A new assay for furin

Following her presentation to the Gordon Research Conference on proprotein convertases in 2010, Gemma has now published a manuscript describing the details of our new assay for the activity of the enzyme furin. Appearing in the 1st February edition of the Journal of Immunological Methods, the paper describes the protocol and provides extensive characterisation of the properties of the assay.

Furin, like other members of the proprotein convertase family, plays an important role in the maturation of a wide variety of proproteins, including receptors, cytokines, enzymes and integrins. Successful processing is pre-requsite for the activity of these target proteins.

During her PhD studies in the Graingerlab, Sarah Griffiths studied the processing one furin substrate, the insulin receptor, and postulated that inadequate furin activity (resulting in incomplete receptor processing) might be a contributory pathogenic mechanism in type II diabetes.

Extending this work, Gemma has investigated other furin substrates, and together we have postulated that aberrant processing of a whole range of furin substrates could plausibly underpin the development of Metabolic Syndrome, a clustering of traits including aberrant lipoprotein metabolism, hypertension and impaired glucose handling) that is itself a substrate for diabetes and cardiovascular disease.

Unfortunately, the lack of a sufficiently sensitive and specific assay for furin activity has for a long time hampered progress towards testing this proposition experimentally. Existing assays, which rely for their specificity on the sequence of the fluorogenic substrate, have poor specificity over other proprotein convertases (and perhaps other proteases
too). By introducing an immunocapture step, followed by assay of activity of the captured enzyme using the fluorogenic substrate, we have overcome this issue and generated an assay that shows good specificity for furin, while retaining sufficient sensitivity to detect furin activity in normal cells. Furin is expressed at very low levels in normal cells (levels too low to detect with antibodies in Western blotting, for example), requiring a high sensitivity assay to quantify its activity.

With this new tool now published, we can begin to investigate whether furin really does play a pathogenic role in diabetes. In parallel, we hope our assay will also facilitate other groups in the proprotein convertase field to increase their understanding of the cell biology of this fascinating enzyme.