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Article
Non-vesicular phosphatidylinositol transfer plays
critical roles in defining organelle lipid composition
Yeun Ju Kim 1,JoshuaGPemberton 1, Andrea Eisenreichova2,AmritaMandal
1, Alena Koukalova1,
Pooja Rohilla1,MiraSohn 1,AndreiWKonradi
3,TracyTTang 3, Evzen Boura2& Tamas Balla 1✉
Abstract
Phosphatidylinositol (PI) is the precursor lipid for the minor phos-
phoinositides (PPIns), which are critical for multiple functions in all
eukaryotic cells. It is poorly understood how phosphatidylinositol,
which is synthesized in the ER, reaches those membranes where PPIns
are formed. Here, we used VT01454, a recently identified inhibitor of
class I PI transfer proteins (PITPs), to unravel their roles in lipid
metabolism, and solved the structure of inhibitor-bound PITPNA to
gain insight into the mode of inhibition. We found that class I PITPs
not only distribute PI for PPIns production in various organelles such
as the plasma membrane (PM) and late endosomes/lysosomes, but
that their inhibition also significantly reduced the levels of phospha-
tidylserine, di- and triacylglycerols, and other lipids, and caused pro-
minent increases in phosphatidic acid. While VT01454 did not inhibit
Golgi PI4P formation nor reduce resting PM PI(4,5)P
2
levels, the
recovery of the PM pool of PI(4,5)P
2
after receptor-mediated hydro-
lysis required both class I and class II PITPs. Overall, these studies
show that class I PITPs differentially regulate phosphoinositide pools
and affect the overall cellular lipid landscape.
Key words Phosphatidylinositol; Non-Vesicular Lipid Transport;
Membrane Contact Sites; Phospholipase C; Golgi Complex
Subject Categories Membranes & Trafficking; Organelles
https://doi.org/10.1038/s44318-024-00096-3
Received 11 September 2023; Revised 12 March 2024;
Accepted 21 March 2024
Introduction
Eukaryotic cells organize their metabolic processes in membrane-enclosed
organelles that all feature unique lipid compositions. While most lipids are
synthesized in the endoplasmic reticulum (ER), there is a steady-state
enrichment of specific lipids in other organelle membranes, and, therefore,
lipids must be selectively transported out from the ER to other organelles.
While lipid transport through vesicular membrane trafficking can deliver
bulk lipids between organelles, non-vesicular lipid transport has proven to
be an important means by which cells can rapidly distribute membrane
lipids (Lipp et al, 2020;Prinzetal,2020). In particular, while
phosphoinositides (PPIns), which are the phosphorylated derivatives of
phosphatidylinositol (PI), represent a quantitatively small fraction of total
membrane phospholipids, they play critical roles in controlling many
aspects of membrane dynamics and cellular signaling (Balla, 2013). Most
recently, PI 4-phosphate (PI4P) gradients, which are formed between
adjacent membrane compartments, have been identified as important
drivers for the non-vesicular transport of structural lipids, including
cholesterol (Mesmin and Antonny, 2016), phosphatidylserine (PS)
(Chung et al, 2015; Maeda et al, 2013; Moser von Filseck et al, 2015),
and perhaps other lipid classes. Whereas PI4P, which is produced by four
different PI 4-kinase (PI4K) enzymes that are selectively localized to
specific organelles (Boura & Nencka, 2015;Waugh,2019) was initially
believed only to serve as the precursor of the important plasma membrane
(PM)-enriched PPIn lipid, PI 4,5-bisphosphate [PI(4,5)P
2
], it is now
understood that PI4P also regulates vesicular trafficking at the Golgi
complex and in endosomes (Baba and Balla, 2020;D’Angelo et al, 2012;
Waugh, 2019). Consequently, all these transport processes must depend
on the production of PI in the ER and its subsequent delivery to the
organelle membranes, where the PI4K enzymes locally convert it to PI4P.
It has been widely assumed that PI-transfer proteins (PITPs)
deliver PI from the ER to the various other organelles. PITPs were first
identified in the late 1960s, when it was shown that soluble tissue
extracts contain proteins that can exchange phospholipids between
membranes of the mitochondria and microsomes in vitro (Wirtz and
Zilversmit, 1969). The preferred lipids transferred by these proteins
were PI and, to a lesser degree, phosphatidylcholine (PC), hence the
initial name of PI/PC transfer proteins, or PITPs. PITPs were soon
purified from the bovine brain (Helmkamp et al, 1974) and
subsequently cloned (Dickeson et al, 1989). PITPs were functionally
identified as soluble protein factors that were necessary to restore
GTPγS-stimulated phospholipase C (PLC)-mediated production of
inositol phosphates in permeabilized HL60 cells (Thomas et al, 1993).
Parallel studies also found PITP as one of three soluble factors required
for priming secretory vesicles for exocytosis in PC12 cells (Hay and
Martin, 1993). However, despite the extensive work done on PITP
proteins, including their functional homolog, Sec14 in yeast (Ashlin
et al, 2021; Cockcroft and Garner, 2011;Grabonetal,2019;Grabon
et al, 2015; Lev, 2010), direct proof that PITPs are indeed responsible
for delivering PI to various organellar membranes has been lacking,
partially due to functional redundancies between the various PITPs
that are found in higher organisms.
1Section on Molecular Signal Transduction, Eunice Kennedy Shriver, National Institute of Child Health and Human Development, Natio nal Institutes of Health, Bethesda, MD
20892, USA. 2Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nam. 2., 166 10 Prague 6, Czech Republic. 3Vivace Therapeutics,
San Mateo, CA 94404, USA. ✉E-mail: ballat@mail.nih.gov
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Mammalian PITPs belong to one of two classes: class I PITPs are
small proteins (~30 kDa) encoded by two separate genes, PITPNA
and PITPNB, with the latter producing two splice forms that differ
at their C-termini (Ashlin et al, 2021). Class II PITPs, such as Nir2
and Nir3 (or PITPNM1 and PITPNM2), are larger proteins that
also have a canonical PITP domain at their N-termini, which is
analogous to the class I PITP-fold and followed by several
additional domains that mediate interactions with membranes as
well as other proteins (Raghu et al, 2021). Importantly, genetic
silencing of the individual PITPs in model organisms yielded
phenotypes that could not be unequivocally traced to specific
defects in PI-transfer (Alb et al, 2003;Albetal,2002; Alb et al,
2007;Huangetal,2018;Xieetal,2018;Zhaoetal,2023;Zhaoetal,
2017). In a similar fashion, cellular studies with genetic targeting of
individual PITPs yielded very subtle phenotypes and little or no
change in PPIns levels (Alb et al, 2002;Carvouetal,2010;Kim
et al, 2022). Ideally, PITPs would need to be inactivated in a rapid
fashion to allow for assessment of their direct contributions to the
regulation of various subcellular lipid pools. The recently identified
natural compound microcolin B and its more active derivative,
VT01454, which selectively inhibits the class I PITPs (Li et al,
2022), now permits these types of studies for the first time.
In the present study, we report on the effects of acute,
pharmacological inhibition of class I PITPs on the various PPIn-rich
membrane compartments as well as on the overall cellular lipidome
using intact cells. We specifically show that acute pharmacological
inhibition of class I PITPs not only leads to profound changes to the
PI4P levels in the PM and late endosomes/lysosomes, but also results
in the accumulation of selected molecular species of phosphatidic acid
(PA) as well as causes a reduction of cellular diacylglycerol (DAG),
triacylglycerol (TAG) and PS. Contrary to expectations, inhibition of
class I PITPs failed to alter PI4P levels in the Golgi complex, or impact
the resting levels of PI(4,5)P
2
in the PM, while also only having small
effects on PI3P in early endosomes. Moreover, our pharmacological
studies show that the recovery of PI(4,5)P
2
within the PM after a
strong PLC activation required both the class I and class II PITPs.
Finally, we solved the structure of the human PITPNA bound to the
inhibitory compound, VT01454, and performed structural analysis to
better understand the molecular mechanism that governs the
interaction of the class I PITP proteins with cellular membranes.
These results show that PITPs do play important roles in the specific
maintenance of the PI4P pools in the PM and late endosomes, but our
pharmacological approach also highlights the tight coupling between
non-vesicular PI-transport processes and the integrated activity of the
lipid synthetic and distribution machineries.
Results
Pharmacological inhibition of class I PITPs selectively
affects distinct subcellular PPIns pools
Individual knockout (K/O) of either PITPNA or PITPNB in
HEK293 cells (Kim et al, 2022) failed to appreciably change the size
of the PI4P or PI(4,5)P
2
pools, as assessed by prolonged labeling
with myo-[3H]inositol (Fig. 1A). Selective PITP knockout also did
not alter the recovery rate of these PPIn lipids after PLC activation
by angiotensin II (AngII) stimulation (Kim et al, 2022). With the
availability of the PITP inhibitor, VT01454, we wanted to test how
acute inhibition of both class I PITPs affects the various subcellular
pools of PPIns. Treatment of HEK293 cells prelabeled with myo-
[3H]inositol for 24 h with VT01454 for 30 min substantially
reduced the signal for total PI4P but not in PI(4,5)P
2
(Fig. 1A).
To understand the effects of the inhibitor on the PI lipids of various
organelles, we used genetically encoded lipid binding probes
(Hammond et al, 2022) and analyzed the localization of these
sensors both by confocal microscopy and bioluminescence
resonance energy transfer (BRET)-based measurements (Fig. 1B)
(Toth et al, 2016) in HEK293-AT1 cells that stably express the rat
AT1 AngII receptors (Hunyady et al, 2002). Importantly, BRET
analysis allows for the assessment of organelle-specificchangesin
selected lipids at the cell population level. Briefly, a protein module
with specific lipid recognition (or lipid binding domain, LBD) is
fused with a Super Renilla (or other forms of) luciferase (sLuc) and
transfected together with an mVenus protein targeted to the
organelle of choice (usually from a single vector). The presence of
the lipid in the targeted membrane attracts the LBD, which brings
sLuc in close proximity to the organelle-anchored mVenus and, in
the presence of a suitable sLuc substrate, allows for energy transfer
between these labels to yield excitation of mVenus. The extent of
energy transfer depends on the level of the particular lipid in the
organelle membrane in question (Fig. 1B and see (Toth et al, 2019)
formoredetailsonBRET).
First, we tested the effects of VT01454 on the PM levels of PI.
For this, we used the bacterial BcPI-PLCH82A probe described earlier
in the BRET analysis (Pemberton et al, 2020). In that study, we
showed that the low level of PI in the PM of resting cells can be
acutely increased when the PM-resident PI4KA enzyme is inhibited
using the selective inhibitor, GSK-A1 (Bojjireddy et al, 2014).
