doi: 10.1021/acscentsci.7b00224.
Epub 2017 Oct 25.
Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity
Affiliations
- PMID: 29202016
- PMCID: PMC5704294
- DOI: 10.1021/acscentsci.7b00224
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Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity
ACS Cent Sci.
.
Abstract
Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.
Conflict of interest statement
The authors declare no competing financial interest.
Figures
Proposed activation pathway
and domain structure of Nrf1. (A) (1)
Full length Nrf1 is glycosylated in the ER lumen (Pro-Nrf1) and subsequently
retrotranslocated to the cytosol by VCP/p97. (2) ER membrane-bound Pro-Nrf1 is de-N-glycosylated by NGLY1. (3)
The protease DDI2 cleaves Nrf1 between W103 and L104 and releases the active p95 form into the cytosol. (4) Nrf1 is immediately
degraded by the proteasome and thus maintained at low levels in the
cell. (5) In cells with insufficient proteasome capacity due to chemical
inhibition or an overload of misfolded proteins, active Nrf1 accumulates
and migrates to the nucleus, where it heterodimerizes with cofactors
(small Maf proteins), binds to chromosomal
targets, and activates the synthesis of PSMs. (B) Domain structure
of Nrf1 with ER transmembrane domain, site of DDI2 proteolysis, and possible N-glycosylation sites labeled
in red. The N-terminal domain (NTD) contains the transmembrane sequence
that anchors Nrf1 within the ER membrane and the proteolytic cleavage
site for DDI2 between W103 and L104. The transactivation
domain (TAD) comprises the two acidic domains (AD1, AD2) and the Asn/Ser/Thr-rich
region (NST) with eight predicted N-glycosylation sites (orange).
The serine-rich region (SR) was found to be multiply O-GlcNAcylated,
and its glycosylation status dictates the ubiquitination of the transcription
factor. The Nrf2-ECH homology 6-like
domain (Neh6L) is conserved in two relatives of Nrf1, Nrf2 and Nrf3. The DNA binding domain comprises the cap ’n’
collar (CNC) and the basic leucine zipper domain (bZIP), which enable
heterodimerization with Maf proteins before binding to the DNA. The
C-terminal domain (CTD) also contributes to transcription factor activity.
Nrf1
processing is altered by genetic or chemical disruption of
NGLY1 activity. (A) Schematic of the mechanism of N-glycan cleavage
by NGLY1 (dark gray). (B) WT and Ngly1–/– MEFs were treated with the proteasome inhibitor carfilzomib (100
nM) for 2 h prior to harvest, cell lysis, and subsequent immunoblotting.
Nrf1 was visualized by incubating the blot with a monoclonal antibody
raised against the region surrounding aa129, followed by a HRP-conjugated
secondary antibody. The unprocessed and glycosylated form of Nrf1
is seen as multiple bands between 100 and 120 kDa (p120) whereas de-N-glycosylated
processed Nrf1 appears at approximately 95 kDa (p95). (C) HEK293 cells
overexpressing human Nrf1 engineered with a C-terminal 3xFLAG-tag
were treated with the NGLY1 inhibitor Z-VAD-fmk (20 μM) or the
pan-caspase inhibitor Q-VD-OPh (50 nM) for 5 h prior to treatment
with carfilzomib (100 nM) for another 2 h. The cells were allowed
to recover in fresh medium for 2 h and then lysed and analyzed by
immunoblotting as above. (D) Chemical structures of carfilzomib, a
proteasome inhibitor; Z-VAD-fmk, an NGLY1 inhibitor with pan-caspase
inhibitor activity; Q-VD-OPh, a pan-caspase inhibitor that does not
inhibit NGLY1. (E) WT and Ngly1–/– MEFs were treated with the proteasome inhibitor carfilzomib (200
nM) for 12 h prior to harvest, cell lysis, denaturation, and treatment
with Endo H (15000 U) for 16 h before immunoblotting as in 2B. (F)
WT and Ngly1–/– MEFs were
treated with a premixed solution of plasmid DNA and Lipofectamine
2000 for 44 h. The medium was replaced with fresh medium containing
carfilzomib (50 nM) for an additional 4 h. The cells were washed,
harvested, and lysed before analysis by immunoblotting with anti-NGLY1
and anti-Nrf1 primary antibodies. EV: empty vector. W: wild-type NGLY1.
Mut: NGLY1 C309S.
