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A Nanopore Approach for Analysis of Caspase-7 Activity in Cell Lysates

Bach Pham1, Scott J Eron1, Maureen E Hill1

  • 1Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts.

Biophysical Journal
|August 21, 2019
PubMed

Insights

Researchers developed a novel nanopore method to measure caspase-7 protease activity. This technique distinguishes between closely related caspases, enabling precise activity profiling.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Nanotechnology

Background:

  • Caspases are critical proteases regulating inflammation and programmed cell death.
  • Caspase-3 and caspase-7 share similar substrate specificities, making individual activity assessment difficult.

Purpose of the Study:

  • To develop a method for individually assessing the activity of proteases with overlapping specificities, specifically targeting caspase-7.
  • To demonstrate the utility of engineered nanopores for protease activity profiling.

Main Methods:

  • Engineered an outer membrane protein G (OmpG) nanopore with an integrated caspase substrate sequence (DEVDG).
  • Measured changes in nanopore electrical current upon substrate cleavage by caspase-7.
  • Applied the method to determine caspase-7 activity in Escherichia coli cell lysates.

Main Results:

  • Cleavage of the DEVDG substrate within the OmpG nanopore produced a distinct signal.
  • Successfully determined caspase-7 activity in complex biological samples (E. coli lysates).

Conclusions:

  • The OmpG nanopore assay provides a sensitive method for detecting caspase-7 activity.
  • This approach offers a potential framework for activity-based profiling of proteases with similar substrate specificities.

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