However, after VT01454 (100 nM) pretreatment for 30 min, the rise
in PM level of PI after blocking PI4KA was almost completely
abolished. (Fig. 1C). Next, we analyzed the PI4P pools within the
PM using the PI4P sensor, (2x)P4M (Hammond et al, 2014)using
BRET. This analysis showed that the PM pool of PI4P was rapidly
reduced after the addition of VT01454 with a near-maximal effect
at a concentration of 100 nM. VT-treatment was almost as effective
in reducing PM levels of PI4P as the inhibition of PI4KA (Fig. 1D).
The basal level of PI(4,5)P
2
was only minimally affected by
treatment with VT01454 in spite of the large decrease observed in
the PM level of PI4P (Fig. 1E). These BRET results were consistent
with those using myo-[3H]inositol labeling.
Next, we investigated the effects of VT01454 on the intracellular
pools of PPIns. Earlier studies have shown that PI4P is present in
Rab7-positive endosomes (Baba et al, 2019; Hammond et al, 2014),
while PI3P is enriched in Rab5-positive early endosomes (Simonsen
et al, 1998). When the effect of VT01454 on the PI4P pool
associated with the Rab7-positive compartment was studied, the
true effect of the inhibitor was initially masked by the release of the
PI4P sensor from the PM and its redistribution to endomembrane
compartments, which resulted in an increased BRET signal [as
shown in (Baba et al, 2019) when the PI4KA was inhibited].
However, this signal did not remain elevated, but instead showed
a sharp decline below the resting BRET levels (Fig. 1F, left panel).
This decline suggested that the Rab7 compartment is losing most
of its PI4P in the presence of the PITP inhibitor. This was
further examined using a different approach. We have previously
reported that inhibition of OSBP-mediated PI4P/cholesterol
exchange by the OSBP inhibitor, OSW1, caused a rapid increase
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in the level of PI4P in the Rab7-positive compartment (Baba et al,
2019). However, when the cells were pretreated with VT01454
(100 nM) for 30 min and then treated with OSW1 (30 nM), the
increase was greatly inhibited (Fig. 1F, right panel). In contrast,
only a minor and slow effect of VT01454 treatment was observed
on the PI3P pool that is present in Rab5-positive compartments,
which was apparent when the effect of VT01454 was compared to
the change caused by inhibition of the resident PI 3-kinase, Vps34
(Fig. 1G).
These data collectively suggested that PI delivery by class I
PITPs is critical for the maintenance of the PI4P levels in the PM
and Rab7-positive endosomes, two compartments where steady-
statePIlevelsarefoundtobelow.Incontrast,PI3Plevelsinthe
Rab5-positive endosomes are more resistant to PITP inhibition,
and the resting PI(4,5)P
2
levels in the PM can be maintained even
at very low levels of PM PI4P, as already reported earlier when
PI4KA was acutely inhibited (Bojjireddy et al, 2014;Gulyasetal,
2022; Hammond et al, 2012).
GF
EDC
B
PI4P in Rab7 PI3P in Rab5
0 1000 2000 3000 4000
0.98
1.00
1.02
1.04
1.06
Time (sec)
VT 1 PM
VT 10 nM
VT 100 nM
Normalized BRET
0 1000 2000 3000 4000
1.00
1.05
1.10
1.15
OSW1 after VT 100 nM
OSW1 after DMSO
Normalized BRET
Time (sec)
VT OSW1
0 1000 2000 3000 4000
0.90
0.95
1.00
IN1
VT 10 nM
VT 100 nM
VT 1 PM
Normalized BRET
Time (sec)
VT or IN1
PM PI(4,5)P2
PM PI4PPM PI
VT 100 nM
VT 10 nM
VT 1 PM
0 1000 2000 3000 4000
0.7
0.8
0.9
1.0
Normalized BRET
Time (sec)
Carbachol
VT or CCh
Normalized BRET
0 1000 2000 3000 4000
0.6
0.7
0.8
0.9
1.0
VT 10 nM
GSK-A1
VT 100 nM
VT 1 PM
Time (sec)
VT or GSK-A1
0 1000 2000 3000 4000
0.98
1.00
1.02
1.04
VT 100 nM + GSK-A1
DMSO + GSK-A1
Time (sec)
Normalized BRET
GSK-A1
VT01454
parental PITPNA KO PITPNB KO parental PITPNA KO PITPNB KO
-+-+-+-+-+-+
PI
lyso-PI
PI4P
PI(4,5)P2
A
PI4P
PI
PI4KA LBD
sLuc
Venus
PITPDE
organelle membrane
LBD
sLuc
VT01454
GSK-A1
Yeun Ju Kim et al The EMBO Journal
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Inhibition of class I PITPs does not acutely reduce P4P
levels in the Golgi complex
IthasbeenwidelyassumedthatPITPsdeliverPItotheGolgi
complex for the maintenance of PI4P levels (Grabon et al, 2019).
However, unexpectedly, the Golgi-specific pool of PI4P was not
affected by the VT01454 compound (100 nM) during a 30-min
treatment regardless of which Golgi-restricted PI4P sensor
(FAPP1-PH, FAPP2-PH, CERT-PH, or GOLPH3) was used
(Fig. 2A–K). BRET analysis of the Golgi compartment has been
notoriously difficult, most likely because of the dynamic cycling of
the Golgi targeting sequences between the Golgi complex and the
other membrane compartments, including the ER, endosomes as
well as PM. Therefore, for these experiments, the quantification of
PI4P levels was based on kinetic recordings captured by confocal
microscopy. Some of the PI4P binding domains, including FAPP2-
PH, CERT-PH, or GOLPH3, actually showed a slightly enhanced
Golgi localization following the VT01454 treatment (Fig. 2G,I,K).
To determine whether the localization of these PI4P binding
proteins requires constant conversion of PI to PI4P, we used an
inhibitor of PI4KB, MI-14 (5 µM) (Mejdrova et al, 2015), and tested
its effect on FAPP1-PH localization. As reported previously, the
levels of PI4P at the Golgi showed oscillations after PI4KB
inhibition, which is thought to be due to the contribution of
PI4P generated by PI4K2A (Mesmin et al, 2017). Indeed, MI-14
(5 µM) caused a decrease in FAPP1-PH localization to the Golgi,
with many cells showing an oscillatory pattern (Fig. 2D). In
PI4K2A knockout cells (Baba et al, 2019), MI-14-treatment caused
a larger overall decrease in FAPP1-PH localization, but some cells
still showed oscillatory changes (Fig. 2E). To determine whether
VT01454 treatment limits the ability of the Golgi complex to
furtherincreasePI4Plevelsevenifthesteady-statelevelofPI4Pis
not reduced by the inhibitor, we subjected VT01454-treated cells to
treatment with OSW1. As described above, OSW1 inhibits the
OSBP-mediated transport of PI4P from the Golgi complex to the
ER and, hence, increases the Golgi level of PI4P in a manner that
still requires PI phosphorylation (Mesmin et al, 2017). Treatment
with OSW1 (30nM) was still able to further increase the levels of
GolgiPI4PasassessedbytheFAPP1-PHdomain,evenincells
pretreated with VT01454 [100 nM, (Fig. 2C)].
These data collectively suggested that while Golgi-associated
PI4P depends on the continued conversion of PI to PI4P by Golgi-
localized PI4Ks, inhibition of class I PITPs does not acutely
decrease Golgi PI4P levels during the period examined (up to
40 min). Since it has been previously suggested that PITPs are
required for PI4K enzymes to effectively utilize their PI substrate
(Schaaf et al, 2008), another important corollary of our findings is
that PI4K enzymes in the Golgi still can efficiently convert PI to
PI4P when the class I PITPs are inhibited.
Expression of drug-resistant PITP mutants can rescue
the lipid changes caused by VT01454
Since VT01454 has been proposed to covalently react with Cys94 in
class I PITPs (Li et al, 2022), we examined whether a mutation of the
C94 position (C94S) would yield drug-resistant PITP variants. It is
important to note that residue C94 has previously been implicated in
supporting PC binding, and both the C94A and C94T mutants were
shown to be defective in PC transfer in vitro (Carvou et al, 2010)[but
not in (Tremblay et al, 2001)]. To minimize the steric alteration, we
mutated C94 and examined the ability of the various C94 mutant
PITPNAs to bind PI and PC. For this, we used recombinant PITPNAs
mutated either in the 94 position (C94S and C94T) or in the 58
position (T58E), the latter substitution that is well known to eliminate
PI-binding, and determined their ability to extract lipids from crude
membranes prepared from cells prelabeled with [14C]-acetate. These
experiments showed that all mutants of PITPNA, including the PI
binding T58E mutant, showed impaired PC binding, with the C94T
substitution having the strongest effect, while also confirming the
inability of the T58E mutant to bind PI. Notably the C94S or C94T
mutants showed stronger PI binding compared to the wild-type
protein (Fig. EV1A,B).
Figure 1. Different cellular phosphoinositide pools are affected by inhibition of PITPs by VT01454.
(A) TLC analysis of lipid extracts from HEK293-AT1 cells labeled with myo-[3H]inositol for 24 h and treated with VT01545 (100 nM) for the last 30 min of the labeling
period. Note that knockout (K/O) of individual PITPs show no obvious effect on the labeling of any of the PI lipids, and that VT01454 strongly inhibited the labeling of PI4P
but not PI or PI(4,5)P
2
in each of the cell lines. (B) Cartoon depicting the principle of lipid detection in specific membranes using the BRET principle. Cells are transfected
with a luciferase enzyme (sLuc) conjugated to a lipid binding domain (LBD) specific to the lipid in question, together with the Venus protein targeted to a specific organelle
membrane. Resonance energy transfer occurs between the sLuc and Venus in the presence of a luciferase substrate, only when the lipid in the membrane attracts the LBD-
sLuc conjugate. This method can monitor lipid changes in specific organelle membranes using cell populations. (C) Accumulation of PI in the PM after the addition of the
PI4KA inhibitor, GSK-A1 (100 nM) (blue), and its inhibition by 30 min VT01454 (100 nM) treatment (red). BRET analysis using the bcPI-PLCH82A (Pemberton et al, 2020)
as the LBD and values normalized in each case to the respective DMSO-treated controls. Grand average ± SEM from three independent experiments performed in
triplicates. Source data are available online for this figure. (D) Dose-dependent inhibition of PI4P in the PM by VT01454 or GSK-A1 (100 nM). BRET analysis using the (2x)
P4M as the LBD to monitor PI4P. BRET values are normalized to the DMSO-treated control. Grand average ± SEM from three independent experiments, each performed in
triplicates. Source data are available online for this figure. (E) Lack of inhibitory effect of VT01454 on PM PI(4,5)P
2
levels. BRET analysis using the PLCδ1-PH domain as the
PI(4,5)P
2
LBD. BRET values were normalized to the DMSO-treated control. For comparison, we plotted the response to stimulation of cells expressing the M1 muscarinic
receptor with carbachol (gray). Grand average ± SEM from three independent experiments, each performed in triplicates. Source data are available online for this figure.
