelve: Enzymatic Assays for Studying Intramembrane Proteolysis

• Abstract
• 1 Introduction
• 2 Detergent-Solubilized Assay
• 3 Proteoliposome Assay
• 4 Inducible Proteoliposome Assay
• 5 Conclusions
• Acknowledgments

Chapter Thirteen: Methods for Structural and Functional Analyses of Intramembrane Prenyltransferases in the UbiA Superfamily

• Abstract
• 1 Introduction to the UbiA Superfamily
• 2 Biological Function and Enzymatic Activity of the Subfamilies
• 3 Predicting the Functional Role of Intramembrane Prenyltransferases by Sequence Clustering Analysis
• 4 Analysis of Enzymatic Activity
• 5 Structural Studies of the UbiA Superfamily
• Acknowledgments

Chapter Fourteen: Functional Study of the Vitamin K Cycle Enzymes in Live Cells

• Abstract
• 1 Introduction
• 2 Cell-Based Assay for Functional Study of Vitamin K Cycle Enzymes
• 3 Functional Study of VKOR Using TALENs-Mediated Gene Knockout Reporter Cells
• 4 Functional Study of GGCX Using CRISPR-Cas9-Mediated Gene Knockout Reporter Cells
• 5 Concluding Remarks

Chapter Fifteen: Activity Assays for Rhomboid Proteases

• Abstract
• 1 Introduction
• 2 In Vivo Assays
• 3 In Vitro Assays
• Acknowledgments