Development of Nanobodies as first-in-class inhibitors for the NEDP1 deNEDDylating enzyme

Abstract

Protein NEDDylation emerges as an important post-translational modification and an attractive target for therapeutic intervention. Modification of NEDD8 onto substrates is finely balanced by the co-ordinated activity of conjugating and deconjugating enzymes. The NEDP1/DEN1/SENP8 protease is a NEDD8 specific processing and deconjugating enzyme that regulates the NEDDylation mainly of non-cullin substrates. Here, we report the development and characterisation of nanobodies as first-in-class inhibitors for NEDP1. The nanobodies display high-affinity (low nM) against NEDP1 and specifically inhibit NEDP1 processing activity in vitro and NEDP1 deconjugating activity in tissue-culture cells and in cell extracts. We also isolated nanobodies that bind to NEDP1 with high-affinity but do not affect NEDP1 activity. The developed nanobodies provide new tools to study the function of NEDP1 and to prevent deNEDDylation in cell extracts used in biochemical assays.

Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution

“…Critically, the data in C. elegans demonstrate that the elimination of ALS-related SGs upon NEDP1 inhibition is correlated with restoration of animal motility, providing the proof-of-principle for targeting NEDP1 for therapeutic intervention. This notion is strongly supported with the use of Nbs that potently and specifically inhibit NEDP1 activity (Abidi et al, 2020) and fully reproduce the findings on aberrant SG dissolution using genetic approaches for NEDP1 inhibition.…”

“…For this, we transiently expressed an anti-NEDP1 Nanobody (Nb9) that displays nM binding affinity and specifically inhibits NEDP1 activity (Abidi et al, 2020). Characterisation in vitro and in tissue culture cells, shows that Nb9 potently inhibits both the processing and deconjugating activity of NEDP1, resulting in the accumulation of NEDD8 conjugates to a level similar to that achieved upon NEDP1 knockout (Abidi et al, 2020). Consistent with this notion, expression of Nb9 increases protein NEDDylation and accelerates SG assembly, similarly to what is observed upon deletion of the NEDP1 gene (Figure 1D-F).…”

“…In addition, we also assessed the effect of transient inhibition of NEDP1 on SG assembly upon stress. For this, we transiently expressed an anti-NEDP1 Nanobody (Nb9) that displays nM binding affinity and specifically inhibits NEDP1 activity (Abidi et al, 2020). Characterisation in vitro and in tissue culture cells, shows that Nb9 potently inhibits both the processing and deconjugating activity of NEDP1, resulting in the accumulation of NEDD8 conjugates to a level similar to that achieved upon NEDP1 knockout (Abidi et al, 2020) Based on these data we also determined the role of NEDP1 in the disassembly of SGs during the recovery period.…”

“…For this, we transiently expressed an anti-NEDP1 Nanobody (Nb9) that displays nM binding affinity and specifically inhibits NEDP1 activity (Abidi et al, 2020). Characterisation in vitro and in tissue culture cells, shows that Nb9 potently inhibits both the processing and deconjugating activity of NEDP1, resulting in the accumulation of NEDD8 conjugates to a level similar to that achieved upon NEDP1 knockout (Abidi et al, 2020) Based on these data we also determined the role of NEDP1 in the disassembly of SGs during the recovery period. Alleviation of stress in control cells causes a progressive decrease in the levels of NEDD8 conjugates induced upon stress, indicative of the recovery process (Figure 2A).…”

Targeting the NEDP1 enzyme to ameliorate ALS phenotypes through stress granule disassembly

“…In addition, we assessed the effect of transient inhibition of NEDP1 on SG dynamics. For this, we transiently expressed an anti-NEDP1 Nb (Nb9) that displays nanomolar binding affinity and specifically inhibits NEDP1 activity (53). Characterization in vitro and in tissue culture cells shows that Nb9 potently inhibits the deconjugating activity of NEDP1, resulting in the accumulation of NEDD8 conjugates (53).…”