TNIP1 inhibits selective autophagy via bipartite interaction with
LC3/GABARAP and TAX1BP1
François Le Guerroué
1
,
Eric N Bunker
1
,
William M Rosencrans
1,2
,
Jack T Nguyen
2
,
Mohammed A Basar
3
,
Achim Werner
3
,
Tsui-Fen Chou
2
,
Chunxin Wang
1
,
Richard J Youle
1,*
1
Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National
Institutes of Health, Bethesda, MD, 20892, USA
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA
3
Stem Cell Biochemistry Unit, National Institute of Dental and Craniofacial Research, National
Institutes of Health, Bethesda, MD, 20892, USA
Summary
Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-
inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy
induction is well known, the regulation of the components is less clear. Here, we demonstrate that
TNIP1 knock out in HeLa cells accelerates mitophagy rates, and that ectopic TNIP1 negatively
regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved
LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP
family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that
phosphorylation appears to regulate its association with the ULK1 complex member FIP200,
allowing TNIP1 to compete with autophagy receptors, providing a molecular rationale for its
inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative
regulator of mitophagy that acts at the early steps of autophagosome biogenesis.
eTOC :
Le Guerroué
et al
show that TNIP1 acts as an inhibitor of mitophagy by binding with TAX1BP1
and FIP200 in a competitive manner. While binding to TAX1BP1 is essential for its inhibitory
*
Correspondence to youler@ninds.nih.gov.
Author’s contribution
ENB analyzed live cell imaging data, ubiquitin foci and quantified immunofluorescence data, WMR and JTN performed and analyzed
fluorescence polarization and dynamic light scattering experiments, FLG conceived the study, performed all other experiments,
analyzed and interpreted all other data. MAB produced and purified TNIP1 recombinant protein. AW performed mass spectrometry
and analyzed MS data. CW provided reagents and cell lines. FLG and RJY wrote the manuscript with input from all authors. RJY, AW
and TFC supervised the project and acquired funds.
Declaration of Interest
The authors declare no financial conflicts and assure that this manuscript is original and has not been published nor is currently under
consideration for publication elsewhere.
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Author manuscript
Mol Cell
. Author manuscript; available in PMC 2024 March 16.
Published in final edited form as:
Mol Cell
. 2023 March 16; 83(6): 927–941.e8. doi:10.1016/j.molcel.2023.02.023.
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role, they further demonstrate that phosphorylation regulates binding to FIP200, providing a
molecular rationale for TNIP1’s function.
Graphical Abstract
Intro
Macroautophagy, one of the main cellular degradation pathways, sequesters cytosolic
components inside a double membrane structure called an autophagosome, before
catabolizing its content by fusing to lysosomes. The molecular mechanisms of the formation
and maturation processes are well described and revolve around a set of proteins called
the ATG conjugation system
1
. Initially described as a non-selective degradation process
triggered when cells face starvation, more recent work shows autophagy can selectively
eliminate certain proteins, protein aggregates and organelles
2
,
3
. Selective autophagy
specifically sequesters cytosolic structures via autophagy receptors. The main feature of
these autophagy receptors is their capacity to bind to mATG8 (mammalian ATG8 proteins)
as well as FIP200 and to ubiquitin in most cases
4
. One of the best characterized selective
autophagy processes is the degradation of mitochondria via mitophagy
5
, where autophagy
receptors such as NDP52 or OPTN are recruited to mitochondria ubiquitinated by parkin via
their ubiquitin-binding domains, allowing the autophagy machinery to assemble and degrade
the mitochondria in a wholesale fashion
6
,
7
.
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TNIP1 (TNFAIP3-interacting protein 1), also called ABIN1 (A20-binding inhibitor of
NF-
κ
B activation 1) participates in the NF-
κ
B pathway, where it negatively regulates
NF-
κ
B activation, maintaining immune homeostasis
8
,
9
. Structurally, TNIP1 possesses a
Ub-binding domain (UBAN domain – Ubiquitin binding of ABIN and NEMO) common
among ABIN proteins, 3 Abin homology domains (AHD) and a NEMO binding domain
(NBD)
8
,
10
. Although the AHD3 domain function is currently not described, AHD1 mediates
binding with the ubiquitin editing enzyme Tumor necrosis factor alpha-induced protein
3 (TNFAIP3, A20) and AHD4 mediates the interaction with OPTN
11
. Being involved
in immune responses, dysregulation of TNIP1 was shown to be implicated in various
human diseases through a number of genome-wide associated studies (GWAS)
12
. TNIP1
single nucleotide polymorphisms (SNP) have been strongly associated with autoimmune
diseases
13
and cross ethnic genetic studies identified the
GPX3-TNIP1
locus to associate
with amyotrophic lateral sclerosis (ALS)
14
. However, a later study concluded that this locus
was less likely to contribute to ALS risk
15
. In addition, a recent GWAS study identified
TNIP1 as the locus of a newly identified risk allele for Alzheimer’s disease (AD)
16
.
Here we describe a role of TNIP1 as a LC3/GABARAP-interacting protein, that triggers
mitophagy when ectopically targeted to mitochondria, and that endogenously negatively
inhibits early stages of mitophagy. Furthermore, we show that physical interactions of
TNIP1 with LC3/GABARAP proteins and TAX1BP1 drive mitophagy inhibition. This
inhibition appears to be regulated by phosphorylation of TNIP1, promoting binding with
FIP200 for a counteracting effect on inhibition. Our study suggests that TNIP1 thus acts as a
security check to fine tune the rate of mitophagy.
Results
Ectopic localization of TNIP1 to the mitochondria induces mitophagy.
TNIP1 possesses a UBAN domain similar to that in OPTN
17
,
18
and was suggested to be an
autophagy substrate
19
. Most autophagy receptors are defined by a ubiquitin-binding domain
and an LC3 interacting region (LIR). We thus investigated if TNIP1 contained an LIR motif.
In silico searches of LIR
20
motif using the bioinformatic tool iLIR
21
website revealed two
potential canonical LIR domains in TNIP1 (Figure 1A). A Blast alignment with 4 other
higher eukaryote TNIP1 proteins showed that LIR2 is conserved but not LIR1 (Supp Figure
1A). In order to determine whether TNIP1 LIR1 and LIR2 are functional LIR domains, we
mutated LIR1 Phe83 and Leu86 to Ala and LIR2 Phe125 and Val128 to Ala (Supp Figure
1B). We also made a LIR1 and LIR2 double mutant construct that carries all 4 mutations.