Loss of
NGLY1 activity reduces nuclear localization of Nrf1 in
response to carfilzomib treatment. (A) Immunofluorescence microscopy
of WT and Ngly1–/– MEFs
grown on coverslips and treated with carfilzomib or vehicle for 2
h. The cells were recovered in fresh medium for 1 h prior to fixation
and imaging. Cells were incubated with a polyclonal antibody recognizing
the middle region of Nrf1 (aa 191–475) followed by an Alexa
Fluor 647 conjugated secondary antibody. Nrf1 immunoreactivity is
indicated in white, autofluorescence is shown in green, and DAPI stained
nuclei are in blue. (i, ii) Vehicle-treated WT and Ngly1–/– MEFs, respectively. (iii, iv) Carfilzomib
(100 nM)-treated WT and Ngly1–/– MEFs, respectively. (B) Quantitation of Nrf1 staining was accomplished
by calculating the overlap of the white channel (Nrf1) with the blue
channel (nucleus) and comparing it to the overall Alexa Fluor 647
signal, which was set to 100%. The difference gave the amount of Nrf1
staining outside the nucleus (green bar) and inside the nucleus (blue
bar). Quantitation was performed using 4 images (125 × 75 μm)
per condition and averaged. (C) Immunofluorescence microscopy images
of HEK293 cells overexpressing human C-terminal 3xFLAG-tagged Nrf1
that were treated with NGLY1 inhibitor Z-VAD-fmk or the caspase inhibitor
Q-VD-OPh for 5 h prior to treatment with carfilzomib. (i, ii, iii)
Cells with no treatment, Z-VAD-fmk (100 μM), or Q-VD-OPh (50
nM). (iv, v, vi) Cells treated as panels i, ii, and iii with carfilzomib
(20 nM, 2 h). The cells were recovered in fresh medium for 1 h prior
to fixation and imaging. (D) Quantitation of Nrf1 staining was performed
using 4 images (125 × 75 μm) per condition and averaged,
as described in panel B. Scale bars = 10 μm. Error bars represent
one standard deviation from the mean. *p < 0.05,
***p < 0.0005, ns = not significant.
Immunofluorescence staining
of Nrf1 in WT or Ngly1–/– MEFs with or without treatment with
carfilzomib. (A) MEFs (WT or Ngly1–/–) were treated with vehicle or carfilzomib (100 nM) and stained for
calnexin (red, ER localized), Nrf1 (white), and DAPI (blue), as described
in Figure 3. Autofluorescence
(green) is shown for full cell visualization. (B) Quantitation of
Nrf1 localization was done by calculating the overlap of the white
channel (Nrf1) with the blue (nucleus) or red (calnexin/ER) channels
and comparing it to the overall Nrf1 signal, which was set to 100%.
Quantitation was performed in 4 images (125 × 75 μm) per
condition and averaged. Scale bars = 10 μm. Error bars represent
one standard deviation from the mean with regard to ER overlap. ***p < 0.0005.
NGLY1 activity is required for Nrf1 to
initiate the proteasome
bounce-back response. (A) WT and Ngly1–/– MEFs were transiently transfected overnight with a plasmid expressing
firefly luciferase under the control of three copies of the human
antioxidant response element (ARE). The next day, the cells were treated
with carfilzomib (200 nM) for 12 h and bioluminscence was measured.
(B) HEK293 cells overexpressing human C-terminal 3xFLAG-tagged Nrf1
were transfected with the same reporter plasmid overnight and then
treated with Z-VAD-fmk (20 μM) or Q-VD-OPh (50 nM) for 5 h prior
to treatment with carfilzomib (20 nM) for 12 h. Bioluminescence was
then measured. (C) WT and Ngly1–/– MEFs were treated with carfilzomib (200 nM) for 12 h. mRNAs corresponding
to proteasome subunits PSMA7, PSMB7, and PSMC4 were quantitated by
qPCR. Statistical significance is similar for each qPCR measurement
between WT and Ngly1–/– MEFs.
Error bars represent one standard deviation. **p <
0.005, ****p < 0.00005, ns = not significant.
NGLY1 knockdown increases sensitivity of cells to carfilzomib.
K562 (A) and HeLa (B) cells transduced with sgGAL4-4, sgNrf1, or sgNGLY1
were treated with carfilzomib for 48 h, and their viability was compared
to vehicle-treated cells using the CellTiter-Glo assay. Cell survival
assays were performed with 3 and 4 replicates for K562 and HeLa cells,
respectively. Error bars represent one standard deviation from the
mean. Inset: The LD50s of carfilzomib for K562 and HeLa
cells were calculated by 4-variable nonlinear regression. Error bars
represent standard error. **p < 0.005, ***p < 0.0005, ****p < 0.00005.
