HtrA1 Proteolysis of ApoE in Vitro Is Allele Selective

Article abstract of DOI:10.1021/jacs.6b03463

Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport. Humans have three different APOE alleles, APOE e2, APOE e3, and APOE e4, and genetic studies identified ApoE4 as the strongest genetic risk factor for Alzheimer's disease (AD). People who are homozygous for ApoE4 (i.e., ApoE4/E4) are an order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3 in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as Tau.

Full text of DOI:10.1021/jacs.6b03463

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Article
HtrA1 Proteolysis of ApoE in vitro is Allele Selective
Qian Chu, Jolene K Diedrich, Joan M. Vaughan, Cynthia J.
Donaldson, Michael F. Nunn, Kuo-Fen Lee, and Alan Saghatelian
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03463 • Publication Date (Web): 05 Jul 2016
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HtrA1 Proteolysis of ApoE in vitro is Allele Selective
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Qian Chu , Jolene K. Diedrich , Joan M. Vaughan , Cynthia J. Donaldson , Michael F. Nunn ,
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Kuo-Fen Lee , and Alan Saghatelian
† The Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology, 10010 N. Torrey
Pines Rd, La Jolla, CA 92037, USA
‡
USA
Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037,
ABSTRACT: Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport.
Humans have three different APOE alleles—APOE ε2, APOE ε3 and APOE ε4—and genetic studies identified ApoE4 as the
strongest genetic risk factor for Alzheimer’s disease (AD). People who are homozygous for ApoE4 (i.e. ApoE4/E4) are an
order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between
ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3
in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the
nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly
than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin
had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the
amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition
assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an
in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as
Tau.
INTRODUCTION
ApoE is a 299 amino acid protein with three different
22
isoforms in humans, ApoE2, ApoE3, and ApoE4 . The
three isoforms differ at two amino acid positions, 112 and
An aging populous will lead to a larger percentage of
individuals with Alzheimer’s disease (AD). Research into
AD hopes to fight this outcome by identifying targets that
can treat or prevent AD. Genetic studies of families with
early-onset (i.e. familial) AD led to the identification of
mutations of amyloid precursor protein (APP) and the
1
58. ApoE2 has two cysteines at these positions, ApoE4
has two arginines at these positions, and ApoE3 has a
cysteine at 112 and an arginine at 158 . ApoE4 is the
23,24
25,26
least stable of all three isoforms , and one hypothesis is
that this instability leads to accelerated ApoE4
degradation. Indeed, analysis of human tissue from
homozygous ApoE3 and ApoE4 genotypes revealed
increased ApoE proteolytic fragments in ApoE4
background . Whether the loss of ApoE4 or the
generation of ApoE4 fragments are related to AD is not
known
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,2
presenilins (PSEN1 and PSEN2) . Mechanistic studies
revealed that the presenilin processing of APP led to the
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-5
formation of the amyloid beta peptide (Aβ) , which is
the major constituent of plaques in the post-mortem
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6-8
brains of AD patients . Increased production of Aβ is
associated with AD, and, therefore, AD drugs under
development
Unfortunately, early results have been disappointing
In addition to research into early onset AD, which
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,10
.
seek
to
target
this
pathway
.
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.
We became interested in trying to identify a candidate
protease that might be responsible for ApoE4 breakdown.
One study using cultures of primary rat neurons
demonstrated that an extracellular serine protease is
responsible for ApoE4 processing, though the enzyme was
not identified . High-temperature requirement serine
peptidase A1 (HtrA1) belongs to the serine peptidase
family. Biochemical experiments have revealed that
amyloid precursor protein (APP) and Tau are both HtrA1
substrates . Oligomerization of hyperphosphorylated
Tau to form neurofibrillary tangles is another hallmark of
AD, and in vitro experiments with HtrA1 showed that this
protease can disentangle Tau fibrils and cleave Tau .
Here, we examined the possibility that ApoE4 may also be
an HtrA1 substrate.