Lysates of HeLa cells transfected with TNIP1 wild type (WT), LIR1 mutant, LIR2 mutant
and LIR1+LIR2 mutant were subjected to GST pull down experiments, an assay previously
used to assess binding to mATG8 proteins (i.e. MAP1LC3A, MAP1LC3B, MAP1LC3C,
GABARAP, GABARAPL1 and GABARAPL2)
22
,
23
. As a positive control, SQSTM1/p62
was demonstrated to bind strongly to all mATG8 proteins (Supp Figure 1C). Conversely,
as a negative control we employed the TNIP family member TNIP3 where no LIR motif
was detected when using iLIR search and accordingly no LC3/GABARAP binding was
observed (Supp Figure 1D). TNIP1, on the other hand, was able to bind to all mATG8
proteins, with an apparent preference for LC3C and GABARAP, and a weaker binding to
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LC3A, LC3B and GABARAPL2 (Figure 1B). LIR1 mutations diminished LC3C binding
while LIR2 mutations diminished GABARAP binding. LC3A and LC3B binding was lost
with both constructs. However, the double LIR1+LIR2 mutations resulted in a total loss of
all 6 mATG8 binding indicating that TNIP1 is a
bona fide
mATG8 binding protein, where
LIR1 seems to preferentially bind LC3C and LIR2 to GABARAP, although both LIR seem
to be able to compensate for each other’s loss of function.
Next, we took advantage of the chemically induced dimerization (CID) assay in cells
expressing mitochondrial-targeted mKeima (mt-mKeima)
24
(Figure 1C). We fused the
protein FRB to the C-terminus tail of the outer mitochondrial protein FIS1 and TNIP1 to
the soluble protein FKBP. Adding the small molecule Rapalog induces dimerization of FRB
and FKBP, conditionally localizing TNIP1 to the mitochondria. The A/C heterodimerizer
Rapalog is an analog of the autophagy-induction drug Rapamycin, however, Rapalog’s
interaction is specific for mutated FRB and is thus unable to inhibit mTor and therefore
incapable of inducing autophagy
25
. We then gauge mitophagy using Fluorescent Activated
Cell Sorting (FACS) by measuring the ratio of mt-mKeima excited by 488 nm or 561 nm to
determine mitochondria in neutral and acidic pH environments, respectively. Because cells
are gated to the same intensity levels for the mKeima and GFP channels, we ultimately
measure cells with the same level of overexpressed protein
26
. Interestingly, localizing TNIP1
to mitochondria for 24 h induced robust mitophagy, and the UBAN loss of function mutation
(D472N)
27
was able to trigger mitophagy to levels similar to WT TNIP1, indicating that
the ubiquitin binding domain of TNIP1 is not necessary for inducing mitophagy when
TNIP1 is artificially targeted to mitochondria (Figure 1D). We next investigated whether
the LIR motifs have any role in TNIP1 to triggered mitophagy. While LIR1 mutations
had a relatively minor effect and retained an ability to substantially trigger mitophagy,
CID with LIR2 mutant displayed an approximately 55% defect compared to WT TNIP1,
indicating the importance of the LIR2 domain in TNIP1 mitophagy activity (Figure 1E).
No additional effect was observed when using the LIR1+LIR2 double mutant construct.
Immunocytochemistry recapitulated the mitophagy FACS data, with LIR1 showing no
defect in recruiting LC3B, while LIR2 mutant construct showed a significant defect but
still retained the ability to recruit LC3B to the mitochondria. LIR1+LIR2 double mutant,
however, showed no additional defect in LC3B recruitment (Supp Figure 1E). On the other
hand, GABARAP recruitment showed a different pattern, with LIR1 and LIR2 constructs
showing no defect in recruitment, while the double LIR1+LIR2 construct displayed a
small but significant decrease in recruitment (Supp Figure 1F). Based on these results we
hypothesize that TNIP1’s function on mitochondria does not depend only on binding with
mATG8 proteins, but probably also interacts with other autophagy machinery proteins as the
LIR1 and LIR2 double mutant construct is still able to trigger mitophagy. An interesting
observation is the clumping of the mitochondria upon Rapalog treatment as is typically
observed upon mitochondrial depolarization in Parkin-expressing cells, due to the clustering
of p62/SQSTM1
28
. A similar mechanism might occur when ectopically expressing TNIP1 to
the mitochondria.
Mammalian cells possess 3 TNIP homologs, TNIP1, TNIP2 and TNIP3, sharing certain
ABIN homology domains (AHDs) that characterize them (Figure 1F). CID and FACS
analysis with TNIP2 and TNIP3 revealed that they were not able to induce mitophagy as
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substantially as TNIP1, indicating the singular capacity of TNIP1 in mitophagy. To further
characterize the important domains of TNIP1 other than LIR2, we carried out CID with
versions of TNIP1 missing individual AHD domains (Figure 2A and Supp Figure 2A).
Looking at their localization, all constructs displayed an even, cytosolic distribution in
puncta similar to the full-length construct (Supp Figure 2B). AHD1 deletion had no effect
on mitophagy, suggesting that binding to A20 is not necessary for its capacity in mitophagy.
TNIP1 with the AHD3 deletion showed a strong reduction in its ability to induce mitophagy,
while deletion of AHD4 domain in TNIP1 showed moderate inhibitory effect on mitophagy
(Figure 2B). Since we discovered that the LIR2 mutant and AHD3 deletion mutant TNIP1
displayed the strongest deficiency in mitophagy, we combined both and made an AHD3
deletion and LIR2 mutant construct and observed that this construct completely abolished
TNIP1’s ability to induce mitophagy (Figure 2C). We conclude that the LIR2 motif and
AHD3 domain of TNIP1 are both involved and possibly have distinct roles in mitophagy.
Both N-terminal and C-terminal fragments of TNIP1 (Figure 2D) proved incapable of
inducing mitophagy, indicating that more than just LIR and AHD3 domains are required for
mitophagy, and likely a fully folded protein is necessary.