(F, Left) Dose-dependent effect of VT01454 on PI4P monitored in the Rab7 compartment. BRET analysis using the (2x)P4M PI4P sensor paired with Rab7-targeted
mVenus. BRET values were norma lized to the DMSO-treated controls. Grand average ± SEM from thre e independent experiments performed in triplicates. Source data are
available online for this figure. Note the initial increase in the BRET signal that is due to the liberation of the PI4P reporter from the PM and its association with the Rab7
compartment. However, this rise is transient, followed by a rapid decrease indicating the loss of PI4P due to the lack of PI delivery to the Rab7 compartment. (F, Right),
PI4P in the Rab7 compartment is increased after blocking the PI4P/cholesterol exchanger, OSBP, with OSW1 (30 nM). This increase is greatly reduced in cells pretreated
with VT01454 (100 nM) for 30 min. BRET analysis was as described for F, left panel. Grand average ±SEM from three independent experiments, each performed in
triplicates. Source data are available online for this figure. (G) Dose-dependent inhibition of PI3P generation in the Rab5-positive compartment by VT01454 or by the
Vps34 inhibitor, IN-1 (300 nM). BRET analysis using the (2x)FYVEHrs as the PI3P LBD, paired with the Rab5-targeted Venus. BRET values normalized to the DMSO-treated
control. Grand average ± SEM from three (VT01454) or four (IN-1) independent experiments, each performed in triplicates. Source data are available online for this figure.
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0 min 30 min
VT
Golgi FAPP1-PH intensity
(normalized to time 0)
FAPP1-PH
Time (min)
0102030
0.0
0.5
1.0
1.5
2.0
Golgi FAPP2-PH intensity
(normalized to time 0)
FAPP2-PH
VT
0 102030
0.0
0.5
1.0
1.5
2.0
Time(min)
0 min 30 min
0 102030
0.0
0.5
1.0
1.5
2.0
Time (min)
GOLPH3
Golgi GOLPH3 intensity
(normalized to time 0)
VT
OSW1 after VT
Golgi FAPP1-PH intensity
(normalized to time 0)
FAPP1-PH
0 102030
0.5
1.0
1.5
2.0
2.5
3.0
Time (min)
0102030
0.0
0.5
1.0
0 102030
0.5
1.0
Time (min) Time (min) Time (min) Time (min)
Fraction of initial
Golgi intensity (It / I0)
Parental HEK293-AT1 PI4K2A KO HEK293-AT1
MI14 MI14MI14 MI14
average individual average individual
FAPP1-PH
GOLPH3
0 min 30 min
CERT-PH
FAPP1-PH
0102030
0.0
0.5
1.0
1.5
2.0
Time (min)
Golgi CERT-PH intensity
(normalized to time 0)
VT
CERT-PH
ABC
DE
G
HI
JK
0 min 30 min
FAPP2-PH
F
Fraction of initial
Golgi intensity (It / I0)
0.0
0 102030
0.0
0.5
1.0
0 102030
0.0
0.5
1.0
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We then tested the effects of the drug-resistant (C94S) forms of
PITPs on the maintenance of the PM pool of PI4P, as this readout
showed the most prominent changes in response to acute VT01454
treatment (Fig. 3A). Overexpression of the wild-type PITPNA or
PITPNB altered the kinetics of the inhibitory effects associated with
VT01454 treatment (100 nM; Fig. 3B), which was attributed to a
reduction to the effective concentration of VT01454 by the over-
expressed proteins. Alternatively, adding back PITPNA-C94S
(Fig. 3C) or PITPNB-C94S (Fig. 3D) were both able to rescue the
VT01454-induced reduction of PI4P levels in the PM. There was no
major difference between the C94A, C94S, and C94T mutant in
their ability to reverse the effects of VT01454. In contrast, the PI-
binding mutant forms of the PITPNB-C94S, including either T58A
or T58E mutants, showed minimal or no rescue, respectively
(Fig. 3D). This agreed with earlier in vitro studies that reported a
small residual PI-transfer activity for the T58A and a complete loss
of PI-transfer activity for the T58E mutant (Tilley et al, 2004). We
also examined whether the defects in PI delivery to the Rab7-
positive compartment could similarly be rescued with the
expression of the VT01454-resistant C94S mutant PITP proteins.
Expression of either the inhibitor-resistant PITPNA-C94S or
PITPNB-C94S restored the ability of Rab7-positive endosomes to
accumulate PI4P when exposed to OSW1 (30 nM) after pretreat-
ment of VT01454 (100 nM; Fig. 3E,F). Once again, the PI-binding
mutant, T58E, was unable to rescue PITP functions in this assay.
Since all C94A, C94S, and C94T mutant PITPNAs were effective in
rescuing both the PM and Rab7-associated PI4P levels (Fig. 3C,D,F),
it was concluded that PC transfer is unlikely to be critical for
delivering PI for the PI4KA enzymes that are active in the PM and
the Rab7-positive endosomes. It must be noted, though, that none
of the C94 mutations completely eliminated the PC binding of the
PITPNA variants tested (Fig. EV1A,B).
Structural basis for the inhibition of PITPNA-mediated
lipid transfer by VT01545
To gain a detailed structural insight into the inhibition of PITPNA
by VT01454, we solved the crystal structure of the human PITPNA
bound to VT01454. Briefly, for these studies, we prepared purified
recombinant PITPNA and incubated it with VT01454 for 1 h
before starting the crystallization trials to allow this covalent
inhibitor to react with the Cys94 of PITPNA via the Thia–Michael
reaction (Berne et al, 2022). We obtained crystals that diffracted to
2.3 Å resolution and solved the structure using molecular replace-
ment with the structure of the PC-bound rat PITPNA (PDB ID:
1T27) as the search model before refining it to good geometry and
R-factors (Table 1) (see more details in the Method section).
We could trace the entire polypeptide chain except for the first
methionine and the last 17 amino acid residues at the C-terminus. The
overall structure of the human PITPNA protein conformed with the
previously described mouse, rat, and human PITP structures (Schouten
et al, 2002; Tilley et al, 2004; Yoder et al, 2001); with the overall fold
resembling a splayed β-sheet that is formed by eight β-strands and
covered by α-helices. The lipid binding site is created by the cleft between
the β-sheet and the α-helices (Fig. 4). The inhibitor is well resolved from
its electron density (Fig. 4B,C). In addition to binding covalently with the
reactive Cys94 residue, VT01454 also forms hydrogen bonds or water
bridges with other residues that contribute to its binding. These include
Gln22, which forms a hydrogen bond directly with VT01454, while
residues such as Tyr18, Glu85, Thr96, His115, Lys194, and Glu217 all
bind VT01454 through a water bridge (Fig. 4B).
In the previous study describing VT01454, the inhibitor was
docked in the lipid binding pocket in silico using existing structures as
a template, which correctly identified the interaction with Cys94 (Li
et al, 2022). This approach, however, could not predict the overall
structure of the PITP-fold when bound with the inhibitor as it
modeled the inhibitor bound to the closed conformation of the
molecule. The structure presented here, however, clearly shows its
almost perfect superpositions of the VT01454-bound structure with
the lipid cargo-free (apo) PITPNA structure (Schouten et al, 2002).
This suggests that VT01454 binding can occur while the lipid is leaving
the PITPNA in an open conformation. In this configuration, the lipid
exchange α2 helix swings out, away from the lipid binding pocket
(Fig. 4D). Notably, this inhibitor-bound conformation is virtually
identical to the published unliganded (Apo) conformation (RMSD =
0.332 Å). Nevertheless, superpositions of the inhibitor-bound struc-
ture with those of the PI- and PC-bound PITPNA also confirmed that
VT01454, indeed, occupies the space where the PI or PC molecule
would be located, which explains the strong inhibitory effect associated
with VT01454 treatment (Fig. 4E). Comparison of the inhibitor-bound
structure with those of the PI- and PC -bound PITPs showed
significantly higher RMSDs (0.616 Å and 0.546 Å for PI and PC loaded
structures, respectively) and demonstrated that the positions of helices
α1 and α7, similarly to helix α2, adopt the open conformation in the
inhibitor-bound state (Fig. 4E).
These findings were important since the only available structure of
the Apo-PITPNA showed the lipid exchange α2 helix in a position that
was proposed to directly mediate membrane association, which
actually resulted from the α2 helix engaging in hydrophobic
interactions with another PITPNA molecule, as part of an intimate
dimer (Schouten et al, 2002). The perfect superimposition of the
VT01454-bound structure with this apo-PITPNA structure suggests
that the α2 helix, indeed, assumes a position that is consistent with its
penetrating in the membrane. Earlier studies using molecular
Figure 2. PI4P levels at the Golgi complex are not reduced by VT01454.
Association of several PI4P-recognizing protein modules such as FAPP1-PH (A–E), FAPP2-PH (F,G), GOLPH3 (H,I), and CERT-PH (J,K) with the Golgi compartment was
followed after VT01454 (100 nM) treatment in live cells using confocal microscopy. For details of image quantification, see Methods. Note that none of these proteins
showed decreased localization after treatment, and in some cases, an increase rather than a decrease was observed. (C) OSW1 (30 nM)-induced increases in Golgi-
associated PI4P as a result of inhibition of PI4P transport from Golgi to ER, was still obvious even after VT01454 treatment. For comparison, the change in Golgi
localization of the FAPP1-PH was tested using an inhibitor of PI4KB (MI-14, 5 µM) both in parental (D) and in PI4K2A K/O HEK293-AT1 cells (E). Note the rapid drop in
FAPP1-PH signal in the Golgi, that was partial and showed oscillatory behavior in the parental cells (shown in D, right panels with a selected individual traces). The inhibitor
caused a more complete depletion of PI4P in the PI4K2A K/O cells, but some cells still showed oscillatory changes (E). Data Information: Grand averages ± S.E.M or range
from four (B), seven (D,E), three (G,I), or two (C,K) independent dishes (5–15 cells, each) are shown. Representative traces of individual cells are shown on the right
panels in panels (D) and (E) from one of these experiments. Scale bars 10 µm. Source data are available online for this figure.
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DE
DE
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dynamics simulations of PITPNA have also shown that in the cargo-
free conformation, the designated lipid exchange loop penetrates into
the membrane, and it was also suggested that the large movement of
this short α2 helix during cargo binding reflects the lifting of the lipid
cargo from the membrane and into the lipid binding cavity (Grabon
et al, 2017; Schouten et al, 2002). These findings prompted us to
further analyze the structural aspects that contribute to the membrane
interactions of PITPs.