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comprises up to 5% of total AD cases , genetics has also
looked for clues for late-onset AD (LOAD), and these
experiments identified ApoE4 as the strongest risk factor
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4-16
for LOAD . People that are homozygous for APOE ε4
allele are 12 times more likely to suffer from AD at the age
of 65 than corresponding non-APOE ε4 carriers . ApoE is
predominantly produced and secreted from astrocytes
and this protein is responsible for trafficking cholesterol
to neurons in the form of lipoprotein particles
Furthermore, research into the role of APOE ε4 and AD
has revealed several potential mechanisms that might
explain the role of this gene in AD.
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.
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kDa that are not observed for ApoE3 (Figure 2B). For
example, we identified fragments between amino acids 1-
25, and 222-299 that are unique to ApoE4 and not
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RESULTS AND DISCUSSION
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ApoEs are HtrA1 substrates. We tested whether HtrA1 is
able to mediate the proteolysis of ApoE4. These initial
experiments were carried out with purified recombinant
ApoE proteins and HtrA1. We incubated recombinant
ApoE3 and ApoE4 with various doses of human HtrA1 (10-
observed in the ApoE3 proteolysis (Figure S1).
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0 µg/mL). Degradation of ApoE3 and ApoE4 was
measured by SDS-PAGE followed by Coomassie staining
to visualize remaining full-length ApoE protein and ApoE
proteolytic fragments. Upon proteolysis of ApoE, we
observed decreased levels of full-length ApoE with
corresponding appearance of lower molecular weight
proteolytic fragments (Figure 1). Higher concentrations of
HtrA1 led to increased proteolysis of ApoEs and their
proteolytic fragments (i.e. 20 kDa and 25 kDa in ApoE4
degradation). Mutation of the HtrA1 catalytic serine
residue (Ser328) to alanine (HtrA1
ApoE proteins (Figure 1), which rules out any
contaminating proteases as the source of the degradation.
Thus, HtrA1 can cleave ApoEs in vitro.
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S328A
) is unable to cleave
Figure 1. In vitro degradation of ApoE proteins by human
HtrA1. Recombinant ApoE3 or ApoE4 (2 µg) was incubated with
purified recombinant human ΔN-HtrA1 (aa 156-480, wild type or
S328A mutant) for 2 hours at 37°C. Full-length ApoE proteins and
their proteolytic fragments were visualized by SDS-PAGE
followed by Coomassie staining.
Figure 2. Kinetic study of in vitro degradation of ApoE
proteins by recombinant HtrA1 enzyme. Recombinant A)
ApoE3 and B) ApoE4 protein (5 µg) were incubated with 10
µg/mL human ΔN-HtrA1 (aa 156-480, 15 µL total volume) at 37°C
for indicated times. ApoE full-length protein and its proteolytic
fragments were Coomassie stained. C) Full-length proteins were
quantified and used to calculate the degree of degradation. The
percent degradation was determined by dividing band intensity
of the full-length ApoE at each time point by the band intensity
at the start of the experiment (i.e., t=0). The kinetic curves were
fitted by nonlinear regression and the dash lines indicated the
time where 50% of the ApoE was degraded.
HtrA1 cleavage of ApoE is allele selective. Comparison
of the processing of ApoE3 and ApoE4 indicated that
ApoE4 is a better substrate for HtrA1 than ApoE3. At
every dose of HtrA1 there is less ApoE4 than ApoE3 and
cleavage of ApoE4 generates more proteolytic fragments
than ApoE3 (Figure 1).
A plot of degradation percentage against incubation
time showed that cleavage of ApoE4 was much faster than
ApoE3. At 8 hours, for instance, over 80% of the starting
ApoE4 was degraded while less than 40% ApoE3 had
undergone proteolysis (Figure 2C). And after 22 hours,
almost all ApoE4 was cleaved, while only slightly more
than 60% of the ApoE3 was gone. To quantitate the
differences between ApoE alleles, we defined the half-life
as the time where 50% of the ApoE was degraded. With a
half-life of about 2 hours for ApoE4 and a half-life of
nearly 13 hours for ApoE3, HtrA1 has a clear allele
selectivity for ApoE4 over ApoE3 (Figure 2C). We also
calculated reaction rates by a linear fit of the first four
time points, and found that the reaction rate for ApoE4
proteolysis is about 7.4 times faster than that for ApoE3
We quantified any preference for ApoE4 by measuring
the percent degradation of ApoE3 and ApoE4 in the pres-
ence of HtrA1 over a 22-hour period. Over this time
period, we observed more ApoE4 than ApoE3 degradation
(Figure 2A and 2B). ApoE3 and ApoE4 share three major
proteolysis fragments at ~25, ~20, and ~12 kDa.
Proteomics experiments identified these fragments as
amino acids 1-195 (~25 kDa), 139-299 (~20 kDa), and 196-
299 (~12 kDa) (Figure S1). With ApoE3 these fragments
appear stable and continue to accumulate throughout the
experiment (Figure 2A). By contrast, the similar ApoE4
fragments are less stable and the levels of most ApoE4
fragments decrease between 10 and 22 hours indicating
further proteolysis of these fragments (Figure 2B).
Furthermore, ApoE4 proteolysis results in an entire set of
proteolytic fragments between 10-20 kDa and less than 10
-
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-1
(5.72 and 0.77 × 10 M•s respectively, Figure S2).
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our biochemical studies indicate that HtrA2 cannot cleave
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either ApoE3 or ApoE4 (Figure 3B and 3C). We also found
no proteolysis of ApoE4 in the presence of the 26S
proteasome (Figure S5). The fact that HtrA2 and 26S
proteasome are not ApoE degrading enzymes indicates
that ApoE4 degradation not general.
Furthermore, trypsin cleaves both ApoE3 and ApoE4,
but we found no allele specificity with trypsin (Figure
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D). Comparison of HtrA1 and trypsin indicates unique
features about HtrA1-ApoE4 interaction, and indicates
that ApoE4 specific degradation is more complicated than
ApoE4 being less stable than ApoE3. In addition, the
proteolytic fragments of ApoE3 and ApoE4 generated by
trypsin differ from HtrA1, indicating that these enzymes
use different cleavage sites, which could be part of the
explanation of the allele selectivity.
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Figure 3. Comparison of ApoE proteolysis by human HtrA1
and HtrA2. A) Structural overlay of human HtrA1 (Wheat, PDB
id: 3NZI) and HtrA2 (Cyan, PDB id: 1LCY) catalytic domains. The
three catalytic triad residues are highlighted in stick
representation. H220, D250 and S328 of HtrA1 are shown in
green, and H198, D228 and S306 of HtrA2 are shown in magenta.
B) Recombinant ApoE3 and C) ApoE4 were incubated with
purified human ΔN-HtrA1 (aa 156-480) or human ΔN-HtrA2 (aa
1
(
34-458) for 2 hours at 37°C. D) Recombinant ApoE3 and ApoE4
2 µg) were incubated with indicated amount of trypsin for 30
min at 37°C. ApoE full-length protein and its proteolytic
fragments were visualized by Coomassie staining.
Specificity of HtrA1-ApoE4 enzyme-substrate inter-
action. In addition to the knowledge that ApoE4 is a
better HtrA1 substrate than ApoE3, we wanted to know
whether ApoE4 is a better substrate than other proteins
too. We tested this by performing the cleavage reaction in
the presence of bovine serum albumin (BSA, 0.1 mg/mL).
If HtrA1 binds to and/or cleaves BSA, we would expect to
observe a slowing in the ApoE4 processing. Comparison
of the ApoE4 proteolysis by HtrA1 in the presence and
absence of BSA showed little difference in ApoE4
proteolysis (Figure S3), indicating that HtrA1 recognizes
specific features of ApoE4 protein.
Figure 4. In vitro degradation of ApoE proteins by HtrA1
transfected HEK293T cells. A) Expression of full-length HtrA1
(wildtype and S328A mutant) in HEK293T cells indicated that the
majority of HtrA1 protein is secreted. B) Recombinant ApoE4
protein (5 µg) were incubated with conditioned media (C.M.)
from HEK293T cells transfected with wildtype full-length HtrA1
or S328A mutant at 37 °C for 4 hours. C) Recombinant ApoE3 and
ApoE4 (2 µg) were incubated with indicated amount of
conditioned media (C.M.) from HEK293T cells transfected with
wildtype full-length HtrA1 for 2 hours at 37 °C. ApoE full-length
protein and its proteolytic fragments were visualized by western
immunoblotting.
ApoE is degraded by HtrA1 in cell culture. Next, we
tested whether HtrA1 can process ApoE4 in cell culture.
We transfected full-length HtrA1 with a C-terminal myc
tag into HEK293T cells. HtrA1 has a validated N-terminal
signaling sequence that directs this enzyme through the
Next, we wanted to know whether other peptidases are
also able to cleave ApoE4. We tested HtrA2, the 26S
proteasome, and trypsin. HtrA2 has an almost identical
catalytic domain to HtrA1 (Figure 3A and S4), however,
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secretory pathway, and western blot analysis revealed that inhibitor at different doses (0-5 µM) and measuring
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most of the myc-tagged HtrA1 is secreted into the cell
culture media (Figure 4A). Incubation of ApoE4 with the
cell lysate or conditioned media showed that ApoE4 is
only degraded in the conditioned media, which contains
the highest levels of HtrA1 (Figure 4B and S6).
ApoE4 degradation. HtrA1 inhibition blocked ApoE4
degradation to indicate that HtrA1 is the primary enzyme
responsible for ApoE4 proteolysis in U-87 conditioned
media (Figure 5C).
To control for the possibility that another extracellular
protease is responsible for ApoE4 proteolysis, we repeated
the experiment using a catalytically inactive HtrA1 mutant
S328A
S328A
(
HtrA1
). HtrA1
was highly expressed in HEK293T
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cells and, like wildtype HtrA1, was predominately secreted
from the cell (Figure 4A). Conditioned media and cell
S328A
lysate from HEK293T cells transfected with HtrA1
had
no activity indicating that HtrA1 is responsible for the
ApoE4 proteolysis (Figure 4B and S6). Exogenous HtrA1
expression and secretion is sufficient to mediate ApoE4
cleavage in cell culture, and given that ApoE4 is secreted
from astrocytes supports a potential interaction between
these two proteins in tissues.
In addition, we compared ApoE3 and ApoE4 cleavage
using conditioned media from HEK293T cells transfected
with wildtype full-length HtrA1. Both ApoE3 and ApoE4
showed a dose-dependent degradation, but ApoE4 has
more proteolytic fragments (Figure 4C). The fragments
are similar in size as degradation in vitro by recombinant
ΔN-HtrA1. We also performed ApoE degradation over a
Figure 5. In vitro degradation of ApoE proteins by U-87
conditioned media. A) Recombinant ApoE3 and ApoE4 proteins
(2 µg) were incubated with U-87 conditioned medium (C.M.) at
3
7 °C for 3 hours. B) Chemical structure of the HtrA1 inhibitor. C)
Recombinant ApoE4 (2 µg) was incubated with U-87 conditioned
medium (1 mg/mL) in presence of indicated amount of HtrA1
inhibitor for 3 hours at 37 °C. ApoE full-length protein and its
proteolytic fragments were visualized by western blotting.
26-hour time course by full-length HtrA1 from HEK293T
conditioned media (Figure S7). Similarly as ΔN-HtrA1
cleavage, several ApoE4, but not ApoE3, fragments
underwent further proteolysis.
ApoE4 inhibits Tau proteolysis by HtrA1. One AD
associated pathology is the accumulation of neuro-
fibrillary tangles generated by abnormal aggregation of
Tau proteins. In vitro biochemical studies into the
regulation of Tau led to the identification of HtrA1 as a
Tau degrading enzyme. HtrA1 has recently been reported
to have a non-proteolytic activity that enables it to
disentangle Tau neurofibrillary tangles, suggesting that it
Next, we wanted to test whether endogenous HtrA1 is
sufficient to induce allele selective degradation of ApoE
proteins. U-87 MG human brain glioblastoma cells have
the highest HtrA1 mRNA level according to Human
Protein Atlas . We observed robust HtrA1 secretion in
conditioned media from U-87 cells (Figure S8).
Incubation of ApoE proteins with U-87 conditioned
media showed a dose-dependent proteolysis for ApoE4,
while ApoE3 cleavage was limited, and the cleavage
pattern was similar as purified ΔN-HtrA1 and full-length
HtrA1 from HEK293T conditioned medium (Figure 5A).
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can regulate the levels and aggregation of Tau .
Since ApoE4 is
a major genetic risk factor for
Alzheimer’s disease and HtrA1 is able to cleave both
ApoE4 and Tau, we wondered whether ApoE4 might
compete with Tau as a substrate for HtrA1. To test this
hypothesis, we incubated recombinant Tau protein with
pure HtrA1 enzyme in the presence of ApoE proteins and
analyzed the results at various times (Figure 6). Without
ApoE present, HtrA1 degraded Tau leading to a steady
decrease in full-length Tau levels over time. And after 90
minutes all of the Tau protein is gone. In the presence of
We then sought to block in vitro ApoE degradation by
an HtrA1 inhibitor (HtrA1 Boronic Acid Inhibitor, HBAI)
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(Figure 5B), which has an IC50 about 0.21 µM . We chose
chemical inhibition over siRNA because this was certain
to block the majority of HtrA1 activity. We validated the
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specificity of HBAI by activity-based proteomics . We
added HBAI at several doses (5-100 uM) to mouse brain
lysate in the absence or presence of recombinant ΔN-
HtrA1 (for easier analysis) followed by addition of the
activity based probe FP-TAMRA. Analysis of the reactions
by SDS-PAGE revealed HtrA1 was inhibited by HBAI
(Figure S9), but no other proteins showed reduced FP-
TAMRA labeling which indicates that HBAI specifically
inhibits HtrA1 in the brain proteome. HBAI was also able
to block in vitro ApoE degradation by recombinant ΔN-
HtrA1 (Figure S10). Next, we used HBAI to test whether
HtrA1 is responsible for ApoE4 in U-87 by adding the
ApoE proteins, we observed
a slowdown of Tau
degradation, but ApoE4 was a better inhibitor than
ApoE3. After 90 minutes, about 40% of full-length Tau
protein was still intact in the presence of ApoE4, while
only about 20% remained in the ApoE3-containing
reaction. A great deal more work needs to be performed
in vivo to determine whether ApoE4 and Tau are
endogenous substrates of HtrA1. Nevertheless, the
concept that ApoE4 proteolysis, which is more prevalent
than ApoE2 or ApoE3, is competing with the degradation
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of AD-related proteins like Tau is compelling whether or
not HtrA1 is involved.
is secreted to extracellular space in vitro and in vivo ,
where it could interact with HtrA1. Therefore, by
elucidating this possible biochemical link between ApoE4
and Tau, this work has the potential of introducing a new
mechanistic avenue to investigate in vivo. In pursuing
these biochemical hypotheses, we will increase our
understanding of the regulation and function of ApoE4 in
late-onset AD, and this may eventually lead to new
opportunities for treating the oncoming AD epidemic.
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EXPERIMENTAL SECTION
Materials
Recombinant human HtrA2 (aa 134-458) and human 26S
Proteasome Protein were purchased from R&D Systems
(1458-HT-100 and E-365). Recombinant Tau protein
(2N4R) was obtained from rPeptide (T-1001-2).
Plasmids
ApoE3 cDNA clone was kindly provided by Dr. Zhijiang
Chen at the Salk Institute. The ApoE4 cDNA was obtained
by site-directed mutagenesis of the ApoE3 sequence using
QuikChange II kit (Agilent Technologies). ApoE3 and
ApoE4 coding sequences were then subcloned into pMAL
vector (New England Biolabs) with a TEV cleavage site
between ApoE protein and MBP fusion for bacterial
expression. Mammalian expression constructs of HtrA1
cDNA clones with a C-terminal myc tag were obtained
from Origene. Human ΔN-HtrA1 (aa 156-480) was
Figure 6. Inhibition of Tau degradation by ApoE. A)
Recombinant Tau (600 nM) was incubated with ApoE3 or ApoE4
(
600 nM) and purified human ΔN-HtrA1 (156-480, 30 nM) for
indicated times at 37℃. Full-length Tau protein was visualized by
silver staining. B) The percent degradation was determined by
dividing band intensity of the full-length Tau at each time point
by the band intensity at the start of the experiment (i.e. t=0) and
plotted against incubation time.
subcloned into pET21a vector with a C-terminal His -tag
for bacterial expression. HtrA1
6
S328A
mutant was generated
by site-directed mutagenesis using QuikChange II kit
Agilent Technologies).