To determine the molecular mechanisms of TNIP1 in mitophagy, we performed CID-
induced mitophagy in different cellular backgrounds: FIP200 KO, WIPI2 KO, A20 KO and
pentaKO
24
cells (5KO: NDP52, OPTN, TAX1BP1, SQSTM1 and NBR1). 5KO cells were
previously described to be deficient in mitophagy, owing to the dependence on recruitment
of autophagy receptors to recruit the autophagy machinery. To our surprise, TNIP1 was
able to mediate mitophagy in 5KO cells at a level similar to that in WT cells (Figure 2E
and Supp Figure 2C). In FIP200 KO and WIPI2 KO cells, no mitophagy was observed
(Figure 2E and Supp Figure 2C), accordingly with their essential roles as general autophagy
machineries. Because of the tight role TNIP1 plays with A20 in the NF-
κ
B pathway, we
explored mitophagy activation in an A20 KO background (Figure 2E and Supp Figure 2C).
Consistent with the results seen with the AHD1 deletion mutant, A20 KO did not alter the
mitophagy response, and revealing a new activity of TNIP1 distinct from prior work on its
role in inflammation.
To clarify the role of TNIP1 requirement in mitophagy, we conducted different CID
experiments in TNIP1 KO cells. Unfortunately, due to the size of FIP200, we could not
produce a functioning FKBP-GFP-FIP200 construct. Consequently, we assessed whether
other autophagy machinery proteins (i.e., WIPI2 and ATG16L1), as well as A20 were
able to trigger mitophagy when placed on mitochondria in WT or TNIP1 KO cells. As
previously reported, CID with ATG16L1 showed a strong mitophagy response in control
cells
7
. TNIP1 KO did not impair that response, and even slightly enhanced it, although not
significantly (Figure 2F and Supp Figure 2D). Similarly, CID with WIPI2 displayed a robust
mitophagy response and this response was significantly stronger in TNIP1 KO cells (Figure
2F and Supp Figure 2D). Finally, CID with A20 displayed a weak mitophagy response
that was abolished in TNIP1 KO cells (Figure 2F and Supp Figure 2D). This could be
explained by a weak recruitment of TNIP1 mediated by A20 to the mitochondria, and the
subsequent recruitment of the autophagy machinery. In light of these results, we conclude
that TNIP1-dependent mitophagy induction does not depend on autophagy receptors, but
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relies on early autophagy machinery. However, TNIP1 is dispensable for these autophagy
machinery proteins to induce mitophagy.
TNIP1 is a negative regulator of mitophagy
The CID results showed that TNIP1 has the potential to trigger mitophagy and allowed
us to identify the domains of TNIP1 required for this function. Since TNIP1 possesses
a ubiquitin-binding domain as well as a LIR motif, we speculated that it could act as
an autophagy receptor. To test this hypothesis, we compared TNIP1 to known autophagy
receptors in mitophagy flux assays. When subjected to mitophagy induced by mitochondrial
depolarization, receptors are degraded in an autophagy-specific manner, together with their
cargo. Blocking autophagosomal degradation with the V-ATPase inhibitor Bafilomycin
A1 (BafA1) is a common way to estimate substrates specifically degraded in lysosomes.
Combining the mitochondrial ATP synthase and complex III inhibitors Oligomycin A and
AntimycinA1 (OA), respectively, is an established treatment for triggering mitophagy.
Autophagy receptors are selectively degraded during mitophagy, as can be seen with
TAX1BP1, NDP52 and OPTN (Supp Figure 3A). Double treatment of cells with BafA1 and
OA promotes mitophagy but prevents lysosomal degradation of the encapsulated proteins
(Supp Figure 3A). However, similar to the ULK1 complex protein FIP200, TNIP1 levels
were not influenced by Bafilomycin or OA treatment. Interestingly, TNIP1 was recently
found to be an autophagy substrate in an autophagosome profiling content screening
19
,
and thus would appear to be an autophagy substrate. Consequently, its apparent lack of
degradation upon mitophagy or absence of accumulation upon Bafilomycin A1 treatment
may be due to a fast turnover rate. Therefore, we performed OA treatments combined with
cycloheximide (CHX) to investigate TNIP1 degradation (Supp Figure 3B). Remarkably,
TNIP1 seems to be a long-lived protein, as no degradation of TNIP1 was seen after 4 hours
of CHX treatment. Interestingly, under steady state conditions, FIP200 is degraded rapidly,
but counterintuitively, is slightly stabilized during mitophagy induction. Consequently,
TNIP1 is very likely not degraded en masse via mitophagy, as is also the case for
FIP200. Immunocytochemistry allowed observation of TNIP1 localization under steady state
condition as well as upon mitophagy. To trigger mitophagy, HeLa cells stably expressing
HA-Parkin were treated for 4 h with OA (Supp Figure 3C). Under steady state conditions,
endogenous TNIP1 appeared as puncta, in the vicinity of the mitochondria, with some
colocalization events at the periphery of mitochondria, in a manner reminiscent of autophagy
receptors. However, upon OA treatment, TNIP1 was found to coalesce in the perinuclear
region and not to substantially relocate to mitochondria, as can be seen with FIP200,
contrary to what is seen with the autophagy receptor TAX1BP1, prompting us to reconsider
the role of TNIP1 as a mitophagy receptor.