Diacylglycerol promotes membrane association of the
class I PITPs
Based on structural studies, it has been suggested that the “open”,
cargo-free structure of PITPNA represents the membrane-bound state
of the protein (Grabon et al, 2017;Schoutenetal,2002). Moreover,
deletion of the C-terminal 11 amino acids in recombinant rat PITPNA
was shown to strongly increase its membrane binding affinity, while
also yielding a more relaxed conformation of the protein (Tremblay
et al, 1998; Voziyan et al, 1996). This raises the question of whether the
C-terminal truncation increases membrane binding via membrane
penetration of the α2 helix, which would favor the open conformation
of the PITP-fold. We compared the basal localizations of the full-
length as well as the C-terminally truncated forms of PITPNA
(PITPNA-Δ5), which were EGFP- or mRFP-tagged at their N-termini,
and also monitored their response to AngII (100 nM) stimulation in
HEK293-AT1 cells. Expressed wild-type PITPNA showed no
discernible membrane localization and did not respond to AngII
stimulation in any significant ways (YJK and TB, unpublished
observations). In contrast, PITPNA-Δ5, while also mostly cytoplasmic,
showed minor perinuclear membrane enrichment in resting cells.
More importantly, this mutant showed prominent translocation to the
PM after AngII stimulation (Fig. 5A,B). A similar localization pattern
was observed with the PITPNB-Δ6 mutant, except that this construct
also showed a prominent Golgi and some ER localization in the resting
state, as well as a strong PM localization after AngII addition
(Fig. 5C,D). The kinetics of this AngII-induced PM translocation were
reminiscent of the DAG accumulation in the PM that has been
documented in our previous studies (Kim et al, 2015). Co-localization
of the PITPNA-Δ5 or PITPNB-Δ6 mutant with our DAG sensor [C1
ab
domain from mouse PKD (Kim et al, 2015)] was also prominent in the
PM during AngII stimulation (Fig. EV2A,B). That the membrane
levels of DAG are the primary determinants of the translocation of
PITPs to the PM was further supported by the finding that inhibition
of the conversion of DAG to PA in the PM by a DAG-kinase inhibitor
during AngII stimulation further promoted the membrane association
of the truncated forms of PITPNA and PITPNB (Fig. 5A–D).
Accordingly, the DAG-mimic PMA also increased the PM localization
of PITPNA-Δ5 (Fig. 5F).
Structural features of class I PITPs contribute to their
membrane binding
Given the DAG-driven localization of the truncated forms of
PITPs, we aligned the sequences of PITPs with the DAG-binding
C1 domains of several PKCs. We found that a proline-threonine-
phenylalanine (PTF) sequence located in the α2 helical lipid-
exchange loop, and conserved across all PITPs, showed a good
alignment with a similar sequence found within the C1 domains of
PKCs, which is located just ten residues upstream of an essential
Figure 3. Expression of VT01454-resistant PIT P variants overcomes PI4P depletion induced by VT01454 treatment in the PM and Rab7-positive compartments.
(A) A cartoon showing the experimental setup for (B–D). BRET analysis was used to monitor PM PI4P using the (2x)P4M PI4P sensor paired with the PM-targeted Venus.
BRET values normalized to the DMSO-treated control cells (dotted lines). mRFP-tagged PITP wild type or the indicated mutants were co-expressed with the BRET sensor,
and the mRFP-C1 empty vector was used as control. The effect of the inhibitor on non-transfected cells (blue traces) is also plotted for reference in all panels. (B) Even
wild-type versions of PITPNA and PITPNB proteins showed a partial rescue, as observed as a delay to the inhibition, simply because of buffering the available inhibitor. (C)
PITPNA mutated in the C94 residue to either Ala, Ser, or Thr, all completely counter the inhibition. (D) Drug-resistant C94S mutant PITPNB also rescues the PI4P
depletion. PITPNB-C94S that is mutated in the T58 position to decrease (T58A) or eliminate (T58E) PI binding shows impaired rescue. Grand average ± SEM from three
independent experiments, each performed in triplicates. Source data are available online for this figure. (E) A cartoon showing the molecular targets of VT01454 and
OSW1. (F) BRET analysis using the (2x)P4M PI4P sensor paired with Rab7-targeted Venus. Cells were pretreated with DMSO or VT01454 (100 nM) for 30 min before the
addition of OSW1 (30 nM). BRET values were normalized to the control that received DMSO during preincubation as well as in place of OSW1. Data Information: Grand
average ± SEM from three independent experiments performed in triplicates. The non-transfected DMSO (blue) and VT01454-treated (red) traces shown for reference are
the same curves used in Fig. 1F, right panel. Only the PI-transfer competent PITPs can reverse the inhibition. Source data are available online for this figure.
Table 1. Statistics of crystallographic data and refinement.
Crystal PITPNA + VT01454
PDB accession code 8PQO
Data collection and processing
Space group P 2 2
1
2
1
Cell dimensions
a, b, c (Å) 50.33, 81.91, 94.70
α,β,γ(°) 90, 90, 90
Resolution range (Å) 25.42–2.30 (2.38–2.30)
No. of unique reflections 17 995 (1760)
Completeness (%) 95.55 (83.47)
Multiplicity 13.7 (13.8)
Mean I/σ(I) 21.5 (1.9)
R-merge 0.127 (1.364)
R-meas 0.131 (1.416)
CC
1/2
(%) 99.9 (95.6)
Structure solution and refinement
R-work (%) 23.51 (33.78)
R-free (%) 28.27 (44.87)
Ramachandran favored/outliers (%) 98.38/0
R.m.s.d.
bonds (Å) 0.005
angles (°) 0.74
Numbers in parentheses refer to the highest resolution shell.
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membrane-oriented tryptophan residue (Fig. 5E, yellow) in the
PKCs (see Fig. 5G for the positions of these residues in the
respective structures). Residues at the +2and+3 positions
downstream from the conserved proline are highly hydrophobic
in all PITPs including the class II members (Fig. 5E). Mutation of
the two hydrophobic residues in this position to alanine (F71, V72
to AA) in the Δ5 form of PITPNA (or in the Δ6formofPITPNB),
completely eliminated the membrane binding in response to AngII
stimulation (Fig. 5H, upper panels). These data together showed
that C-terminal truncations promoted the membrane penetration
of the α2 helix, and it does so in DAG-rich membrane domains.
Two conserved tryptophane residues (W201, W202) previously
identified (Tilley et al, 2004) were also required for membrane
localization, as their mutation to alanine also completely eliminated
the AngII-induced membrane translocation of the C-terminally
truncated PITPs (Fig. 5H, lower panels).
After identifying the residues that are critical for membrane
interaction, it was of interest to perform rescue experiments using
PITPNB variants with mutations in the membrane interface
prepared in the C94S background. Deletion of the last 6 residues
in PITPNB, which compromises the retention of cargo in PITPs,
greatlyreducedbutdidnoteliminate the ability of the protein to
maintain PM levels of PI4P following VT01454 treatment (Fig. 6A).
In contrast, mutation of the double Trp residues (W201A, W202A),
completely prevented rescue. In contrast, mutation of the FV
residues (F71, V72 to AA) within the lipid exchange loop of the
Figure 4. Structural description of the PITPNA-VT01454 complex.
(A) Structure of the PITPNA-VT01454 complex. VT01454 is shown in white in stick representation, while PITPNA is colored blue. (B) Detailed view of the VT01454
inhibitor in the lipid binding cavity. An identical color scheme is used as in panel (A). VT01454-interacting residues are represented as sticks. Water molecules engaged in
the interaction are depicted as red spheres. Selected hydrogen bonds are depicted as gray dotted lines. (C) The electron density of VT01454 (white sticks) is shown in the
simple Fo-Fc omit map contoured at 3D and colored dark green. (D) Structural alignment of mouse apo-PITPNA (light yellow, PDB ID:1KCM) and PITPNA-VT01454 (blue).
Note that the inhibitor-bound protein assumes the open conformation. (E) Structural alignment of PITPNA-VT01454 (blue) and the PC- (light green, PDB ID:1T27) and PI-
bound PITPNA (pink, PDB ID:1UW5) from rat and human, respectively. PyMol (Schrodinger, LLC) was used to create this Figure. Source data are available online for this
figure.
Yeun Ju Kim et al The EMBO Journal
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AngII 5 min AngII + DGKi
control
AngII + DGKi
control
GFP-PITPNA-'5
GFP-PITPNB-'6
AB
C
cytoplasmic intensity
(normalized to 0 min value)
Time (min)
AngII DGKi
Time (min)
cytoplasmic intensity
(normalized to 0 min value)
AngII DGKi
PITPNB-'6
GFP-PITPNA-'5
control PMA
PKD-C1a
LLMSKVKVPHTFVIHSYTRPTVCQFCKKLLKGLFRQGLQCKDCRFNC
PKD-C1b
PKCtheta-C1b
PITPNA
PKCtheta-C1a 151
223
136
268
50
PITPNB 50
PHHGNGQFTEKRVYLNSKLPSWARAVVPKI-FYVTEKAWNYYPYTIT
RdgBb 49
GPGGSGQYTHKVYHVGSHIPGWFRALLPKAALQVEEESWNAYPYTRT
Nir2 51
GPGGSGQYTHKVYHVGMHIPSWFRSILPKAALRVVEESWNAYPYTRT
Nir3 51
GPGGNGQYTKKIYHVGNHLPGWIKSLLPKSALTVEEEAWNAYPYTRT
dmRdgB 51
TFEDFQIRPHALFVHSYRAPAFCDHCGEMLWGLVRQGLKCEGCGLNY
KERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQGLKCDACGMNV
QAKIHNVKCHEFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAI
KDGEKGQYTHKIYHLQSKVPTFVRMLAPEGALNIHEKAWNAYPYCRT
KDGEKGQYTHKIYHLKSKVPAFVRMIAPEGSLVFHEKAWNAYPYCRT
GFP-PITPNA-'5, FV/AA
AngII 5 mincontrol
D
EF
G
H
GFP-PITPNA-'5, WW/AA
AngII 5 min
AngII 5 min
control
0.6
0.8
1.0
02.5 5 7.5 10
02.5 5 7.5 10
0.6
0.8
1.0
PITPNA-'5
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full-length PITPNB, did not impair its ability to reverse the effects
of VT01454 on the PM levels of PI4P (Fig. 6A). While these latter
data were surprising, they were in agreement with a previous report
regarding the reduced functionality of the C-terminally truncated
PITPNA (Hara et al, 1997) but not the F72A mutant PITPNA
protein in an assay based on reconstituting PLC activity in
permeabilized cells (Tilley et al, 2004).
VT01454 facilitates the interaction of class I PITPs
with membranes
Given that VT01454 blocks cargo binding, and the solved structure
suggests that it stabilizes the “open”conformation of PITPs (see
Figs. 4D, 5G), which, in turn, enhances membrane binding, we next
examined how VT01454 affects the localization of the wild-type
PITPNA and PITPNB proteins. The addition of VT01454 to cells
expressing the mRFP-tagged PITPs showed a minor effect, with
some cells showing weak ER localization of the PITPs. However,
after AngII stimulation, the VT01454-bound PITPs showed a clear
association with both the PM and the ER, although this latter may
only become more obvious as the cytoplasmic signal decreases. The
overall membrane association appeared to be stronger in the case of
PITPNB (Fig. 6B) and also required the two surface Trp residues
(W201, W202, Fig. 6C). Importantly, mutation of the Cys94 residue
that is critical to react with VT01454 to serine (C94S), completely
prevented the VT01454-induced membrane localization as well as
the AngII-induced translocation of the otherwise wild-type
PITPNA protein to the membranes (Fig. 6D).
Taken together, these data showed that VT01454 can promote
stabilization of the open conformation of class I PITP proteins,
facilitating their interactions with the membrane. This increased PITP-
membrane interaction was not, however, as large as those seen after
C-terminal truncations were performed. Surprisingly, however, despite
their presumably impaired ability to close in on their lipid cargo,
C-terminally truncated PITPs still remain partially active. In contrast,
the hydrophobic F71, V72 residues in the lipid-exchange loop that
confer DAG recognition appear to be dispensable, at least under our
overexpression assay conditions.
Complementary roles for class I and class II PITPs in
supporting PI(4,5)P
2
maintenance during PLC activation
It has been previously shown that the class II PITP, Nir2, transports
PI from the ER to the PM (Chang and Liou, 2015,2016; Kim et al,
2013; Kim et al, 2022) in exchange for PA, which is returned to the
ER from the PM (Kim et al, 2015; Yadav et al, 2015). This process
was found to be important for the maintenance of the signaling
pool of PI(4,5)P
2
in the PM when PLC was activated (Fig. 7A).
Given the complete lack of effect of the VT01454 compound on
resting PI(4,5)P
2
levels (Fig. 1E), we wanted to assess the extent to
which class I PITPs contribute to the replenishment of the PI(4,5)
P
2
levels in the PM during strong PLC activation. For this, we used
BRET analysis and challenged the cells with the Gq-coupled
muscarinic, M1 receptor agonist, carbachol, and followed the
recovery kinetics of PI(4,5)P
2
after terminating the response by
adding the antagonist, atropine. For comparison, we also used the
PI4KA inhibitor, GSK-A1 (100 nM), which, strongly inhibits the
recovery phase of PI(4,5)P
2
replenishment upon terminating PLC
activation without decreasing the resting PI(4,5)P
2
levels (Ham-
mond et al, 2012;Tothetal,2016).AsshowninFig.7B,
pretreatment of the cells with VT01454 (100 nM) did not affect
resting PI(4,5)P
2
levels, and had only a minor effect on the PI(4,5)
P
2
recovery rate, which contrasted the sizable inhibitory effects of
GSK-A1 (Fig. 7B). We noted that both inhibitors reduced the
relative size of PI(4,5)P
2
drop, and the reason for this change is still
under investigation. Regardless, the finding that PI4KA inhibition
had a larger inhibitory effect on PI(4,5)P
2
recovery following
carbachol stimulation than VT01454 treatment, suggested that
PI4KAinthePMreceivesPIfromasourceotherthantheclassI
PITPs. Obvious candidates for such a PI delivery role are the class
II PITPs, including the ubiquitously expressed Nir2. To test this
possibility, we compared Nir2 K/O cells with parental HEK293-
AT1cellsandanalyzedtheeffectsofVT01454andGSK-A1
treatment on the level and recovery rate of PI(4,5)P
2
following the
carbachol-atropine treatment regime. Pretreatment of Nir2 K/O
cells with VT01454 reduced the rate of PI(4,5)P
2
recovery after
atropine almost to the same level observed in cells pretreated with
GSK-A1 (Fig. 7C). This suggested that class I PITPs made a
significant contribution to PI delivery to the PM for PI(4,5)P
2
synthesis when Nir2 was eliminated.
These findings prompted us to compare the PI4P and PA
changes in parental and Nir2 K/O cells in response to inhibition by
VT01454 or GSK-A1 using BRET analyses. As shown in Fig. 7D,
pretreatment of parental cells with VT01454 (100 nM) for 30 min,
reduced PI4P levels to a very low level that was comparable to what
was found after GSK-A1 (100 nM) treatment (also see Fig. 1D).
Accordingly, carbachol stimulation caused only a small additional
decrease, and the recovery of PI4P levels after atropine addition was
Figure 5. C-terminal truncated PITPs recognize DAG-rich membranes.
(A) Representative images showing the localization of EGFP-tagged PITPNA truncated at the five C-terminal amino acids (EGFP-PITPNA-Δ5) expressed in HEK293-AT1
cells. AngII (100 nM) stimulation caused rapid association of the protein with the PM also causing a drop in the cytoplasmic EGFP intensity (middle panel). The addition of
a DAG kinase inhibitor, R59022 (50 µM) causes a further increase in membrane localization (right panel). (B) The cytoplasmic intensity of EGFP-PITPNA-Δ5 was
quantified in regions of interest outside the nucleus. Translocation to the PM is reflected in a decrease in cytoplasmic fluorescence. The Intensity values were normalized
to the 0 min values and expressed as ratio cha nges over time Grand averages ± SEM are shown from four independent experiments (8–18 cells monitored in each). (C,D)
Same as panels A,B using EGFP-PITPNB-Δ6. Note the more prominent Golgi and some ER localization even before AngII stimulation. (E) Sequence alignment of C1 domains
from PKC and PKD and the PITP domains of class I and class II PITPs. Note the conserved “P-T/A-F/V”sequence (red) in the two groups of molecules. Also, note that the
double Trp residues (W201,202) of PITPs is not covered in this alignment. Their functional role is analogous to that of the conserved Trp (yellow on gray background)
found in C1 domains. Also shown is the VT01454-reactive C94 residue (white on a gray background) that is not conserved in the class II proteins. (F) Cellular distribution
of EGFP-PITPNA-Δ5 was monitored after PKC activation after PMA (100 nM) treatment. Note the translocation of the protein to the PM. (G) Structural similarities
between the PKC C1 domain and PITPs in their hydrophobic interactions with the membrane. PDB IDs: 7L92 and 1KCM for PKCδC1b and PITPNA, respectively. (H)
Membrane association of EGFP-PITPNA-Δ5 after AngII stimulation was eliminated when the two hydrophobic residues (F71, V72) localized in the lipid exchange loop or
the double Trp (W201, W202) were mutated to alanine. Scale bar: 10 µm. Source data are available online for this figure.
Yeun Ju Kim et al The EMBO Journal
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small but still reached the pre-stimulatory low levels in VT01454-,
but not in GSK-A1-pretreated cells. Nir2 K/O cells showed a
slightly slower recovery of PI4P from the carbachol-induced
decrease compared to the parental cells (Fig. 7E), and both
VT014154 and GSK-A1 pretreatment yielded comparably low PI4P
levels, which were not appreciably changed further by either
carbachol or atropine treatment (Fig. 7E).
Since PLC activation also results in the production and
accumulation of PA in the PM [see e.g. (Kim et al, 2022)], limiting
the supply of PI is expected to impact the amount of PA that
accumulates in the PM during stimulation of PLC. In spite of its
small effect on PI(4,5)P
2
recovery, in parental HEK293-AT1 cells,
VT01454 treatment had a major impact on the basal level of PA
and its accumulation at the PM during carbachol treatment
(Fig. 7F). However, GSK-A1 treatment still had a stronger
inhibition in these parental cells (Fig. 7F). Nir2 K/O cells showed
a somewhat reduced PA response to carbachol stimulation and a
slower rate of PA clearance from the PM after the addition of
atropine (Fig. 7G). The slower PA clearance after atropine
treatment in Nir2 K/O cells was consistent with the reported role
of Nir2 in recycling PA from the PM to the ER for use in PI
resynthesis (Kim et al, 2015). Again, the difference between the
inhibitory effects of VT01454 and GSAK-A1 on the accumulation
of PA in the PM was reduced in Nir2 K/O cells compared to that of
parental cells, although GSK-A1 was still more effective than
VT01454 in the Nir2 K/O cells (Fig. 7F,G).
Collectively, these data showed that class I and class II PITPs are
both involved in the supply of PI to the PM for PI4P and PI(4,5)P
2
generation during the high demand following PLC activation.
Notably, however, even though the overall flux through PI4P and
PI(4,5)P
2
during PLC activation is greatly reduced in cells treated
with VT01454, which is best judged by the changes to PA
accumulation in the PM, the restoration of PI(4,5)P
2
levels
following stimulation is more efficiently supported by the class II
PITPs that are specifically recruited to ER-PM contact sites during
PLC activation.
Inhibition of class I PITPs affects several other lipid
classes beyond PPIns
Next, we performed lipidomic analyses on HEK293-AT1 cells
treated with VT01454 (100 nM) for 90 min. While most of our
BRET and confocal experiments covered shorter treatment times,
we chose 90 min treatment for this measurement to ensure that
with longer times the changes are larger and, hence can be
evaluated with more certainty. This analysis showed the largest
changes in PA, but strikingly, increases only in the levels
of unsaturated PA species, including the mono-unsaturated 32:1
and 34:1 forms, and, to a smaller extent, di-unsaturated 34:2
molecular forms, while the saturated PA species remained
unchanged (Fig. 8A). A moderate decrease was observed in DAG,
which was restricted only to two short chain-length and saturated
DAG species, namely 30:0 and 32:0 (Fig. 7B). Reduced levels were
observed in several species of PS, the largest reduction being in the
34:1 and 36:1 forms (Fig. 8D) as well as a slight elevation in the 38:4
form of PI (Fig. 8C). Cholesteryl esters were generally increased
while several forms of TAG showed a slight decrease (Fig. 8E,F). No
significant changes were observed in the levels of PC,
0 1000 2000 3000 4000
0.6
0.7
0.8
0.9
1.0
No transfection
mRFP-C1
PITPNB-C94S/'6
PITPNB-C94S
PITPNB-C94S/WW/AA
PITPNB-C94S/FV/AA
Time (sec)
Normalized BRET
P4M2x in PM
mRFP-PITPNB-C94S
mRFP-PITPNBwt
mRFP-PITPNB-WWAA
+AngII 5 minVT 10 min
D
B
C
A
VT
+AngII 5 min
+AngII 5 min
control before VT
control before VT
control before VT
VT 10 min
VT 10 min
Figure 6. PITPs treated with VT01454 associate with membranes.
