ADAMTS7 Substrate: A Great Mystery

Background: ADAMTS7 is a metalloprotease which is involved in a variety of conditions from coronary artery disease (CAD) to fertility to arthritis and more. ADAMTS7 is involved in angiogenesis, potentially by breaking down the angiogenesis inhibitor Thrombospondin 1. The angiogenesis connection is important to understanding why ADAMTS7 may be involved in such a spectrum of diseases or disorders. Atherosclerosis leading to CAD is the best documented condition associated with ADAMTS7. Thirty-two percent of deaths worldwide are from cardiovascular diseases, and in the United States 7.2% of adults over 20 years of age have CAD. An inactivating SNP in the ADAMTS7 gene has been shown to mitigate atherosclerosis and coronary artery disease in animal and human studies. ADAMTS7 is an enzyme whose substrate has yet to be defined; finding the substrate for ADAMTS7 will provide more information on the purpose of ADAMTS7.

Methods: Our experiment looks at potential substrates through TurboID – a protein technology by which proteins in close proximity to ADAMTS7 will be biotinylated. A biotinylating protein is fused with the active domains of ADAMTS7 or a variation. The protein is then grown in cell culture and biotinylated proteins are purified using streptavidin agarose which binds biotin. Isolated proteins are then sent for identification via mass spectrometry.

Results: Data from the mass spectrometry experiment will be analyzed from which candidate substrates will be selected. From here, assays of candidate substrates will be performed by mixing them with ADAMTS7. These reactions will then be analyzed by SDS-PAGE and densitometry analysis looking for cleavage of the enzyme.

Conclusion: Once a suitable substrate has been identified, a FRET assay for ADAMTS7 activity can be developed and inhibitors searched for to help combat coronary artery disease and possibly other disorders.