Therefore, we asked if TNIP1 KO cells and such cells rescued with FKBP-GFP-TNIP1
(Figure 3A) show any alteration in PINK1/Parkin mediated mitophagy. HeLa cells
expressing mt-mKeima and HA-Parkin were treated for 6 h with OA and analyzed by
FACS. This treatment induced an almost complete mitophagy response in WT cells and
the same response in TNIP1 KO cells (Figure 3B). In contrast, re-expression of TNIP1
in TNIP1 KO cells showed a substantial defect in mitophagy. These results indicate that
TNIP1 may be a negative regulator of mitophagy and prompted us to investigate earlier time
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points when in mitophagy was submaximal in WT cells. Indeed, at 2 h of OA treatment, we
saw a stronger mitophagy induction in TNIP1 KO cells, a response that could be reversed
by re-expression of TNIP1 (Figure 3C). To further validate TNIP1’s role in inhibiting
mitophagy, we overexpressed TNIP1 in HeLa cells instead of rescuing in TNIP1 KO cells
and monitored mitophagy using mt-mKeima and FACS. Similarly, overexpression of TNIP1
in WT HeLa cells induced a robust inhibition of mitophagy (Figure 3D). Over-expression
of AHD domain deletion mutants in WT cells was then used to compare to the full length
TNIP1 and assess the deletion mutant’s abilities to inhibit mitophagy. While the ΔAHD1
construct showed a mitophagy response similar to WT TNIP1, in accordance with a lack of
function in previous CID experiments, ΔAHD3 was less inhibitory, while ΔAHD4 showed
an intermediate response compared to ΔAHD3 and WT TNIP1. We also carried out rescue
experiments in TNIP1 KO cells with the LIR mutant construct in PINK1-Parkin dependent
mitophagy. Similar to the observations with the AHD3 deletion construct, the inhibition of
mitophagy was drastically reduced with the LIR mutant construct (Figure 3E). Therefore,
we conclude that the LIR motif and the AHD3 domain of TNIP1 are both contributing to
its role in inhibiting mitophagy. These results are consistent with the results obtained with
CID, where we identified the AHD3 domain and LIR motif as being important for TNIP1
function.
We also performed the same over-expression experiments with TNIP2 and TNIP3 (Supp
Figure 3D). While TNIP2 overexpression had no effect, TNIP3 overexpression showed a
mild inhibition of mitophagy, but much less than seen with TNIP1 over-expression. This
may be explained by the presence of an AHD3 domain in TNIP3 but not in TNIP2.
We next performed a complementary mitophagy assay by monitoring the degradation of
the mitochondrial matrix protein MTCO2 (COXII) and outer membrane protein MFN2.
In WT cells expressing BFP-Parkin, the outer mitochondrial protein MFN2 is rapidly
degraded via the proteasome, while the mitochondrial electron transport chain COXII
is degraded at later time points in the lysosome. A robust degradation of COXII was
observed after 15 h, progressing further after 24Hrs of mitochondrial depolarization
(Figure 3F). When GFP-TNIP1 is overexpressed, the steady state level of COXII is higher
than in non-overexpressing cells. 15 h after mitophagy induction, COXII degradation is
impaired in GFP-TNIP1 overexpressing cells. 24 h after mitophagy induction, however,
COXII levels are similar in non-overexpressing and overexpressing cells, reflecting the
observation with the FACS data where TNIP1 reduces the rate of mitophagy but does
not abolish it. As we wondered whether TNIP1’s function was specific for mitophagy,
we investigated non-selective autophagy. Ectopic expression of TNIP1 did not influence
starvation-mediated autophagy as degradation of p62/SQSTM1 was not altered when GFP-
TNIP1 was overexpressed (Supp Figure 3E). We also used a modified version of the steady
state autophagic flux probe YFP-LC3B_RFP-LC3BΔG reporter
29
, where autophagic flux
can be estimated by measuring the YFP/RFP signal ratio. Because we use the YFP and RFP
channels for our imaging experiments, we overexpress TNIP1 with a BFP tag (Supp Figure
4A) and observed that it had no effect on the autophagic flux upon starvation (Supp Figure
4B). We next investigated whether TNIP1 was also involved in inhibition of other selective
autophagy pathways by monitoring aggregate clearance using puromycin treatment
30
. In
cells that ectopically express GFP-TNIP1, we observed a defect in aggregate clearance
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when puromycin was washed out compared to non-overexpressing cells (Supp Figure 4C).
Additionally, we explored an alternative mitophagy pathway triggered by iron depletion
31
.
We treated cells with the iron chelator Phenanthroline (Phen) for 16 h and observed the
mitophagy response by FACS in WT and TNIP1 KO cells expressing the mt-mKeima
reporter. To our surprise and contrary to results seen with mitophagy triggered by membrane
depolarization, TNIP1 KO cells displayed a small defect in mitophagy response compared to
WT cells (Supp Figure 4D). The lack of inhibition of Phenmediated mitophagy may relate
to the lack of ubiquitination in this pathway
32
, in contrast to Parkinmediated mitophagy
and aggrephagy. Together, these data indicate that TNIP1 inhibits some forms of selective
autophagy but not non-selective autophagy.
Binding of TAX1BP1 to the AHD3 domain is required for TNIP1’s inhibition of mitophagy
To obtain mechanistic insights into the function of TNIP1 as a negative regulator of selective
autophagy, we performed an unbiased mass spectrometry (MS) screen to identify specific
interactors of the AHD3 domain. We immuno-precipitated (IP) HA-TNIP1 full length,
TNIP1 ΔAHD3 and TNIP1 ΔAHD4 and determined high confidence interaction partners
(HCIPs) by MS followed by Comparative Proteomics Analysis Software Suite (compPASS)
analysis as previously described
33
,
34
(Figure 4A, Table S1). Gene Ontology (GO) analysis
revealed autophagy components as the only significantly enriched class of proteins. In
particular, RAB11FIP5, the autophagy receptors TAX1BP1, OPTN and to a smaller extent
CALCOCO2 (NDP52) as well as the ULK1 complex component RB1CC1 (FIP200) were
the most abundant HCIPs.
Consistent with previous reports
11
, we observed that the TNIP1-OPTN interaction occurs
via the AHD4 domain by IP/MS and validated this by IP/immunoblotting (Figure 4A and
4B). We also confirmed binding of TNIP1 to TAX1BP1
35
, which we found to require
the AHD3 domain. As a control, we used TNIP3, that also possesses an AHD3 domain
and observed binding to TAX1BP1 but not to OPTN. To test the possibility that TNIP1’s
function in mitophagy might depend on TAX1BP1 binding, we further characterized
the interaction between TNIP1 and TAX1BP1. We overexpressed HA-tagged TAX1BP1
full length, coiled-coil (CC), ΔSKICH or Δzinc-finger (ZF) constructs in 293T cells
stably expressing GFP-TNIP1 followed by anti-GFP IPs (Figure 4C). These experiments
revealed that TNIP1 interacts with full length TAX1BP1 and ΔSKICH TAX1BP1, but not
with TAX1BP1 CC and ΔZF constructs indicating that TNIP1 binds via the TAX1BP1
ubiquitin binding domain ZF. We performed FACS experiments to assess the involvement of
TAX1BP1 in TNIP1’s role in mitophagy inhibition (Figure 4D). No reduction in mitophagy
was observed in TAX1BP1 KO cells compared to WT cells, in accordance to previously
published data
24
. Furthermore, overexpressed TNIP1 in TAX1BP1 KO cells did not inhibit
mitophagy to levels similar to those in WT cells. Overall, TNIP1 binds the ZF domain of
TAX1BP1 via its AHD3 domain, and this binding is critical for its inhibitory action.
