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TGN resident proteases include carboxypeptidases, prohormone convertase (PC) family members, and β-site amyloid precursor protein (BACE) family members. This process of pro-protein maturation involves recognition and cleavage of unique sequences in target protein prodomains by specific proteases. Proteases involved in maturation of secretory proteins typically reside in the Trans-Golgi Network (TGN) where they proteolytically process newly formed proteins from the endoplasmic reticulum before packaging into secretory vesicles. Proteolytic processing is modulated both temporally and positionally and contributes to protein activation and sub-cellular localization. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.Ĭellular proteases perform diverse critical functions crucial to proper execution of physiological processes including development, hormone maturation, immunity, blood clotting, pathogenesis of viral and bacterial diseases, and programmed cell death. BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease.

Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu.