Download Nuclear Translocation and Degradation of Target Proteins Using

Nuclear Translocation and Degradation of Target
Proteins Using Engineered Intracellular Antibodies
Shiyao Wang, Yong Ku Cho
Chemical and Biomolecular Engineering Department
University of Connecticut
Storrs, CT
[email protected]
Abstract: Manipulation of genetic information through
modifying DNA and RNA has become a widely used tool for
basic scientific studies and a promising therapeutic means for
treating genetic disorders. However, these approaches are
blind to downstream events that cause proteopathy, such as
protein
misfolding
or
improper
post-translational
modifications (PTMs) that consequently impair cellular
function. One viable approach to address proteopathies may be
to direct these species for degradation, using endogenous
cellular machineries. In mammalian cells, proteins with
biosynthetic errors are mainly degraded using the ubiquitinproteasome system (UPS). In this study, we have applied
intracellular antibodies as targeting domains to direct binding
partners for proteasomal degradation. We discovered that
when intracellular antibodies are fused with the E3 ubiquitin
ligase adapter domain of Speckle-type POZ protein (SPOP),
the target protein is translocated to the nucleus, in which
proteasomal degradation occurs. We found that the nuclear
translocation activity is mediated by a nuclear localization
signal (NLS) within the SPOP. The modularity of this design
was tested by replacing the targeting domain with intracellular
antibodies to different target proteins. We also demonstrate the
capability to control the efficiency of target protein
translocation and degradation by modifying the NLS and
changing the epitope of the intracellular antibody. This
approach may provide a tunable mechanism for modulating
target protein levels in cells, and enable the selective removal of
misfolded or post-translationally modified target proteins.
I. INTRODUCTION
Protein degradation via the ubiquitin proteasome system
(UPS) is a major pathway in mammalian cells to remove
proteins that have biosynthetic errors including mutaions,
improper post-translational modifications, or misfolding [1].
Therefore, it is plausible that directing improperly folded or
modified proteins to the UPS for degradation may be a viable
approach for removing these unwanted species. Previous
studies have shown that E3 ubiquitin ligase adapter domains
can be fused with a targeting domain in a modular fashion to
mediate ubiquitination of the target [2-5], leading to its
degradation. The E3 ligase adapter domain of Speckle-type
POZ protein (SPOP), which localizes at nuclear speckles, is
one such example that was effective in degrading target
proteins [2]. However, it was previously thought that SPOP
was only effective in degrading nuclear proteins. Here we
report that intracellular antibody fused with SPOP enables
nuclear translocation of the target protein and results in its
degradation. We found that a nuclear localization signal
Funding for this work was provided by the Brain & Behavior Research
Foundation.
(NLS) in SPOP leads to effective nuclear translocation of
normally cytosolic proteins, leading to their degradation
within the nucleus. The degradation of the target protein
leaves speckled localization pattern of the target, when
visualized using fluorescent proteins as a tag. Changing the
epitope of the targeting intracellular antibodies led to the
formation of higher order assembly of nuclear speckles,
suggesting increased accumulation of undegraded target.
Preliminary results show that changing the efficiency of the
SPOP NLS by replacing it with NLS from other proteins
leads to significant decrease in the level of target
accumulation in speckles while mediating target degradation.
We also show that this modular design enables nuclear
translocation and degradation of other target proteins.
Preliminary results show that our engineered intracellular
antibody-SPOP fusion constructs enable efficient nuclear
translocation and potentially degradation of human
microtubule associated protein tau, which plays an essential
role in stabilizing microtubules in neurons [6]. This approach
may enable PTM-specific segregation and degradation of tau,
potentially leading to elucidation of its role in progression of
neurodegenerative disorders such as Alzheimer’s disease
(AD).
II. RESULTS
A. Nuclear translocation and degradation of target proteins
using antibody-SPOP fusion constructs
In order to assess the ability of intracellular antibodySPOP fusion constructs to degrade target proteins, we fused
a camel single domain antibody (nanobody) (cAbGFP4) [7]
that binds enhanced green fluorescent protein (EGFP) to the
E3 ligase adapter domain of SPOP. When this construct was
transfected in a human embryonic kidney (HEK) 293T cell
line expressing EGFP, we observed nearly complete
elimination of EGFP from the cytosol, with nuclear specklelike localization patterns. When these cells were treated with
MG-132, a specific inhibitor of proteasome, EGFP was
detected in the entire nucleus, instead restricted in speckles,
suggesting that the nuclear speckle pattern of EGFP is due to
proteasomal degradation after being translocated to the
nucleus. Considering the fact that EGFP can passively
diffuse into the nucleus, we hypothesized that deleting the
NLS of SPOP will result in loss of EGFP fluorescence only
in the nucleus, leaving cytosolic EGFP intact. Indeed, when
we expressed cAbGFP-SPOP without the NLS (cAbGFPSPOPΔNLS), EGFP fluorescence was exclusively detected
in the cytosol. When these cells were treated with MG-132,
the EGFP was detected in the entire cell. These results
indicate that SPOP mediates nuclear translocation of the
target, and subsequent ubiquitination, leading to proteasomal
degradation.
In order to assess the modularity of targeting, we
replaced the antibody with a nanobody specific to mCherry
(LaM4) [7], creating LaM4-SPOP and co-expressed it with
mCherry in HEK 293 FT cells. In these cells, we observed
similar nuclear speckle pattern of mCherry, similar to that
found in EGFP cell lines expressing cAbGFP4-SPOP. We
also co-expressed cAbGFP4-SPOP with EGFP fused with
human microtubule associated protein tau (EGFP-tau).
Without cAbGFP4-SPOP, EGFP-tau is primarily cytosolic,
but upon co-expression with cAbGFP4-SPOP, it is nearly
exclusively found in nuclear speckles. We have also tested a
single chain antibody fragment specific to phosphorylated
tau at T231 (pT231 scFv [8]) fused with SPOP. Preliminary
results
show
that
pT231
scFv-SPOP
mediates
phosphorylation-selective nuclear translocation of EGFP-tau,
and potentially degradation. These results demonstrate that
the intracellular antibody-SPOP construct is a modular
design for targeting of cellular proteins for nuclear
translocation and degradation.
B. Target epitope selection affects the degree of nuclear
speckle assembly
Since substrate ubiquitination mediated by SPOP is
known to be dependent on nearby residues, we hypothesized
that using targeting domains (nanobodies) with different
epitopes may affect the degradation efficiency of the target.
In order to test this, we replaced cAbGFP4 with EGFP
targeting nanobodies that bind three distinct epitopes on
EGFP (LaG2, LaG16, and LaG41) [7]. When these
constructs were transfected in the HEK 293T EGFP cell
line, we observed a striking change in the pattern of nuclear
EGFP accumulation, in some cases leading to higher order
linear structures in the nucleus. Such higher order assembly
was also observed in HEK 293FT cells co-expressing
EGFP-Tau and LaG2-SPOP. These results suggest that the
epitope in the target protein affects its degradation
efficiency, and the need for target epitope optimization.
C. Changing the NLS allows reduction of target protein
accumulation in nuclear speckles
Based on the observation that the degradation mediated
by intracellular antibody-SPOP constructs requires nuclear
translocation, we hypothesized that modulating the
efficiency of nuclear transport may impact the steady state
level of target protein in the nucleus. To test this hypothesis,
we replaced the original NLS of SPOP with four previously
characterized NLS variants that showed difference in
nuclear transport efficiencies [9]. When these NLS variants
were co-expressed in the HEK 293T EGFP cell lines, one
construct (cAbGFP4-SPOPTus) resulted in significantly less
accumulation of EGFP in nuclear speckles, compared to that
found from other constructs. When this variant was coexpressed with EGFP-tau, it nearly completely eliminated
nuclear speckle localization of the target. However, in these
cells EGFP-tau was observed at higher levels in the cytosol,
compared to that in cells co-expressing cAbGFP4-SPOP
with more efficient NLS. These results suggest that the
nuclear translocation efficiency may be tuned to result in
target degradation while avoiding large degree of
accumulation in the nucleus.
III. CONCLUSIONS
We report the development of an intracellular antibodyE3 ligase adaptor domain fusion construct that enables
nuclear translocation and degradation of target proteins. We
find that the design provides a modular means of targeting
proteins for degradation. We found that the binding epitope
of the targeting domain impacts the degree of target protein
accumulation in the nucleus, potentially by affecting
ubiquitination efficiency. By tuning the efficiency of nuclear
transport, the degree of nuclear accumulation of target
proteins could be greatly reduced.
ACKNOWLEDGMENT
Funding for this work was provided by the Brain and
Behavior Research Foundation (NARSAD Young
Investigator Grant).
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