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. 2017:584:229-253.
doi: 10.1016/bs.mie.2016.10.025. Epub 2016 Dec 7.

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

R P Baker  1 , S Urban  2
Affiliations

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

R P Baker et al. Methods Enzymol. 2017.

Abstract

Intramembrane proteases are an ancient and diverse group of multispanning membrane proteins that cleave transmembrane substrates inside the membrane to effect a wide range of biological processes. As proteases, a clear understanding of their function requires kinetic dissection of their catalytic mechanism, but this is difficult to achieve for membrane proteins. Kinetic measurements in detergent systems are complicated by micelle fusion/exchange, which introduces an additional kinetic step and imposes system-specific behaviors (e.g., cooperativity). Conversely, kinetic analysis in proteoliposomes is hindered by premature substrate cleavage during coreconstitution, and lack of methods to quantify proteolysis in membranes in real time. In this chapter, we describe a method for the real-time kinetic analysis of intramembrane proteolysis in model liposomes. Our assay is inducible, because the enzyme is held inactive by low pH during reconstitution, and fluorogenic, since fluorescence emission from the substrate is quenched near lipids but restored upon proteolytic release from the membrane. The precise measurement of initial reaction velocities continuously in real time facilitates accurate steady-state kinetic analysis of intramembrane proteolysis and its inhibition inside the membrane environment. Using real data we describe a step-by-step strategy to implement this assay for essentially any intramembrane protease.

Keywords: Alzheimer's disease; Cancer; Cell signaling; ER-associated degradation; Gamma-secretase; Malaria; Membrane protein; Parkinson's disease; Presenilin; Protease; Regulated intramembrane proteolysis; Site-2 protease.

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Figures

Figure 1
Figure 1
Schematic diagram of the inducible reconstitution and fluorogenic intramembrane protease assay. Pure rhomboid protease and FITC-TatA substrate in detergent micelles are mixed with liposomes at low pH to inactivate the protease during reconstitution. Detergent is removed from the protein/lipid/detergent complexes by means of dilution followed by ultracentrifugation. Resuspension of the proteoliposome pellet in neutral reaction buffer re-activates the protease. The FITC fluorophore on the reconstituted substrate is quenched by its proximity to the membrane lipids, allowing detection of a fluorogenic signal as evidence of proteolytic release of the FITC-labeled amino-terminus.
Figure 2
Figure 2
Steady-state kinetic analysis in detergent. FITC-TatA substrate titrations performed under identical reaction conditions except detergent concentration was either 0.1% (left panel) or 0.3% (right panel). While the kinetic parameter, Vmax, was unaffected, KM was approximately double in the higher detergent condition. The increased concentration of detergent also resulted in decreased cooperativity, as evidenced by the lower Hill coefficient.
Figure 3
Figure 3
Fluorogenic FITC-TatA substrate. (A) A peptide comprised of residues 1-33 of TatA is labeled amino-terminally through β-alanine linkage to fluorescein isothiocyanate. The rhomboid cleavage site between two adjacent alanine residues is indicated. (B) FITC fluorescence emission is quenched when the FITC-TatA substrate is reconstituted in proteoliposomes (black line) but robust fluorescence emission is restored when the liposomes are solubilized by 0.2% (w/v) sarcosine detergent (green line).
Figure 4
Figure 4
Key parameters for inducible intramembrane protease assay development. (A) Representative samples taken pre- and post-reconstitution for reactions with either wildtype GlpG or its catalytic mutant (SAHA is GlpG-S201A+H254A) were resolved electrophoretically and imaged for GlpG protease levels by Krypton infrared protein staining followed by Odyssey infrared scanning (upper panel) and for FITC-TatA using a Typhoon fluorescence scanner (lower panel). Reconstitution efficiency of 90-95% was observed for both the protease and substrate. (B) Real-time reaction time courses show a linear increase in FITC-fluorescence over time for wildtype GlpG (black line) compared to a negligible but common ‘drift’ in fluorescence with the catalytic mutant (red line). A robust fluorescence signal is generated when proteinase K is added to the SAHA reaction (green line). (C) SDS gel electrophoresis followed by fluorescent imaging of reaction products confirms that the FITC fluorescence signal detected for wildtype GlpG in real-time corresponds to an intramembrane proteolytic cleavage product (lane 2) that is not detected when the catalytic residues are mutated (lane 3) or when the wildtype enzyme is assayed at pH 4 (lane 1). Smaller reaction products corresponding to cleavage sites outside the membrane were detected in the presence of proteinase K (lane 4).
Figure 5
Figure 5
Kinetic analysis of membrane-immersed proteolysis. (A) Real-time reaction time courses showing an increase in reaction rate with increasing substrate concentration. Note that the two highest concentrations of substrate (black and red points/lines overlay, indicating enzyme saturation by substrate). (B) A plot of initial velocities against FITC-TatA substrate concentration (in mole percent) is modeled well by a Michaelis-Menten enzyme kinetics model, allowing KM and Vmax kinetic constants to be extracted for GlpG intramembrane proteolysis.
Figure 6
Figure 6
Analysis of inhibition kinetics of intramembrane proteolysis. (A) Inhibition of FITC-TatA cleavage by GlpG with increasing concentration of 7-amino-4-chloro-3-methoxy-isocoumarin (JLK6) in proteoliposomes (blue) compared to detergent micelles (black) reveals that the membrane environment decreases efficacy of inhibition approximately 10-fold. (B) Initial velocities plotted against substrate concentration in the absence or presence of increasing concentrations of peptide aldehyde inhibitor (Ac-VRMA-CHO). Non-linear regression analysis using a non-competitive inhibition model (blue solid line) gives a significantly better fit (R2 = 0.98) than a competitive inhibition model (red dashed line).
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