(A) Rescue experiments with PITPNB variants with mutations that affect their
membrane interaction. For BRET analyses, see Legend to Fig. 4. Note that the
two Trp residues are essential for rescue, and the C-terminally truncated
protein is still somewhat functional. In contrast, in the context of the full-length
protein, the mutated FV residues only slightly impair the ability of the protein to
rescue. Grand average ± SEM from for independent experiments performed in
triplicates. Source data are available online for this figure. Also note that the
VT-treatment (blue), PITPNB-C94S (red), and mRFP-C1 rescue (yellow) curves
are shown for reference, which are the same curves that are shown in the
respective panels in Fig. 3. Scale bars: 10 µm. (B) mRFP-tagged wild-type
PITPNB was expressed in HEK293-AT1 cells and treated with VT01454
(100 nM) for 10 min (middle), followed by AngII (100 nM) stimulation (right
panel). VT01454 treatment caused a modest enrichment of the protein in
membranes, whereas AngII stimulation increased the localization of PITPNB
primarily in the PM (right panel). (C) Mutation of W201 and W202 to alanine in
PITPNB abolished their membrane binding after VT01454 and AngII treatment.
(D) VT01454-resistant C94S mutant of PITPNB doesn’t bind to the membrane
after VT01454 and AngII treatments. Source data are available online for this
figure.
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Yeun Ju Kim et al The EMBO Journal
© The Author(s) The EMBO Journal 13
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phosphatidylethanolamine (PE), phosphatidylglycerol (PG), or
sphyngomyelin (SM) (Fig. EV3A–D).
To understand the connection between PITP inhibition and the
changes observed in lipidomic analysis, we performed further metabolic
labeling experiments. Our previous study showed that decreases in the
PM levels of PI4P induced by PI4KA inhibition had a strong and
immediate impact on PS biosynthesis (Sohn et al, 2016). Such decreases in
PI4P in the PM could be responsible for the observed drop in PS levels in
VT01454-treated cells. Therefore, we also investigated whether inhibition
ofclassIPITPsalsoaffectedthePSsyntheticrateusingisotopelabeling
experiments with [14C]-serine. Treatment of cells with VT01454 (100 nM)
strongly inhibited the labeling of both PS and PE in a 2 h incubation,
which was similar in magnitude to the effect of acute PI4KA inhibition
(Sohn et al, 2016) (Fig. EV2C). These data showed that PITPs are
important for the maintenance of thePI4Pgradientthatisestablished
between the PM and the ER, which supports the transport of PS out from
the ER. Together with the strong feedback inhibition exerted by PS on the
PS-synthesizing enzyme, PSS1, a defect in PS export from the ER can
directly cause a significant inhibition of PS synthesis (Sohn et al, 2016).
Acute inhibition of class I PITPs inhibits PI synthesis
The PA species found to accumulate in the lipidomic analysis of
VT01454-treated cells suggested that PI synthesis might be stalled
under conditions when PI is not extracted from the ER, leading to
the accumulation of its precursor, PA. In fact, the PI synthase
enzyme is known to work in the reverse mode and catalyze robust
“inositol exchange activity”(Kim et al, 2022; Lykidis et al, 1997).
Therefore, we tested the effects of VT01454 on the incorporation
rate of myo-[3H]inositol into PI in a 1 h incubation both in control
cells and in cells stimulated by AngII. Note that these relatively
short inositol-labeling experiments primarily monitor the PI
synthetic rate, whereas the longer, close-to equilibrium-labeling
regime that was used in the experiments described earlier and
shown in Fig. 1A, reflects on the total level of PI, which is not
changed during the period of VT01454 treatment used in these
experiments. As shown in Fig. EV2D, knockout of either PITPNA
or PITPNB, had no effect on the basal PI synthetic rate, nor did
they show any effect on the AngII-induced increase in PI synthesis,
which was almost tenfold in all cases. However, in the presence of
VT01454 (100 nM) the incorporation rate was reduced by more
than 50%. These data were consistent with a reduced rate of PI
synthesis in cells where PI transport from the ER by class I PITPs is
inhibited.
Discussion
Extensive studies on mammalian class I PITPs and their functional
yeast homolog, Sec14, led to the general conclusion, that these
proteins deliver PI from the ER to the other organelle membranes.
This includes essential roles in supporting PI(4,5)P
2
production in
the PM to maintain PLC-mediated signaling, and PI4P production
in the Golgi complex to serve the multiple functions within this
organelle (Ashlin et al, 2021; Mousley et al, 2012). It has been an
important and highly debated question whether PITPs simply
transport PI between membranes or serve as instructive context-
specific regulators of PI kinases (Cockcroft and Garner, 2011;
Grabon et al, 2019).
Our studies are the first that were able to investigate the role of
class I PITPs in intact cells using a comprehensive pharmacological
approach, allowing acute and simultaneous inactivation of both
class I PITPs, PITPNA, and PITPNB, which was not possible to
accomplish with previously used genetic means. Our analysis
showed that acute inhibition of class I PITPs did affect PI4P
formation in the PM and Rab7-positive endosomal compartments,
both of which show low steady-state PI levels (Pemberton et al,
2020;Zeweetal,2020). However, treatment with VT01454 had a
much smaller effect on PI3P generation in Rab5-positive early
endosomes and failed to acutely reduce PI4P levels in the Golgi
compartment. Since PI3P in the Rab5-positive compartment was
still largely dependent on the activity of the class III PI3K, Vps34,
we must assume that this compartment receives PI by a mechanism
that is less dependent on class I PITPs. This question needs further
investigation and is currently being pursued in our laboratory. The
high level of PI that is detected in the Golgi complex (Pemberton
et al, 2020;Zeweetal,2020) is the most likely reason why the Golgi
compartment can sustain PI4P production for an extended period
even when the PITPs are inhibited. While the situation might be
very different when the PITP proteins are genetically inactivated,
previous studies also showed minimal or no changes in the Golgi
level of PI4P when the individual PITPs were removed or
downregulated by genetic means (Alb et al, 2002; Cockcroft and
Carvou, 2007).
Another long-standing question that we were able to address
here was the extent of contributions by class I PITPs to the supply
of PI to the PM during PLC-mediated consumption of PI(4,5)P
2
.
Our results showed no effect VT01454 on resting PI(4,5)P
2
levels in
the PM, even though the PM levels of PI4P were drastically
reduced. This was similar to what was already observed when
Figure 7. Contribution of class I and class II PITPs for PI delivery to the PM during PLC activation.
(A) Cartoon describing the components of the “PI-cycle”. Agonists-coupled PLC activation stimulates PI(4,5)P
2
hydrolysis with the generation of inositol 1,4,5-
trisphosphate and DAG, the latter is rapidly converted to PA in the PM. Abbreviation: PIS (PI synthase), CDS (CDP-DG synthase), Sac1 (PPIn phosphatase). PI is rapidly
resynthesized in the ER from PA, but for this to happen, PA needs to be transferred to the ER. Newly synthesized PI also has to be delivered to the PM to maintain the PM
pool of PI(4,5)P
2
. The class II PITP, Nir2 has been shown to perform the PA/PI exchange in ER-PM contact sites during PLC activation (see text for original References). In
parallel pathways, PI4P generated from PI in the PM by the PI4KA enzyme is used to facilitate the transport of PS from the ER to the PM by the ORP5/8 proteins. PI supply
is also critical for the efficient operation of this transport pathway. (B–D) BRET analysis following the PM levels of PI(4,5)P
2
(B,C), PI4P (D,E), and PA (F,G) using the (2x)
P4M, PLCδ1-PH and NES-Spo20, respectively, as LBDs, paired with PM-targeted mVenus. Cells were transfected for 24–28 h with the indicated BRET construct together
with the M1 muscarinic receptor. Carbachol (Cch, 100 µM) was added to stimulate PLC and atropine (Atr, 10 µM) was added to terminate the response (gray areas show
the duration of the stimulation). Cells were pretreated with DMSO, GSK-A1 (100 nM), or VT01454 (100 nM) for 30min before the start of BRET measurements. HEK293-
AT1 parental cells (B,D,F) or their Nir2 K/O version (C,E,G) are shown. Data Information: Grand averages ± SEM from three experiments are shown, each performed in
triplicates. Source data are available online for this figure.
The EMBO Journal Yeun Ju Kim et al
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PA 30:0
PA 30:1
PA 32:0
PA 32:1
PA 32:2
PA 34:0
PA 34:1
PA 34:2
PA 36:1
PA 36:2
mol%
A
PA
DAG 30:0
DAG 30:1
DAG 32:0
DAG 32:1
DAG 32:2
DAG 34:0
DAG 34:1
DAG 34:2
DAG 36:1
DAG 36:2
DAG 36:3
DAG 36:4
DAG 38:2
DAG 38:3
DAG 38:4
DAG 38:5
DAG 40:5
B
mol%
DAG
PI 30:0
PI 30:1
PI 32:0
PI 32:1
PI 32:2
PI 34:0
PI 34:1
PI 34:2
PI 36:1
PI 36:2
PI 36:3
PI 36:4
PI 38:3
PI 38:4
PI 38:5
PI 40:4
PI 40:5
mol%
mol%
C
PI
PS 30:1
PS 32:0
PS 32:1
PS 32:2
PS 34:0
PS 34:1
PS 34:2
PS 36:1
PS 36:2
PS 36:3
PS 36:4
PS 38:3
PS 38:4
PS 38:5
PS 40:4
PS 40:5
D
PS
EF
CE 14:0;0
CE 16:0;0
CE 16:1;0
CE 18:1;0
CE 18:2;0
CE 20:1;0
CE 20:4;0
Cholesteryl ester
0.0
0.1
0.2
0.3
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0
0.5
1.0
1.5
2.0
2.5
TAG 44 :0
TAG 44 :1
TAG 46 :0
TAG 46 :1
TAG 48 :0
TAG 48 :1
TAG 48:2
TAG 50:1
TAG 50:2
TAG 52 :1
TAG 52 :2
TAG 52 :3
TAG 54 :1
TAG 54 :2
TAG 54:3
TAG 54:4
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.00
0.05
0.10
0.15
0.20
0.25
0.30 TAG
Figure 8. Lipidomic analyses of HEK293-AT1 cells treated with VT01454.