Binding of FIP200 to an evolutionarily conserved LIR motif is required for TNIP1’s
inhibition of mitophagy
Other HCIPs in our MS data were FIP200 and other members of the ULK1 complex
(ATG13 and ATG101). We confirmed FIP200 binding by IP/Immunoblotting with TNIP1
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but not with TNIP3 (Figure 4B). FIP200 binding to TNIP1 potentially explains how CID
with TNIP1 induces mitophagy, as it was previously demonstrated that CID with a FIP200-
binding peptide was sufficient for mitophagy induction
7
. Corroborating the MS data that
showed the ΔAHD4 mutant seemed to bind more strongly to FIP200 (Figure 4A), the
ΔAHD4 mutant displayed a much stronger IP interaction with FIP200 (Figure 4B). We next
performed IP/Immunoblotting in TAX1BP1 KO cells and confirmed that TNIP1 was also
able to pull down FIP200 (Figure 4E), implying that TNIP1 binding to FIP200 does not
depend on TAX1BP1. Surprisingly, the increase of FIP200 binding seen with constructs
ΔAHD3 and ΔAHD4 was not observed in TAX1BP1 KO cells, suggesting that TAX1BP1
may mediate this stronger binding. However, while the data in Figure Fig.B was obtained
using 293T cells, Figure 4E was obtained using HeLa cells, thus we cannot exclude that this
observation could be cell type specific.
SQSTM1/p62 was recently demonstrated to bind to FIP200 through its LIR motif
36
. We
therefore examined whether TNIP1 would utilize a similar binding mode and asked whether
the LIR2 mutant construct bound FIP200 (Figure 5A). Confirming our hypothesis, we could
not detect any FIP200 binding with the LIR2 mutant TNIP1. In addition, consistent with our
MS analyses (Figure 4A and B), we did not detect co-immunoprecipitation of TNIP1 with
WIPI2. We also did not detect binding of N-Terminal and C-terminal deletion constructs
of TNIP1 to FIP200 (Supp Figure 5A), which is in line with our CID experiments (Supp
Figure 2C). This is somewhat surprising as we expected the N-terminal construct to still
bind FIP200 as it retains the LIR motif. Truncating TNIP1 may disturb its folding and thus
its ability to bind FIP200. In order to exclude the possibility that TNIP1 could pull down
FIP200 via a secondary interaction with mATG8 binding, we performed CID experiments
coupled with immunocytochemistry in a mATG8 6KO or TAX1BP1 KO background (Figure
5B and Supp Figure 5B). In WT cells, TNIP1 was able to recruit FIP200 upon Rapalog
treatment. The LIR2 mutant TNIP1, however, was less able to recruit FIP200, confirming
that this region is necessary for FIP200 binding. Recruitment of FIP200 to mitochondria
in mATG8 6KO and TAX1BP1 KO cells was similar as in WT cells and the LIR mutant
construct significantly impaired recruitment of FIP200 to the mitochondria. These results
confirm that FIP200 binding to TNIP1 is independent of mATG8 proteins as well as
TAX1BP1. To further characterize the interaction between TNIP1 and FIP200, we explored
the binding region to TNIP1 within FIP200 (Figure 5C). While TNIP1 was able to bind full
length and the C-terminal region of FIP200, it was not able to bind the N-terminal part of
FIP200 (Figure 5C), prompting us to further map the C-terminal region for the interaction
with FIP200. NDP52 was previously shown to interact with the leucine zipper domain
of FIP200, while the CLAW domain of FIP200 was recently identified as responsible for
binding with p62/SQSTM1
36
. TNIP1 was able to pull-down only the CLAW domain of
FIP200, indicating that the CLAW domain is the minimal necessary region for binding to
TNIP1 (Figure 5C).
Phosphorylation of the FIR motif regulates TNIP1’s binding to FIP200 CLAW domain
A consensus FIP200 Interacting Region (FIR) core motif present in several autophagy
receptors was recently reported, showing that this FIR motif overlapped with their LIR
motif
37
and that phosphorylation of a residue immediately preceding the FIR core motif
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increases binding to the CLAW domain of FIP200. Careful examination of the LIR motif of
TNIP1 showed one threonine and two serine are immediately preceding the LIR motif (Supp
Figure 1A). Furthermore, these serine proximal to the LIR domain were recently found
to be phosphorylated, resulting in an increased binding to LC3s
38
. We thus hypothesized
that TNIP1 interacts with FIP200 in a phosphor-FIR-dependent manner and that allows
it to compete with autophagy receptors for interaction with FIP200. To test this, we
generated FITC-labeled peptides unmodified or with either a phosphorylated S122 (pS122)
or phosphorylated S123 (pS123) and monitored their ability to bind recombinant CLAW
domain using fluorescence polarization (FP) as previously demonstrated for the FIR of
ER-phagy receptor protein CCPG1. Phosphorylation at either site on TNIP1 displayed
a stronger binding to the CLAW domain, with a KD of 46μM for unmodified TNIP1
versus a KD of 9.7μM for the pS122 peptide and 3.27μM for the pS123 peptide (Figure
6A). Using unlabeled TNIP1 peptides, we performed a competition assay with the FITC-
labeled pCCPG1 FIR peptide for the binding to the CLAW domain. TNIP1 pS123 peptide
was able to compete with CCPG1 for the binding with the CLAW domain, with an
absolute IC50 of 70.16μM (Figure 6B). This suggests that TNIP1 acts as a competitive
inhibitor and provides an explanation of how TNIP1 inhibits mitophagy, by competing
with autophagy receptors for the binding with the CLAW domain. Of note, the mitophagy
receptor OPTN was previously measured with a KD of 306μM for unmodified and 11.5μM
when phosphorylated
37
, indicating a lower affinity for the CLAW domain than TNIP1,
suggesting that TNIP1 would outcompete OPTN for binding with the CLAW domain. To
our knowledge, TAX1BP1 binding to the CLAW domain of FIP200 has not been estimated.
