Presentation Title

CDP identified as a potential substrate fragment of PERM-1’s NAD-dependent ascarylose biosynthesis

Start Date

November 2016

End Date

November 2016

Location

HUB 302-186

Type of Presentation

Poster

Abstract

Current treatments for helminth infections fail to target parasitic worm embryos because of the permeability barrier (PB), which controls the embryo viability. Previous studies identified PERM-1 from C.elegans as an essential transmembrane protein for the synthesis of ascaroside glycolipids in the PB. It’s been proposed PERM-1 catalyzes a NAD-dependent epimerization/reduction reaction as the final step in the synthesis of cytidine diphosphate (CDP)-L-ascarylose. The actual substrate of PERM-1, however, remains unknown. To confirm the PERM-1’s hypothesized binding with NAD and determine the potential substrates, a truncated recombinant construct containing the cytosolic domain has been expressed in a shuffle cell (E. coli) overexpression system. The recombinant protein of the N-terminal truncated cytosolic domain has been purified using detergent-based purification protocols. The folding of the protein was checked by fluorescence spectroscopy. Isothermal titration calorimetry (ITC) and NanoOrange dye differential scanning fluorimetry (DSF) were used independently to characterize PERM-1’s binding with potential substrates. ITC suggested a weak binding between UDP-Glucose and PERM-1 ( Kd ~ 4.75 mM), and a medium strength binding between NAD and PERM-1 ( Kd ~ 0.1 mM). DSF results indicated that PERM-1’s binding with NAD and CDP increased the protein’s melting temperature by 7°C and 10°C respectively, suggesting that PERM1 protein was stabilized by substrate binding. Our finding confirmed that NAD binds PERM-1 as a cofactor and suggested that CDP, instead of UDP, could be a fragment of the actual substrate.

Keyword: helminth infection, permeability barrier, PERM-1, isothermal titration calorimetry, differential scanning fluorimetry, NanoOrange, NAD binding

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Nov 12th, 1:00 PM Nov 12th, 2:00 PM

CDP identified as a potential substrate fragment of PERM-1’s NAD-dependent ascarylose biosynthesis

HUB 302-186

Current treatments for helminth infections fail to target parasitic worm embryos because of the permeability barrier (PB), which controls the embryo viability. Previous studies identified PERM-1 from C.elegans as an essential transmembrane protein for the synthesis of ascaroside glycolipids in the PB. It’s been proposed PERM-1 catalyzes a NAD-dependent epimerization/reduction reaction as the final step in the synthesis of cytidine diphosphate (CDP)-L-ascarylose. The actual substrate of PERM-1, however, remains unknown. To confirm the PERM-1’s hypothesized binding with NAD and determine the potential substrates, a truncated recombinant construct containing the cytosolic domain has been expressed in a shuffle cell (E. coli) overexpression system. The recombinant protein of the N-terminal truncated cytosolic domain has been purified using detergent-based purification protocols. The folding of the protein was checked by fluorescence spectroscopy. Isothermal titration calorimetry (ITC) and NanoOrange dye differential scanning fluorimetry (DSF) were used independently to characterize PERM-1’s binding with potential substrates. ITC suggested a weak binding between UDP-Glucose and PERM-1 ( Kd ~ 4.75 mM), and a medium strength binding between NAD and PERM-1 ( Kd ~ 0.1 mM). DSF results indicated that PERM-1’s binding with NAD and CDP increased the protein’s melting temperature by 7°C and 10°C respectively, suggesting that PERM1 protein was stabilized by substrate binding. Our finding confirmed that NAD binds PERM-1 as a cofactor and suggested that CDP, instead of UDP, could be a fragment of the actual substrate.

Keyword: helminth infection, permeability barrier, PERM-1, isothermal titration calorimetry, differential scanning fluorimetry, NanoOrange, NAD binding