HEK293-AT1 cells were treated with VT01454 (100 nM) or DMSO for 90 min and prepared for lipidomic analyses, which were performed by Lipotype as described in the
Methods Dark columns show VT01454-treated cells. (A) Phosphatidic acid; (B) Diacylglycerol; (C) Phosphatidylinositol; (D) Phosphatidylserine; (E) Cholesteryl ester; (F)
Triacylglycerol; Blue colors designate lipid species that show minor or no change, whereas orange/red columns highlight lipid species showing the largest changes in
response to the inhibitor treatments. Note that among the PA species, only the unsaturated forms show increases in response to VT01454 treatment. (E,F) Increases in
cholesteryl esters and decreases/increases in selected TAG species were more broadly distributed among the various fatty acyl side chain forms. Source data are available
online for this figure. Data Information: Means ± SEM and the individual data points are shown from three independent biological samples from one experiment that was
repeated with essentially the same results. Source data are available online for this figure.
Yeun Ju Kim et al The EMBO Journal
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inhibiting PI4KA with the specific inhibitor GSK-A1 [e.g.
(Bojjireddy et al, 2014;Gulyasetal,2022)], a puzzling observation
that suggests that the PM levels of PI(4,5)P
2
can be maintained at a
wide range of PI4P concentrations as long as the PI(4,5)P
2
-
consuming PLC enzymes are not highly active. Importantly, when
PLCs are stimulated, recovery of the PM levels of PI(4,5)P
2
were
not significantly impaired by VT01454 pretreatment despite a
substantially reduced flux through the PPIn pools in the PM. Only
in cells lacking the class II PITP, Nir2, did the contribution of class
I PITPs become critical for PI(4,5)P
2
resynthesis. These findings
suggests that class I PITPs may primarily support the maintenance
of the PI4P-driven non-vesicular transport pathways between the
PM and the ER rather than supply the PI(4,5)P
2
synthetic
machinery, which seems to rely upon the PI and PA exchange
functionsoftheclassIINir2/Nir3 PITPs. Our pharmacological
studies could not tell which of the two class I PITPs are more
important for these functions. Reports on platelets deficient in
PITPNA (Zhao et al, 2017) or PITPNB (Zhao et al, 2023) suggested
differences between the two PITPs in supporting various angles of
platelet functions. However, our studies in the HEK293 cell
background were not able to detect a major loss of PI4P or
PI(4,5)P
2
levels in single PITPNA or PITPNB K/O cells [see Fig. 1A
and (Kim et al, 2022)].
We devoted a significant effort to determine the mechanism of
membrane interaction of the class I PITPs. C-terminal truncations
were previously reported to significantly enhance the interaction of
recombinant rat PITPNA with lipid vesicles and reduced their PI or
PC transfer activity by ~60% in vitro (Tremblay et al, 1998). Using
intact cells, we found that both proteins truncated in their last five
(PITPNAΔ5) or six (PITPNBΔ6) C-terminal residues showed
prominent binding to DAG-rich membranes, which required the
hydrophobic residues within the conserved PTFV (PITPNA) or
PAFV (PITPNB) sequence, which is also found in the DAG-sensing
C1 domains of PKCs (Fig. 5E). This sequence is located within the
α2helical“lipid exchange loop”of the protein that dips into the
membrane interface in the open molecular state (Fig. 5G) and
shows the highest conformational change upon binding to lipid
cargoes (Grabon et al, 2017;Schoutenetal,2002; Tilley et al, 2004;
Yoder et al, 2001). Based on these findings, it is tempting to
speculate that DAG-rich membranes could facilitate lipid exchange
in the cargo binding avity of class I PITPs. Since activation of PLC
in the PM consumes PI4P and PI(4,5)P
2
, while also generating
DAG, this mechanism could ensure delivery of PI to the PM under
higher demands.
The reliance of the VT01454 inhibition on covalent interaction
with the Cys94 residue at the base of the lipid binding cavity,
allowed us to analyze features of PITPs that are critical to their
function in rescue experiments using C94S mutant variants. These
experiments are of great interest given the extensive mutational
studies reported in the literature assessing the functions of class I
PITPs in liposome experiments and permeabilized cells [see (Ashlin
et al, 2021) for original citations]. C94S mutations made both
PITPNA and PITPNB resistant to VT01454, and while it had some
effect on the ability of PITPNA to extract PC from membranes, we
found that these mutants were functionally fully competent at least
for the processes that we have examined in live cells. These rescue
studies showed that PI-binding was essential for their functions in
supporting PI4P formation in the PM and Rab7-positive endo-
somes. The rescue experiments were also instructive regarding the
importance of membrane interaction of these proteins in intact
cells. We confirmed the critical importance of the tandem Trp
residues (W201, W202) that were previously described as affecting
the membrane interaction and lipid transfer of PITPs in vitro and
in permeabilized cells (Phillips et al, 2006;Tilleyetal,2004). The
short C-terminal truncation of PITPNB (Δ6) in the C94S
background reduced the activity of the protein by about 50%,
whereas mutation of the FV residues to AA in the lipid exchange
loop in the full-length PITPNB-C94S had only a minor effect (Hara
et al, 1997). All of our rescue data fit very well with the in vitro
behavior of PITPs described earlier by the Cockcroft group (Hara
et al, 1997; Tilley et al, 2004). Importantly, treatment of the cells
with VT01454 caused a slow association of the PITPNA and
PITPNB proteins with both the PM and the ER, which was rapidly
enhanced after PLC activation. This is consistent with the structural
description of the VT01454-bound PITPNA, which shows the
inhibitor stabilizing the open conformation that, in turn, has a
higher membrane affinity. This finding also suggests that PLC
activation increases cargo exchange in the PITPs, most likely
through the generation of DAG, which would help to facilitate
membrane binding of the inhibitor. It is notable that the reactivity
of the C94 residue with the thiol-reactive reagents was found to be
facilitated by membrane binding (Shadan et al, 2008;Tremblay
et al, 2001). The VT01454-induced membrane localization,
however, was less prominent than that of the C-terminally
truncated proteins. The C-terminus of the protein [also termed
the “lid”(Shadan et al, 2008)] likely acts as a latch that traps the
ligand in the binding pocket through stabilization of the long
C-terminal helix in the closed conformation. The fact that the
C-terminal tail of the protein is not resolved in the open structure
[(Schouten et al, 2002) and this study] suggests that additional
interactions with the membrane are likely to control its movements.
Lastly, our lipidomics analyses of cells treated with
VT01454 showed changes in several lipid classes without any
major change in the absolute levels of PI. The most prominent of
these changes was the selective accumulation of PA, which was
remarkably restricted to only the unsaturated acyl-chain forms. In
contrast, moderate decreases in DAG levels were observed that only
affected the short, more saturated acyl-chain forms. The decreased
PS levels were consistent with previous studies showing that
reducing levels of PI4P in the PM results in the rapid inhibition of
the PSS1 enzyme due to the strong product inhibition on PSS1 that
is exerted by the PS that is retained in the ER as a result of the
diminished PI4P gradient that no longer exists between the PM and
the ER (Sohn et al, 2016). This was directly confirmed by isotope
flux studies using [14C]-serine labeling. Similarly, we attributed the
accumulation of PA to the impaired exit of the synthesized PI from
the ER, which, together with the reversibility of the PI synthesizing
machinery, could cause the accumulation of the metabolic
precursor, PA. The reduced myo-[3H]inositol labeling of PI was
also consistent with an inhibition of the conversion of PA to PI.
The fact that only the unsaturated PA species accumulates also
supports the idea that there is a functional compartmentalization of
the PA pools that are directed towards various metabolic fates.
Also, the reduced levels of DAG and TAG, as well as the
accumulation of some forms of cholesteryl esters, suggest that
these additional metabolic changes also influence neutral lipid
storage in the ER. While some of these lipid changes can be traced
back to the inhibition of PI transport by class I PITPs, others, such
The EMBO Journal Yeun Ju Kim et al
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as those in TAG and cholesteryl esters, could be secondary or
possible off-target effects. Addressing these possibilities will require
further studies.
It is of particular interest that VT01454 was identified as part of
a screen for inhibitors of the Hippo signaling pathway (Li et al,
2022), and the most profound inhibitory effects of VT01454 were
on the PI4P levels in the PM. The rapid reductions in PM levels of
PI4P are also observed with the PI4KA inhibitor, GSK-A1. PI4KA
has already been implicated in the control of the Hippo pathway in
Drosophila (Tan et al, 2014; Yan et al, 2011), and PI(4,5)P
2
is
believed to be the primary acidic lipid required for anchoring
Merlin to the PM and assembling the active LATS1/2 complex that
keeps the Hippo pathway off (Chinthalapudi et al, 2018;Hongetal,
2020;Manietal,2011). However, unless PLC was strongly
activated, neither VT01454 nor GSK-A1 treatment significantly
dropped the PI(4,5)P
2
levels in the PM, which raises the possibility
that the Hippo pathway activity may depend on the PM levels of
PI4P and the tightly co-regulated anionic PS as much as it does on
the total amounts of PI(4,5)P
2
.
In summary, our studies present the first comprehensive
analysis of the role of class I PITPs in cellular lipid metabolism
using a pharmacological approach in intact cells. The structure of
PITPNA with the bound to the VT01454 inhibitor reveals an open
conformation that is also associated with increased membrane
binding. While class I PITPs supply several organelles with PI for
sustained production of PPIns, their acute inhibition reveals
important effects on several additional lipid classes and highlights
the central role of these non-vesicular lipid transfer proteins for
orchestrating cellular lipid metabolism.
Methods
Reagents
Angiotensin II (human octapeptide) was from Bachem (Vista, CA).
Atropine and DAG kinase inhibitor I (R59022) were purchased
from Sigma Aldrich (St Louis, MO). Coelenterazine h (1-361301-
200) was purchased from Regis Technologies (Morton Grove, IL).
GSK-A1 inhibitor was described previously (Bojjireddy et al, 2014).
VPS34-IN-1 was obtained from Selleckchem (Houston, TX).
VT01454 has been described previously (Li et al, 2022). MI-14
was a kind gift from Dr. Radim Nencka (Institute of Organic
Chemistry and Biochemistry AS CR, Prague 6, Czech Republic),
and OSW1 was a generous gift from Dr. Matthew Shair
(Department of Chemistry and Chemical Biology, Harvard
University). [14C]-acetic acid and [14C]-serine (SA: 52 and
55 mCi/mmol, respectively) were purchased from American
Radiolabeled Chemicals (St Louis, MO), and myo-[3H]inositol
(SA: 98 Ci/mmol) was from PerkinElmer (Waltham, MA). The
polyclonal rabbit anti-PITPNA (clone “103”) and monoclonal anti-
PITPNB (clone “1C1”) antibodies (Carvou et al, 2010) were kind
gifts of Dr. Shamshad Cockcroft (University College, London, UK).
All other reagents were of the highest molecular biology grade.