To validate that full length TNIP1 binds the CLAW domain of FIP200, we purified
recombinant TNIP1 protein and performed dynamic light scattering (DLS) analysis. When
TNIP1 (red) and CLAW (blue) were measured alone, peaks around 2.5nm were observed,
with a broader distribution for purified TNIP1 alone. Mixing CLAW and TNIP1 at equal
mass-ratios lead to the observation of a second larger peak at 5.5nm (purple), indicating that
TNIP1 and CLAW bind together. Adding the pFIR of CCPG1 in excess (100μM) prevented
TNIP1 binding to the CLAW domain, reducing the formation of the 5.5nm complex,
shifting the apparent peak to 3.1nm (green) (Supp Figure 6A). We noticed the formation
of a precipitate upon mixing TNIP1 and the CLAW domain, suggesting larger particle
formation than is typically measured by DLS. We thus assessed the turbidity, allowing
us to characterize the formation of larger particles when TNIP1 and CLAW were mixed,
confirming the formation of an insoluble complex. Adding excess of pFIR CCPG1 peptide
resulted in perturbation of this complex by competition binding and partially prevented this
precipitation (Supp Figure 6B).
We next performed FACS experiments to assess the contribution of the phosphorylation
to the function of TNIP1 by using a phospho-dead mutant, where Thr121, Ser122 and
Ser123 residues were replaced by Ala (TNIP1-AAA). Ectopic expression of TNIP1-AAA
displayed an even stronger mitophagy inhibition than WT indicating that phosphorylation
counteracts TNIP1’s inhibitory effect on mitophagy (Figure 6C). This likely occurs through
FIP200, as IP/immunoblotting experiments revealed a weaker binding of TNIP1-AAA to
FIP200 compared to WT TNIP1 (Figure 6D). We additionally performed GST pull-down
of TNIP1-AAA mutant and observed that, while LC3B binding was reduced, no such loss
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of binding to GABARAP was observed compared to the LIR2 mutant (Figure 6E vs Figure
1B), indicating that TNIP1-AAA likely impairs binding to FIP200 but not to GABARAP.
In an attempt to mimic phosphorylated TNIP1, we mutated residues TSS_121–123_EDD
and performed IP/immunoblotting. Unfortunately, these mutations did not function as
phospho-mimetic and did not recapitulate the FP data and lacked stronger binding to FIP200
(Supp Figure 6C). We also tried using single mutations, but neither S122E nor S123E
displayed phospho-mimicking abilities (Supp Figure 6D). TANK-Binding Kinase 1 (TBK1)
regulates several autophagy receptors in the autophagy pathway
7
. Furthermore, TBK1 was
previously shown to be recruited to the TNIP1-TAX1BP1 complex
35
. We thus investigated
whether TBK1 was involved in phosphorylating TNIP1. Similar to results seen with the
TNIP1-AAA, cells treated with the TBK1 inhibitor displayed weaker binding to FIP200,
implying that TBK1 is likely the kinase responsible for phosphorylating TNIP1 (Figure
6F). Lastly, in order to compare the affinity of TNIP1’s FIR peptide for FIP200-CLAW
and mATG8 proteins, we performed FP using purified mATG8 proteins (Supp Figure 6E).
As seen with FIP200-CLAW, phosphorylated FIR displayed a stronger binding with all six
mATG8 proteins than unmodified TNIP1, with the strongest binding observed with LC3C
and GABARAP, as observed using GST pulldowns.
Overall, we here show that TNIP1 is a negative regulator of selective mitophagy, by
interacting with autophagy receptors and competing with them for binding with FIP200
via a FIR domain. We therefore speculate that FIR phosphorylation by TBK1 modulates
the affinity of TNIP1 by increasing binding to FIP200, displacing the complex FIP200-
autophagy receptor to a complex comprised of FIP200 and TNIP1 (Figure 6G).
Discussion
Here, we investigated the involvement of TNIP1 in autophagy and identify it as an inhibitor
of mitophagy. Our findings have important implications for our understanding of the
regulation of early events of mitophagy induction, ULK1 complex recycling at the forming
autophagosome, and the development of neurodegenerative diseases.
One of the key findings of this report is the identification of a LIR motif in TNIP1
suggesting that TNIP1 could be driven to the forming autophagosomes by binding to the
LC3/GABARAP proteins, and at the same time, allosterically binding to TAX1BP1 via the
zinc finger domain, preventing the latter from binding to ubiquitinated cargos, occasioning a
defect in mitophagy (See graphical abstract). One of the remaining unelucidated aspects of
selective autophagy is the seemingly small amount of the ULK1 complex proteins degraded
compared to autophagy receptors. A mechanism must thus take place to displace the
complex from the closing autophagosome. Based on the results reported here, we speculate
that upon mitophagy activation, releasing FIP200 from binding with autophagy receptors
via competition with phosphorylated TNIP1 removes FIP200 from the autophagosome,
ultimately allowing FIP200 to be available for further expansion of the autophagosome
(Figure 6G). The MS data with the ΔAHD3 and ΔAHD4 mutants showing a stronger
interaction with FIP200 could reflect this binding competition between TNIP1, TAX1BP1
(or OPTN) and FIP200, as it seems that losing the domains responsible for binding to the
autophagy receptors enhances FIP200 interaction. Additionally, the FIR-binding deficient
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mutant (AAA mutant) seems to bind more strongly to TAX1BP1 than the WT construct
(Supp Figure 6C), exemplifying the binding competition between FIP200 and TAX1BP1.
Although to our knowledge this is the first time that a negative function of mitophagy is
described for TNIP1, it was recently shown to be a signal-induced autophagy receptor in
the context of inflammation
39
. Additionally, phosphorylation of serine residues upstream of
the LIR motif by TBK1, resulting in an increased binding to LC3 proteins was recently
described
38
. Another study implicated TNIP1 as a modulator of mitophagy
40
, where its
loss resulted in a lower relative mitophagy. However, the apparent discrepancy between this
study and our present results can be explained by the fact that the authors looked at a late
stage of mitophagy where not much change can be observed, as we observed in Figure 4A.