Taken together, ApoE and Tau are likely competitive
substrates of HtrA1 enzyme, and the preference of ApoE4
over ApoE3 resulted in delayed degradation of Tau
aggregates, which provides a new possible mechanism
leading to Alzheimer’s disease.
(
Does-dependent ApoE degradation by pure enzymes.
Recombinant ApoE3 and ApoE4 were incubated for 2
hours at 37°C with purified human ΔN-HtrA1 (aa 156-480,
wild type or S328A mutant) or human ΔN-HtrA2 (aa 134-
458) in 50 mM Tris pH 8.0 with total volume of 15 µL.
After reactions were completed, 5 µL of 4×SDS loading
dye were added. Samples were boiled at 95°C for 10 min
CONCLUSION
As the most significant genetic risk factor for late-onset
Alzheimer’s disease, understanding the biochemical,
cellular, and physiological function and regulation of
ApoE4 is of paramount importance. Here, we tested the
possibility that the serine protease HtrA1, which had
previously been linked to APP and Tau, is able to
proteolytically degrade ApoE4. Our biochemical results
indicate that HtrA1 is a candidate ApoE4 regulating
enzyme, but additional work in vivo will be necessary to
test whether this connection takes place in tissues. A
proteomics study using the Tg-SwID mouse model
revealed that HTRA1 is the most upregulated gene
TM
and subjected to SDS-PAGE using Bolt 4-12% Bis-Tris
Plus polyacrylamide gels (Life Technologies). ApoE full-
length protein and its proteolytic fragments were
visualized by Coomassie staining.
Time-dependent ApoE degradation by human HtrA1.
Recombinant ApoE3 and ApoE4 (5 µg) were incubated
with purified human ΔN-HtrA1 (aa 156-480) at 37°C in 50
mM Tris pH 8.0 with total volume of 15 µL. 5 µL 4×SDS
loading dye were added at the time points indicated in
Figure 2A and 2B. Samples were boiled at 95°C for 10 min
3
8
between 3 to 9 months of age , and this presents a
unique model where we can test the role of HtrA1 on
ApoEs in vivo.
TM
and subjected to SDS-PAGE using Bolt 4-12% Bis-Tris
Plus polyacrylamide gels (Life Technologies). ApoE full-
length protein and its proteolytic fragments were
visualized by Coomassie staining.
Furthermore, we observed that ApoE4 and Tau are
competitive substrates for HtrA1. To our knowledge, the
concept of ApoE4 inhibition of Tau or Aβ degradation has
not been explored. Although Tau is predominantly an
intracellular protein, emerging evidence suggests that Tau
ApoE degradation with HEK293T lysate and
conditioned media. HEK293T cells (3×10 ) were seeded
on a 10-cm plate. 24 hours after seeding, cells were
6
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transfected in Opti-MEM (Gibco) with 12 µg of plasmid and Dorothy Dorsett Brown Foundation and Helen
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DNA using Lipofectamine 2000 (Life Technologies)
according to manufacturer’s protocol. 6 hours post-
transfection, media were changed to 10 mL DMEM
without phenol red. 72 hours post-transfection, cell
culture media were collected and centrifuged at 1,000 rpm
for 5 minutes to remove residual cells. The supernatant
was then concentrated to 100 µL using a 10 kDa MWCO
filter (Millipore). Meanwhile, cells were harvested and
lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5%
NP-40, 0.5% sodium deoxycholate. Expression of
corresponding proteins was tested by western blotting of
McLoraine Chair in Molecular Neurobiology (K.-F.L.), the
NCI Cancer Center Support Grant P30 (CA014195 MASS
core, A.S.), the Leona M. and Harry B. Helmsley
Charitable Trust grant (#2012-PG-MED002, A.S.), and Dr.
Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.).
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Supporting Information
Supplementary methods and figures are described. This
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asaghatelian@salk.edu (A.S.)
Notes
The authors declare no competing financial interest.
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is a generous gift from Dr. Natalie Luhtala and Professor
Tony Hunter at the Salk Institute. We also thank
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postdoctoral fellow funded by the George E. Hewitt
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