Dataset Information

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

ABSTRACT: Caspases are an important protease family that coordinate inflammation and programmed cell death. Two closely related caspases, caspase-3 and caspase-7, exhibit largely overlapping substrate specificities. Assessing their proteolytic activities individually has therefore proven extremely challenging. Here, we constructed an outer membrane protein G (OmpG) nanopore with a caspase substrate sequence DEVDG grafted into one of the OmpG loops. Cleavage of the substrate sequence in the nanopore by caspase-7 generated a characteristic signal in the current recording of the OmpG nanopore that allowed the determination of the activity of caspase-7 in Escherichia coli cell lysates. Our approach may provide a framework for the activity-based profiling of proteases that share highly similar substrate specificity spectrums.

SUBMITTER: Pham B

PROVIDER: S-EPMC6731459 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Publications

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

Pham Bach B Eron Scott J SJ Hill Maureen E ME Li Xin X Fahie Monifa A MA Hardy Jeanne A JA Chen Min M

Biophysical journal 20190802 5

Caspases are an important protease family that coordinate inflammation and programmed cell death. Two closely related caspases, caspase-3 and caspase-7, exhibit largely overlapping substrate specificities. Assessing their proteolytic activities individually has therefore proven extremely challenging. Here, we constructed an outer membrane protein G (OmpG) nanopore with a caspase substrate sequence DEVDG grafted into one of the OmpG loops. Cleavage of the substrate sequence in the nanopore by cas ...[more]

PMID: 31427065

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Project description:Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.

Dataset Information

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

Publications

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

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