To date, very few proteins have been characterized as inhibitors of autophagy. The
deubiquitinating enzyme (DUB) USP30 was shown to oppose Parkin by removing ubiquitin
moieties from mitochondria
41
. Most other proteins have been identified to negatively
regulate bulk autophagy and not selective autophagy. We also show that the role of TNIP1
in inhibition is not solely observed in mitophagy, but in aggrephagy as well, a type of
selective autophagy that was previously shown to mainly rely on TAX1BP1
30
. These
results are potentially very interesting considering the importance of aggregate clearance in
neurodegenerative diseases and the recent findings implicating mitochondrial dysfunction in
neurodegenerative disease (ND), and more particularly in Parkinson’s disease (PD) and AD.
One can hypothesize that loss of TNIP1’s function in mitophagy regulation could be a factor
leading to NDs. On the other hand, for PD, efforts are being made to activate mitophagy.
One strategy seen as amenable to pharmacologic manipulation is to inhibit the proteins that
inhibit mitophagy such as USP30. One could consider pharmacologic inhibition of TNIP1 as
an alternate approach.
Limitations of the study
Our inability to purify TNIP1 and TAX1BP1 in substantial amounts prevented us from
performing competition assays assessing the complex formed by FIP200, TAX1BP1 and
TNIP1. Structural studies investigating complex dynamic will be crucial to fully understand
TNIP1’s function. Furthermore, the Kd of the binding with all six mATG8 is smaller than
measured with FIP200-CLAW domain, implying that the binding of the phosphorylated
TNIP1 is stronger with mATG8 proteins than with FIP200, although we only used the
CLAW domain of FIP200 and not the full-length protein like we did with mATG8 proteins.
This makes interpretation of the phosphorylation of the FIR motif of TNIP1 difficult
and therefore we cannot conclude with certainty that phosphorylation of TNIP1 displaces
binding with mATG8 toward FIP200, although our pull-down experiments show that AAA
mutant loses binding to FIP200 but not to GABARAP, indicating that the phosphorylation
more likely influences FIP200 than GABARAP
in vivo
. Lastly, we unfortunately could not
test a phospho-mimicking mutant as this did not seem to bind better to FIP200 as true
TNIP1 phosphopeptides do. This is not uncommon for phosphomimicking mutants to not
always mimic phosphates, and this was already seen with previous LIR motifs
42
.
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Star Methods
Resource availability
Lead Contact—
Further information and requests for resources and reagents should be
directed to and will be fulfilled by the Lead Contact, Richard Youle (youler@ninds.nih.gov).
Materials Availability—
Plasmids and cell lines generated in this study will be available
upon request.
•
High content imaging data is deposited at BioImage Archive and is available
with the accession number
S-BIAD619. Original Western blot data, microscopy
images, raw FACS data, raw MS data, raw FP data and code used in this paper
have been deposited at Mendeley Data and are publicly available as of the date
of publication at
10.17632/jh7h5cx4yh.1
. Mass spectrometry data is deposited at
MassIVE and is available at
MSV000091090.
•
No new code has been generated for this study.
•
Any additional information required to reanalyze the data reported in this paper
is available from the lead contact upon request.
Experimental Model and Subject Details
Cell line—
HEK293T and HeLa cells were purchased from ATCC. HEK293T and HeLa
cells were cultured in Dulbecco’s modified eagle medium (DMEM) with 10% (v/v) Fetal
Bovine Serum (FBS) (Sigma), 1 mM Sodium Pyruvate, 2 mM GlutaMAX. All media and
supplements were from Thermo Fisher. All cells were tested for mycoplasma contamination
every two weeks with PlasmoTest kit (InvivoGen). Reagents used for transfections were,
X-tremeGENE 9 (Roche) for sgRNA transfection, or Polyethylenimine (PEI) (PolySciences)
for all other transfections. The full list of antibodies and reagents are found in Key
Resources Table.
Method Details
Knockout line generation using CRISPR/Cas9 gene editing—
CRISPR gRNAs
were generated to target exon 3 of TNIP1, exon 2 of TNFAIP3 and exon 3 of TAX1BP1.
gRNAs were cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988).
To make KO, HeLa cells were transfected with the gRNA plasmid and treated with 1 ug/ml
puromycin for 2 days to enrich transfected cells, which were then diluted and placed into 96-
well plates for single colonies. Primer set (ggtggaccagcatggagttt and accagggagcttccaactca)
were used for PCR screening of TNIP1 KO clones. Primer set (tcagtacccactctctgccttc and
ctccaagcctcaatgtgctct) were used for PCR screening of TNFAIP3 KO clones. Primer set
(ttatccttgagaaattggatagca and tagtacctaaaaagaaacccactcttc) were used for PCR screening of
TAX1BP1 KO clones.
Cloning, mutagenesis and stable cell line generation—
For lentiviral constructs,
inserts were either amplified by PCR and cloned into pHAGE vector, respectively by Gibson
assembly (New England Labs) or Gateway cloning (Thermo Fisher). Deletion mutants were
generated using Gibson Cloning. Point mutants were generated by site directed mutagenesis
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or Gibson cloning. All constructs used or generated in this study were validated by Sanger
sequencing and complete plasmid sequences and maps are available upon request. Stable
expression of lentiviral constructs in HeLa or HEK293T cells were achieved as follows:
lentiviruses were packaged in HEK293T cells by transfecting constructs together with
appropriate helper plasmids and PEI. The next day, media was exchanged with fresh
media. Viruses were harvested 48 hrs and 72 hrs after transfection and transduced in HeLa
or HEK293T cells with 8 μg/mL polybrene (Sigma). Cells were then directly used in
experiments or optimized for expression by FACS.
Immunoblot Analyses—
Cells seeded into 12-well plates were washed with phosphate
buffered saline (PBS) and lysed with RIPA buffer. The protein concentration was measured
using a BCA kit. Samples were boiled at 99°C for 5 min. 20–50 μg of protein lysate of
each sample was loaded and separated on 4–12% Bis-Tris gels (Thermo Fisher) according
to manufacturer’s protocol. Gels were transferred to polyvinyl difluoride membranes and
immunostained using specific antibodies. For mitophagy measurements by immunoblotting,
cells were treated with 10 μM Oligomycin (Calbiochem), 10 μM Antimycin A (Sigma)
and 10 μM QVD (ApexBio) in growth medium at different timepoints indicated in
figure legends, prior to western blot analysis. For starvation-induced autophagy, by
immunoblotting, cells were washed 3 times with PBS and incubated for the indicated time
with HBSS containing calcium and magnesium.
