13 research outputs found
Identification of Human Islet Amyloid Polypeptide as a BACE2 Substrate.
Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer's disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia
Effect of pH, Temperature, and Salt on the Stability of <i>Escherichia coli-</i> and Chinese Hamster Ovary Cell-Derived IgG1 Fc
The circulation half-life of a potential therapeutic
can be increased
by fusing the molecule of interest (an active peptide, the extracellular
domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal
antibody. For the fusion protein to be a successful therapeutic, it
must be stable to process and long-term storage conditions, as well
as to physiological conditions. The stability of the Fc used is critical
for obtaining a successful therapeutic protein. The effects of pH,
temperature, and salt on the stabilities of <i>Escherichia coli</i>- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order
structure were probed using a variety of biophysical techniques. Fc
molecules derived from both <i>E. coli</i> and CHO were
compared. The IgG1 Fc molecules from both sources (glycosylated and
aglycosylated) are folded at neutral pH and behave similarly upon
heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc
molecules occurs via a multistep unfolding process, with the tertiary
structure and C<sub>H</sub>2 domain unfolding first, followed by changes
in the secondary structure and C<sub>H</sub>3 domain. The acid-induced
unfolding of IgG1 Fc molecules is only partially reversible, with
the formation of high-molecular weight species. The CHO-derived Fc
protein (glycosylated) is more compact (smaller hydrodynamic radius)
than the <i>E. coli</i>-derived protein (aglycosylated)
at neutral pH. Unfolding is dependent on pH and salt concentration.
The glycosylated C<sub>H</sub>2 domain melts at a temperature 4–5
°C higher than that of the aglycosylated domain, and the low-pH-induced
unfolding of the glycosylated Fc molecule occurs at a pH ∼0.5
pH unit lower than that of the aglycosylated protein. The difference
observed between <i>E. coli</i>- and CHO-derived Fc molecules
primarily involves the C<sub>H</sub>2 domain, where the glycosylation
of the Fc resides
The selectivity of tyrosine 280 of human 11β-hydroxysteroid dehydrogenase type 1 in inhibitor binding
Key for mouse IAPP peptide sequences and cleaved fragments.
<p>Key for mouse IAPP peptide sequences and cleaved fragments.</p
Key for human IAPP peptide sequences and cleaved fragments.
<p>Key for human IAPP peptide sequences and cleaved fragments.</p
Key for mutant human IAPP peptide sequences and cleaved fragments.
<p>Key for mutant human IAPP peptide sequences and cleaved fragments.</p
BACE2 modulates human IAPP protein levels in pancreatic beta-cells.
<p>(A) βTC3 cells co-transfected with, or without, hIAPP plasmid DNA (1μg) and varying concentrations of hBACE2 plasmid DNA, hBACE1 plasmid DNA, or empty pCMV DNA, the latter to normalize DNA concentrations across conditions. A commercial source of Myc-DDK tagged hIAPP lysate (5 μg, last lane) was used to confirm identification of the hIAPP bands. The relative intensity of the top hIAPP band (B) and bottom hIAPP band (C) for each condition, as detected by anti-DDK and normalized to β-actin. Results are averaged from five separate experiments and the standard error is shown.</p
BACE2 modulates human IAPP protein levels HEK293 cells.
<p>(A) RNAseq FPKM values for basal expression of human <i>IAPP</i>, <i>BACE2</i>, <i>BACE1</i>, <i>PCSK1</i>, <i>PCSK2</i> and <i>APP</i> in HEK293 cells. (B) HEK293 cells co-transfected with, or without, hIAPP plasmid DNA (1μg) and varying concentrations of hBACE2 plasmid DNA, hBACE1 plasmid DNA, or empty pCMV DNA, the latter to normalize DNA concentrations across conditions. A commercial source of Myc-DDK tagged hIAPP lysate (5 μg, last lane) was used to confirm identification of the hIAPP bands. The relative intensity of the top hIAPP band (C) and bottom hIAPP band (D) for each condition, as detected by anti-DDK and normalized to β-actin. Results are averaged from two separate experiments and the standard deviation is shown. (E) HEK293 cells co-transfected with hAPP, hBACE2, hBACE1 or empty pCMV DNA.</p
Morphometric analysis of pancreas tissue from the chronic dosing study.
<p>Morphometric analysis of pancreas tissue from the chronic dosing study.</p
Evaluation of BACE inhibitors, Compound J and Compound 15, in B6.Cg-Lep<sup>ob</sup>/J mice.
<p>(A) The IC<sub>50</sub>s (μM) for Compound J and Compound 15. (B) Plasma clearance of Compound J and Compound 15 after a single oral gavage administration at 30mg/kg in 8–10 week old male B6.Cg-Lep<sup>ob/J</sup> mice, n = 2 per time point. (C) Blood glucose levels before (0 minutes) and after an intraperitoneal glucose injection (10%/kg body weight) in B6.Cg-Lep<sup>ob/J</sup> mice treated daily for 14 days with vehicle (black diamond, n = 14), Compound 15 (red circle, n = 13), Compound J (blue square, n = 13), or Exendin 4 (green triangle, n = 14). Asterisks indicate statistical significance based on 2-way ANOVA analysis, ** = 0.007, ***≤ 0.0007, **** < 0.0001. Target coverage analysis for BACE2 (D) and Tmem27 (E) on islets isolated from B6.Cg-Lep<sup>ob/J</sup> mice treated with a single dose (30mg/kg, by oral gavage) of Compound J, Compound 15, or vehicle; n = 2 per group and islets were pooled for protein analysis. (F) One day before harvest, animals were injected with BrdU and pulsed for 24 hours prior to termination. Pancreas tissue was collected, stained for insulin and BrdU expression, and evaluated for several parameters to quantitate beta-cell proliferation. The image on the left shows a representative islet stained for insulin (Vulcan Red) and BrdU (DAB); the arrows point to Insulin<sup>+</sup>BrdU<sup>+</sup> cells. The corresponding image demonstrates the morphometric analysis applied to quantitate proliferating beta-cells: individual insulin<sup>+</sup> cells (green), insulin<sup>+</sup>BrdU<sup>+</sup> cells (blue, indicated by arrows).</p