A targeted screen of thiol-reactive compounds led to the discovery
of novel NGLY1 inhibitor WRR139. (A) Warhead variety represented in
the 553-compound library. (B) Schematic of the modified Cresswell
assay. K562 cells stably express a fluorescent Venus protein with
a mutated asparagine N-glycosylation site (ddVenus). Upon translation,
the protein is N-glycosylated, preventing proper folding and thus
fluorescence. The glycoslated ddVenus is shuttled through the ERAD
pathway, and upon de-N-glycosylation the mutated Asn is converted
to Asp, allowing proper folding and thus fluorescence. A proteasome
inhibitor is needed to prevent immediate degradation of the fluorescent
ddVenus. Inhibition of NGLY1 in this cellular assay decreases fluorescence
by preventing proper folding of ddVenus. (C) Structure of the hit
WRR139 as well as related compounds that did not show activity in
the assay. (D) K562 cells expressing ddVenus were incubated with carfilzomib
(1 μM) and either WRR139 or Z-VAD-fmk for 6 h. Fluorescence
was measured by flow cytometry and compared to cells treated with
only carfilzomib. Error bars represent one standard deviation from
the mean.
WRR139 inhibits NGLY1 in vitro and impairs processing
of Nrf1 in cells. (A) Recombinant NGLY1 (3.75 μg) was incubated
with WRR139 or Z-VAD-fmk for 60 min at 37 °C, at which time denatured
and S-alkylated RNase B (1.7 μg) was added. The mixture was
incubated for 60 min at 37 °C before separation by SDS–PAGE
and Coomassie staining. RNase B only = no NGLY1, no inhibitor; Rxn
= no inhibitor; all other lanes: inhibitor + Rxn. (B) HEK293 cells
overexpressing human C-terminal 3xFLAG-tagged Nrf1 were treated with
NGLY1 inhibitor Z-VAD-fmk or WRR139 for 18 h prior to treatment with
carfilzomib (100 nM, 2 h). Nrf1 was visualized as described in Figure 2B. (C) Luciferase
assay as described in Figure 5B using HEK293 cells overexpressing human C-terminal 3xFLAG-tagged
Nrf1. The cells were treated with WRR139 (5 μM) for 5 h prior
to treatment with carfilzomib (20 nM). Data for untreated cells are
recapitulated from Figure 5B for comparison. (D) Immunofluorescence microscopy images
of HEK293 cells overexpressing human C-terminal 3xFLAG-tagged Nrf1
that were treated with WRR139 (20 μM) for 5 h prior to treatment
with vehicle (i) or carfilzomib (20 nM, ii) for 2 h. The cells were
recovered in fresh medium for 1 h prior to fixation and imaging. Nrf1
immunoreactivity is indicated in white, autofluorescence is shown
in green, and DAPI stained nuclei are in blue. Scale bars = 10 μm.
(E) Quantitation of Nrf1 staining was accomplished by calculating
the overlap of the white channel (Nrf1) with the blue channel (nucleus)
and comparing it to the overall Alexa Fluor 647 signal, which was
set to 100%. The difference gave the amount of Nrf1 staining outside
the nucleus (green bar) and inside the nucleus (blue bar). Quantitation
was performed using 5 images (125 × 75 μm) per condition
and averaged. Data for untreated cells are recapitulated from Figure 3D for comparison.
Error bars represent one standard deviation from the mean. ***p < 0.0005, ****p < 0.00005, ns =
not significant.
Inhibition of NGLY1 by WRR139 potentiates cytotoxicity of carfilzomib
against MM and T-ALL cell lines in an NGLY1-dependent manner. (A,
B, C) U266, H929, and Jurkat cells, respectively, were treated with
either vehicle or WRR139 (1 μM) and carfilzomib for 24 h. Remaining
viable cells were compared to vehicle control using the CellTiter-Glo
2.0 assay, n = 3. (D, E, F) HeLa CRISPRi cells with
stably expressing sgGAL4-4, sgNrf1, and sgNGLY1, respectively, were
treated as in panels A, B, and C, n = 4. Error bars
in A–F represent one standard deviation from the mean. Inset:
The LD50 of carfilzomib with cotreatment with vehicle (black)
or WRR139 (gray). Error bars represent standard error. **p < 0.005, ***p < 0.0005, ****p < 0.00005, *****p < 0.000005, ns = not significant.
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