2 research outputs found
Unlocking a Caged Lysosomal Protein from a Polymeric Nanogel with a pH Trigger
A polymeric nanogel
has been used to sequester and turn off a lysosomal
protein, acid α-glucosidase (GAA). The nanogel contains a β-thiopropionate
cross-linker, which endows the nanogel with pH-sensitivity. While
encapsulation of the enzyme fully turns off its activity, approximately
75% of the activity is recovered upon reducing the pH to 5.0. The
recovered activity is ascribed to pH-induced degradation of the β-thiopropionate
cross-linker causing the swelling of the nanogel and ultimately causing
the release of the enzyme. We envision that strategies for sequestering
protein molecules and releasing them at lysosomal pH might open up
new directions for therapeutic treatment of lysosomal storage diseases
Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and Its H‑Bonding Network
Crystal structures of nickel-dependent
superoxide dismutases (NiSODs)
reveal the presence of a H-bonding network formed between the NH group
of the apical imidazole ligand from His1 and the Glu17 carboxylate
from a neighboring subunit in the hexameric enzyme. This interaction
is supported by another intrasubunit H-bond between Glu17 and Arg47.
In this study, four mutant NiSOD proteins were produced to experimentally
evaluate the roles of this H-bonding network and compare the results
with prior predictions from density functional theory calculations.
The X-ray crystal structure of H1A-NiSOD, which lacks the apical ligand
entirely, reveals that in the absence of the Glu17–His1 H-bond,
the active site is disordered. Characterization of this variant using
X-ray absorption spectroscopy (XAS) shows that NiÂ(II) is bound in
the expected N<sub>2</sub>S<sub>2</sub> planar coordination site.
Despite these structural perturbations, the H1A-NiSOD variant retains
4% of wild-type (WT) NiSOD activity. Three other mutations were designed
to preserve the apical imidazole ligand but perturb the H-bonding
network: R47A-NiSOD, which lacks the intramolecular H-bonding interaction;
E17R/R47A-NiSOD, which retains the intramolecular H-bond but lacks
the intermolecular Glu17–His1 H-bond; and E17A/R47A-NiSOD,
which lacks both H-bonding interactions. These variants were characterized
by a combination of techniques, including XAS to probe the nickel
site structure, kinetic studies employing pulse-radiolytic production
of superoxide, and electron paramagnetic resonance to assess the Ni
redox activity. The results indicate that in addition to the roles
in redox tuning suggested on the basis of previous computational studies,
the Glu17–His1 H-bond plays an important structural role in
the proper folding of the “Ni-hook” motif that is a
critical feature of the active site