Functional and structural characterization of neutral cholesterol ester hydrolase 1
Date
2016
Authors
Journal Title
Journal ISSN
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Publisher
University of Delaware
Abstract
Neutral cholesterol ester hydrolase 1 (NCEH1), also known as KIAA1363 and
arylacetamide deacetylase-like 1 (AADACL1), has been found to be upregulated in
several invasive cancers. The ability of this enzyme to act as a biomarker and possible
therapeutic target increased the desire for research that elucidated the endogenous
substrate of NCEH1, which was initially demonstrated to be 2-acetyl
monoalkylglycerol ether (2-acetyl MAGE), a de novo precursor to platelet-activating
factor. Later work also demonstrated a controversial role of NCEH1 in the hydrolysis
of cholesterol esters in macrophages, preventing the rupture of foam cells and thereby
preventing atherosclerosis. In recent years, research on NCEH1 has continued to
portray the enzyme as a pro-tumorigenic, anti-atherosclerotic, regulatory enzyme.
However, its role has also expanded to include participation in the regulation of
platelet aggregation, and it has surfaced as a potential protein engineering target due to
its ability to hydrolyze toxic organophosphate compounds. The structural knowledge
of NCEH1 is limited to structures of homologous serine hydrolases, the existence of
an N-terminal transmembrane helix and posttranslational glycosylation predicted to
occur at residues 270, 287, and 389.
A variety of fusion E. coli constructs were created using a truncated NCEH1
which removed the transmembrane helix. Further truncation removing a second
hydrophobic region was found to have no effect on solubility. It was determined that
NCEH1 requires a large fusion protein, like MBP, to help with the stability of the
enzyme, and cleavage of MBP fusion resulted in degradation of the protein.
Functional characterization of the NCEH1 fusion protein was carried out with paranitrophenyl
acetate and a modified natural substrate 2-thio-acetyl MAGE. Also
reported within this work are the first reported KM and kcat values for the hydrolysis of
PNPA by NCEH1, determined to be 5.3 mM and 4 s-1, respectively.
In an effort to isolate purified enzyme lacking a fusion protein partner,
constructs of the 111-440 NCEH1 truncation were developed for expression in the
eukaryotic yeast system Pichia pastoris. Both intracellular and secreted expression
protocols were developed and generated a stable construct with a small histidine tag.
The construct was further modified to include a TEV protease cleavage site to allow
for the removal of the histidine tag after purification and both expression protocols
yielded purified stable NCEH1. The enzyme was recovered in high purity and
adequate concentration for the screening of optimal crystallization conditions
providing an important first step toward the structural elucidation of NCEH1.
Previous research has indicated glycosylation for functional activity; however
the work of this thesis provides an alternative view of the need for glycosylation.
Instability of NCEH1 in an E. coli expression system supports a more structural role
for glycosylation over functional. Mutation of the three glycosylation residues in the
Pichia pastoris secretory expression system, without a loss in functional ability, also
demonstrates the possible need for glycosylation in folding and stability.
The work compiled in this thesis advances our knowledge of NCEH1. The
development of an expression and purification protocol generating a purified enzyme
advances the ability to functionally characterize NCEH1. In addition, preliminary
crystallization screens provided three potential conditions for future protein crystal
optimization. The NCEH1 structure will ultimately further our knowledge and ability
to elevate the treatments of diseases directly correlated to its function.