Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity.

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Šaway.

Enzyme Replacement Therapy for Mucopolysaccharidosis IIID using Recombinant Human α-N-Acetylglucosamine-6-Sulfatase in Neonatal Mice.

There is currently no cure or effective treatment available for mucopolysaccharidosis type IIID (MPS IIID, Sanfilippo syndrome type D), a lysosomal storage disorder (LSD) caused by the deficiency of α-N-acetylglucosamine-6-sulfatase (GNS). The clinical symptoms of MPS IIID, like other subtypes of Sanfilippo syndrome, are largely localized to the central nervous system (CNS), and any treatments aiming to ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease need to be delivered across the blood-brain barrier. Here, we report a proof-of-concept enzyme replacement therapy (ERT) for MPS IIID using recombinant human α-N-acetylglucosamine-6-sulfatase (rhGNS) via intracerebroventricular (ICV) delivery in a neonatal MPS IIID mouse model. We overexpressed and purified rhGNS from CHO cells with a specific activity of 3.9 × 104 units/mg protein and a maximal enzymatic activity at lysosomal pH (pH 5.6), which was stable for over one month at 4 °C in artificial cerebrospinal fluid (CSF). We demonstrated that rhGNS was taken up by MPS IIID patient fibroblasts via the mannose 6-phosphate (M6P) receptor and reduced intracellular glycosaminoglycans to normal levels. The delivery of 5 µg of rhGNS into the lateral cerebral ventricle of neonatal MPS IIID mice resulted in normalization of the enzymatic activity in brain tissues; rhGNS was found to be enriched in lysosomes in MPS IIID-treated mice relative to the control. Furthermore, a single dose of rhGNS was able to reduce the accumulated heparan sulfate and ß-hexosaminidase. Our results demonstrate that rhGNS delivered into CSF is a potential therapeutic option for MPS IIID that is worthy of further development.

Evaluating p97 Inhibitor Analogues for Potency against p97-p37 and p97-Npl4-Ufd1 Complexes.

We previously found that the p97 cofactor, p47, significantly decreased the potency of some ATP-competitive p97 inhibitors such as ML240 [2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine] and ML241 [2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8 tetrahydroquinazolin-4-amine]. In this study, we aimed to evaluate inhibitor potencies against two additional p97 cofactor complexes, p97-p37 and p97-Npl4-Ufd1. We focused on these two cofactor complexes, because the protein sequence of p37 is 50 % identical to that of p47, and the Npl4-Ufd1 heterodimer (NU) is the most-studied p97 cofactor complex. We screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of p97 alone and of p97-p37 and p97-NU complexes. In contrast to the effect of p47, p37 and NU did not significantly change the potencies of most of the compounds. These results highlight differences among p97 cofactors in influencing p97 conformation and effects of inhibitors on p97 complexes, as compared to p97 alone. Continued efforts are needed to advance the development of complex-specific p97 inhibitors.

Evaluating p97 inhibitor analogues for their domain selectivity and potency against the p97-p47 complex.

We previously found that p97 ATPase inhibitors 2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine (ML240) and 2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8-tetrahydroquinazolin-4-amine (ML241) specifically target the D2 domain of wild-type p97. In addition, one of the major p97 cofactors, p47, decreases their potencies by ∼50-fold. In contrast, N(2) ,N(4) -dibenzylquinazoline-2,4-diamine (DBeQ) targets both the D1 and D2 domains and shows only a four- to sixfold decrease in potency against the p97-p47 complex. To elucidate structure-activity relationships for the inhibitors, we screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of either or both of the D1 or D2 domains, as well for their effects on p47 potency. The selectivity of 29 of these compounds was further examined by eight-dose titrations. Four compounds showed modest selectivity for inhibiting the ATPase activity of D1. Eleven compounds inhibited D2 with greater potencies, and four showed similar potencies against D1 and D2. p47 decreased the potencies of the majority of the compounds and increased the potencies of five compounds. These results highlight the possibility of developing domain-selective and complex-specific p97 inhibitors in order to further elucidate the physiological roles of p97 and its cofactors.