NEDD8 is a small protein involved in essential pathways including cell cycle regulation and DNA-damage-induced apoptosis. It regulates these functions through conjugation to other proteins. In living cells, NEDD8 exists in 3 forms (pools): Conjugated NEDD8 (NEDD8 conjugated to some other protein, of which there are many), Activated NEDD8 (NEDD8 conjugated to NEDD8 enzymes), and Free NEDD8 (unconjugated NEDD8). These pools’ concentrations are kept in balance by neddylation, the conjugation of free NEDD8 to a protein, deneddylation, the separation of conjugated NEDD8 from its target to make free NEDD8, and translation of the NEDD8 gene to manufacture more NEDD8. Maintaining the balance of the two NEDD8 pools is critical to maintaining homeostasis. Chemicals that inhibit neddylating or deneddylating enzymes can potentially be used to treat diseases caused by overexpression of these enzymes. Current NEDD8-related enzyme assays are specialized for NEDD8 conjugated to a specific target and cannot be efficiently scaled up or adapted to be used with other forms of conjugated NEDD8. There is currently no sensor that can detect free NEDD8. Because free NEDD8 is either a substrate or product of all NEDD8-related enzymes, development of such a sensor would enable the study of the ratio between the two NEDD8 pools in cells and enable large-scale drug screening. Our project aims to develop a sensor to measure free NEDD8 concentrations for general NEDD8-related enzyme assays. Such a sensor should have high sensitivity and specificity to NEDD8 and must be equipped with a method for signaling when it has bound to the target. To this end, we are developing a fluorescent biosensor that can be used to quantify the concentration of free NEDD8 in-vitro and in living cells. Our sensor is derived from a genetically modified deneddylase enzyme. It is manufactured by transforming E. coli cells with a cloned plasmid with the gene encoding our sensor. Our current work focusses on optimizing the sensitivity and selectivity of our sensor. We have produced several models of our sensor with different signaling methods and active site mutations. We have characterized one version of the sensor using a binding assay. Its Kd for free NEDD8 is 212 nM, and its Kd for conjugated NEDD8 is 140 μM. These data show us that our sensor has 660 times higher affinity for free NEDD8 than for conjugated NEDD8. Future work will focus on applying the sensor to free NEDD8 assays and drug screening.

Development of biosensors specific for free NEDD8

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