Stem cell factor (SCF) is a cytokine that regulates hematopoiesis and other biological processes. While clinical treatments using SCF would be highly beneficial, these have been limited by toxicity related to mast cell activation. Transmembrane SCF (tmSCF) has differential activity from soluble SCF and has not been explored as a therapeutic agent. We created novel therapeutics using tmSCF embedded in proteoliposomes or lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and improved the revascularization in the ischemic hind limb. Proteoliposomal tmSCF preferentially acted on endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization and recruitment to the site of injury. The type of lipid nanocarrier used altered the relative cellular uptake pathways and signaling in a cell type dependent manner. Overall, we found that tmSCF-based therapies can provide therapeutic benefits without off target effects.

Understanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved globally-available drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.

No abstract

Understanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved alcohol-abuse drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.Summary sentenceParasite-encoded COP9 signalosome is an essential upstream regulator of ubiquitin-proteasome mediated protein degradation, and shows significant potential as a therapeutic target.

The Ubiquitin‐Proteasomal System (UPS) is essential for cell survival because it is responsible for the large majority of protein degradation within the cell. Thus, targeting the UPS has emerged as an attractive approach for combating protozoan parasites. Yet, the regulation of UPS‐mediated protein degradation remains poorly understood in clinically important protozoans. We identified COP9 signalosome to be conserved in multiple pathogenic parasites including Leishmania, Trypanosoma, Toxoplasma and Entamoeba. Here, we show that COP9 signalosome is an essential and druggable parasite target that regulates protein degradation. Using Entamoeba histolytica as a model parasite, we found that genetic disruption of COP9 subunit 5 (CSN5) by two independent but complementary approaches, inhibited parasite cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin1 in a neddylated state disrupting UPS activity and protein degradation. Using virtual screening by molecular docking, we identified zinc dithiocarbamate (ZnDTC), a metabolite of the FDA approved alcohol drug disulfiram, as a potential COP9 inhibitor. We found that ZnDTC acts in a COP9‐dependent manner, phenocopy CSN5 gene disruption, and is active against E. histolytica parasites at nanomolar concentrations. In addition, in‐vivo studies revealed that ZnDTC had oral efficacy in clearing E. histolytica parasites in a mouse model that mirrors human amebic colitis. Parasite clearance was assessed by live bioluminescent imaging, amebic culture and immunohistochemistry. Also, ZnDTC treatment promoted resolution of inflammation and tissue damage. Our findings provide insights into how COP9 signalosome regulates parasite protein degradation, and supports COP9 inhibition as a potential anti‐parasite therapy.