Recombinant protein/peptide production and protein purification—
mATG8
proteins (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2) were first
cloned into pENTR vector using GATEWAY cloning. They were cloned into pDEST60
GST vector using LR clonase. GST expression vector were expressed into DL21 (DE3)
bacteria. 50ml LB broth was inoculated with 2ml pre-culture of the GST constructs. Bacteria
were incubated for ̃1 hr at 37°C with agitation until an optical density of ̃0.6 was reach.
400μM IPTG was used to induce protein production for 4 Hrs. Bacteria were collected and
the pellet was lysed with lysis buffer (20mM TRIS-HCl pH7.5, 10mM EDTA, 5mM EGTA,
150mM NaCl, 0.5% NP40, 1% Triton X-100, Benzonase, 1mM DTT, protease inhibitor,
2mg/ml lysozyme). The lysate was shock-freezed with liquid nitrogen before thawing and
sonication. The samples were centrifuged and cleared lysate was incubated with 100μl of
slurry GST beads, followed by an overnight incubation at 4°C on a rotating shaker. Beads
were subsequently washed (20mM TRIS-HCl pH 7.5, 10mM EDTA, 5mM EGTA, 150mM
NaCl, 1mM DTT) before exchanging the buffer for the storage buffer (20mM TRIS-HCl
pH 7.5, 10mM EDTA, 5mM EGTA, 150mM NaCl, 1mM DTT, 5% glycerol, proteinase
inhibitor).
Production of recombinant His6-TEV-FIP200-CLAW domain was purified from
E. coli
BL21 (DE3) containing the plasmid was grown in TB medium containing 50 μg/L
ampicillin, which was shaken at 37 °C to an OD
600
of 0.5. The cell culture was cooled
down to 20 °C and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) and
harvested 16 h later by centrifugation. The cell pellet (
≈
6 g from 2 L) was suspended
in the 30 mL lysis buffer [100 mM Tris (pH 7.4), 500 mM KCl, 5 mM MgCl
2
, 20 mM
imidazole, 5% glycerol, 2 mM
β
-mercaptoethanol, and protease inhibitor tablet (Roche)].
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The cells (held in an ice bath) were lysed by six 30-s pulses of sonication, separated by
2-min intervals. The lysate was centrifuged at 20,000×
g
for 45 min at 4 °C, and the resulting
supernatant was loaded onto a Ni-NTA column [5-mL suspension, preequilibrated with
wash buffer (50 mL, 50 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl
2
, and 20 mM
imidazole)] and incubated at 4 °C with rotation for 30 min. The column was then flushed
with wash buffer (100 mL), and His6-tagged FIP200-CLAW was eluted by an increased
ration of imidazole elution buffer (500 mM imidazole in wash buffer) to wash buffer.
Fractions containing the CLAW proteins were combined and concentrated with an Amicon
Ultra-15 centrifugal filter unit (nominal molecular weight limit = 10 kDa). The mixture (0.5
mL of 20 mg/mL) was then fractionated with a gel filtration column (Tricorn Superdex 200;
GE Healthcare), eluted with GF buffer [20 mM HEPES (pH 7.4), 150 mM KCl, and 1 mM
MgCl
2
] at 0.5 mL/min flow rate, and fractions corresponding to an apparent molecular
weight of 15–30 kDa were collected and analyzed by 4–12% SDS/PAGE to evaluate
purity (Invitrogen). Fractions that contained FIP200 of ≥95% purity were concentrated to
4 mg/mL, exchanged into storage buffer [20 mM HEPES (pH 7.4), 150 mM KCl, 1 mM
MgCl
2
, 5% glycerol, and 1 mM TCEP], aliquoted, frozen in liquid nitrogen, and stored at
−80 °C.
Full-length TNIP1 cDNA was cloned into pFastbac-HTA vector with an N-terminal 6x His
tag. Bac-to-Bac
®
Baculovirus Expression System (Invitrogen) and pFastbac-HTA-6x His-
TNIP1 were used to generate recombinant baculoviruses. To express TNIP1, 1L of Sf9 cells
(1.5X106 cells/ml) were infected with recombinant TNIP1 baculovirus and incubated in a
28°C-orbital shaker at 100 rpm. After 72 hours of incubation, insect cells were centrifuged
at 1000xg for 20 minutes and cell pellets were flash frozen in liquid nitrogen and stored
at −80°C. For protein purification, the insect cell pellets containing 6xHis-TNIP1 were
resuspended in 25 ml Buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol,
0.1% Triton X, protease inhibitor cocktail). Cells were lysed using a microfluidizer at 15000
psi. The lysate was centrifuged at 50,000xg at 4°C for 35 minutes to clear cell debris. The
supernatant was incubated with 1 mL of Ni-NTA agarose (Qiagen) at 4°C with continuous
rotation for 2h. Ni-NTA agarose beads were washed with Buffer A containing 20 mM
imidazole and His-TNIP1 was eluted in buffer A with 250 mM imidazole. Subsequently,
PD-10 Desalting Columns containing Sephadex G-25 resin was used to remove imidazole
from the His-TNIP1 protein solution. His-TNIP1 was further purified by size exclusion
chromatography using the Superdex
™
200 Increase 10/300 GL column. Purified His-TNIP1
was aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C. All TNIP1 and CCPG1
peptides used in this study were synthesized in vitro by Genescript.
Immunoprecipitation, GST-pull down, GFP-TRAP and HA beads precipitation
—
For GFP-TRAP (Chromotek), HA-beads (Pierce) and GST precipitation experiments,
HEK293T or HeLa cells seeded in 10cm plates were co-transfected with specific constructs
to overexpress proteins of interest for 24 to 48 h, if indicated. IPs were performed following
manufacturer’s instructions.
Briefly, for HA-IP, cells were then lysed using ice cold lysis buffer (25 mM Tris-HCl pH
7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with EDTA-free
cOmplete protease inhibitor (Roche). Samples were incubated on ice with intermittent
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