DNA constructs
The human PITPNA was cloned from human brain cDNA
(Clontech) using PCR amplification and subcloned into pEGFP-
C1 (all of PCR primers are listed in Table EV1). The human
PITPNB (accession number BC018704, clone number 4643509
purchased from Open Biosystems) was also subcloned into pEGFP-
C1, and a ten amino acid residue linker (GGAGGAAAGA) was
inserted between the EGFP tag and the PITPNB protein
(synthesized as an oligomer pair). Truncated mutations (Δ5or
Δ6) of PITPs were generated by side-directed mutagenesis by
introducing of stop codon using the QuikChange mutagenesis kit
from Promega. PITP constructs were also subcloned into mRFP
plasmids using simple restriction digestions to replace EGFP with
mRFP. PITPNA was also subcloned into pET-19b plasmids for
generating recombinant proteins (see below). Human PITPNA and
PITPNB numbering are used throughout the manuscript. The
corresponding residues in the rat and mouse sequences used in
most studies in the literature are one number higher; therefore,
Cys94 of human PITPs corresponds to Cys95 in the rat or mouse
proteins.
The single plasmid-based BRET constructs to monitor PI, PI4P,
PI(4,5)P
2
, and PA in the PM, PI3P in the Rab5 compartment, and
PI4P in the Rab7 compartment have been described previously
(Baba et al, 2019; Kim et al, 2022; Pemberton et al, 2020). EGFP-
FAPP1-PH and EGFP-CERT-PH were described in our previous
study (Toth et al, 2006), and EGFP-FAPP2-PH was kindly provided
by Dr. Maria Antonietta De Matteis. The 3xHA-M1 human
muscarinic receptor was a kind gift from Dr. Jurgen Wess (NIDDK,
NIH). EGFP-GOLPH3 was amplified by PCR amplification from a
human cDNA clone (SC112810, NM_022130.3) purchased from
ORIGENE using primers containing XhoI and EcoRI restriction
sites to insert into the pEGFP-C1 plasmid. All primers used in this
study are listed in Table EV1.
Cell culture
HEK293 cells stably expressing the AT1a rat AngII receptor
(HEK293-AT1(Hunyadyetal,2002)), PITPNA K/O, PITPNB K/O
and Nir2 K/O cells described in (Kim et al, 2022) were maintained
in Dulbecco’smodified Eagle’smedium(DMEM—high glucose,
sodium pyruvate) containing 10% FBS and 1% penicillin-
streptomycin. The cell line has been treated with Plasmocin
prophylactic (InvivoGen, San Diego, CA) at 25 μg/ml for 1 week
after thawing. The subsequent passages were maintained at 5 μg/ml
of Plasmocin.
Live-cell image acquisition, -processing, and analysis
About 350,000 cells were seeded on 30 mm glass bottom culture
dishes (#1.5, Cellvis, Mountain View, CA) pre-coated with 0.01%
poly-L-lysine solution (Sigma-Aldrich). Cells were transfected the
next day with the indicated plasmid DNAs (0.1–0.2 μg/well) using
Lipofectamine 2000 (2–5μL/well; Invitrogen) using the manufac-
turer’s protocol. After 1 day of transfection, the media was replaced
with 1 ml modified Krebs–Ringer buffer (containing 120 mM NaCl,
4.7 mM KCl, 2 mM CaCl
2
,0.7mMMgSO
4
,10mMglucose,and
10 mM Na-Hepes, adjusted to pH 7.4) and cells were observed at
room temperature with a Zeiss confocal microscope (LSM510,
LSM710 or LSM 880 Airyscan) using a 63x Plan-Apochromat oil-
immersion objective (N.A: 1.4). To quantify Golgi-localized EGFP-
taggedlipidprobesinHEK293cells,z-stacksoffields with multiple
cells were acquired in multi-position mode (3–4 positions) as a
Yeun Ju Kim et al The EMBO Journal
© The Author(s) The EMBO Journal 17
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time series from each separate dish. Maximum intensity projection,
generated by the Zeiss Zen software, were then analyzed in Fiji
software. Regions of interest (ROI) were selected after global
thresholding, and the multi-measure function of the time series was
used where the average pixel intensities were multiplied by the
number of pixels covering the Golgi area, and for each individual
cell, the initial values were taken as 100 percent. A typical dish
yielded values obtained from about 30–40 cells that were averaged,
and these values were then used to calculate the grand averages
with S.E.M. from multiple dishes obtained in at least two
independent experiments.
BRET measurement
HEK293-AT1 cells (40,000/well) were seeded in a 200 μL total
volume to white-bottom 96 well plates pre-coated with 0.01% poly-
L-lysine solution (Sigma) and cultured overnight. Cells were then
transfected with 0.1–0.25 μgofthespecified BRET biosensor using
Lipofectamine 2000 (0.5–1μL/well) within OPTI-MEM (40 μL)
according to the manufacturer’sprotocol.After25–27 h of
transfection, the cells were quickly washed before being incubated
for 30 min in 50 µl of modified Krebs–Ringer buffer at 37 °C in
ambient air. After the preincubation period, the cell-permeable
luciferase substrate, coelenterazine h (40µl, final concentration
5 µM), was added, and the signal from the mVenus fluorescence
and sLuc luminescence were recorded using 485 and 530 nm
emission filtersovera4minbaselineBRETmeasurement(15s/
cycle). Following the baseline recordings, where indicated, the
plates were quickly unloaded for the addition of various treatments,
which were prepared in a 10 µl volume of the modified
Krebs–Ringersolutionandaddedmanually using a repeater pipet
to achieve good mixing. Measurements were then continued for the
indicated times. All measurements were performed in triplicate
wells. BRET ratios (mVenus/Luciferase) were calculated for
each well by dividing the 530-nm with the 485-nm intensity
values, which, when indicated, were normalized to the baseline
measurement. To facilitate the pooling of data from individual
wells and between replicate experiments, the raw BRET ratios were
processed using a simple moving average with a four-cycle interval
across the BRET kinetic. The processed BRET ratios obtained from
drug-treated wells were then normalized to internal vehicle
controls.
Lipidomics analyses
HEK293-AT1 cells (600,000 cells/well, passage 6–9) were plated onto
60 mm culture dishes and cultured for 2 days. VT01454 were added for
90 min in DMEM with high glucose medium with 10% serum and then
cells were detached with trypsin-EDTA and centrifuged. The cell pellet
was resuspended in PBS (at 6000 cells/μl)andfrozenondryicefor
shipment to Lipotype GmBH (Drezden, Germany) for mass
spectrometry-based lipid analysis as described in (Surma et al, 2021).
Briefly, lipids were extracted using a chloroform/methanol procedure
(Ejsing et al, 2009). Samples were spiked with internal lipid standard
mixture containing: cholesterol ester 16:0 D7 (CE), diacylglycerol 17:0/
17:0 (DAG), phosphatidate 17:0/17:0 (PA), phosphatidylcholine 15:0/18:1
D7 (PC), phosphatidylethanolamine 17:0/17:0 (PE), phosphatidylglycerol
17:0/17:0 (PG), phosphatidylinositol 16:0/16:0 (PI), phosphatidylserine
17:0/17:0 (PS), sphingomyelin 18:1;2/12:0;0 (SM) and triacylglycerol 17:0/
17:0/17:0 (TAG). After extraction, the organic phase was transferred to an
infusion plate and dried in a speed vacuum concentrator. The dry extract
was resuspended in 7.5 mM ammonium formate in chloroform/
methanol/propanol (1:2:4; V:V:V). All liquid handling steps were
performed using the Hamilton Robotics STARlet robotic platform with
the Anti Droplet Control feature for organic solvent pipetting.
MS data acquisition
Samples were analyzed by direct infusion on a QExactive mass
spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate
ion source (Advion Biosciences). Samples were analyzed in both
positive and negative ion modes with a resolution of Rm/
z = 200 = 280,000 for MS and Rm/z = 200 = 17,500 for MSMS
experiments, in a single acquisition. MSMS was triggered by an
inclusion list encompassing corresponding MS mass ranges scanned in
1 Da increments (Surma et al, 2021). Both MS and MSMS data were
combined to monitor CE, DAG, and TAG ions as ammonium adducts;
PC as formate adducts; and PA, PE, PG, PI, and PS as deprotonated
anions. MS was only used to monitor SM as formate adducts.
Data analysis and post-processing
Data were analyzed with in-house developed lipid identification
software based on LipidXplorer (Herzog et al, 2012;Herzogetal,
2011). Data post-processing and normalization were performed
using an in-house developed data management system. Only lipid
identifications with a signal-to-noise ratio >5, and a signal intensity
fivefold higher than in corresponding blank samples were
considered for further data analysis.
Analysis of myo-[3H]inositol or [14C]-
serine- labeled lipids
HEK293-AT1 cells (500,000 cells/dish) were plated on 12-well plates
andculturedfor2days.Formyo-[3H]inositol labeling, the radioactive
tracer (10 μCi/ml) was added for 1 h in inositol-free DMEM
supplemented with 50 μM unlabeled myo-inositol with or without
AngII (100 nM) and with our without VT01454 (100 nM). The steady-
state level of inositol lipids were measured with 2 μCi/ml myo-[3H]
inositol in 24 h labeling in inositol-free DMEM supplemented with 2%
dialyzed serum and 50 μM unlabeled myo-inositol and VT01454
(100 nM) was treated for the last 30 min during labeling. For [14C]-serine
labeling, cells were incubated with the radioactive tracer (0.5 μCi/ml) for
2 h in serine-free DMEM supplemented with or without VT01454. All
reactions were terminated by the addition of ice-cold perchloric acid (to
afinal concentration of 5%), and cells were kept on ice for 10 min. After
scraping, cells were centrifuged, and the pellet was processed to extract
lipids by an acidic chloroform/methanol extraction (Nakanishi et al,
1995). The resulting lower organic phase was dried by nitrogen gas and
followed by thin layer chromatography (TLC) using each solvent system
either with chloroform/ methanol/ ammonia/water 70:70:4:16 (v/v), for
inositol labeled lipids or chloroform/ methanol/ acetic acid/ water
85:38:5:8 (v/v) for serine-labeled lipids. For inositol lipids separation,
TLC plates (silica gel 60 W, 20 × 20 cm, EMD Millipore) were pre-
impregnated with an impregnating solution (75 mM Na-oxalate, 2 mM
EDTA, 0.5% boric acid) and completely dried at 80’Cinanoven.TLC
plates with [14C]-labeled samples were exposed to X-ray films with
multiple exposures while [3H]-labeled TLC plates were immersed in the
solution (diphenyl-oxazole 5% in diethylether) and air dried to enhance
the [3H]-signal before radiography.
The EMBO Journal Yeun Ju Kim et al
18 The EMBO Journal © The Author(s)
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Protein expression, purification, and crystallization
The protein was expressed and purified as described in Tilley et al,
2004. For functional assays, wild-type PITPNA and its mutants (T58E,
C94A, C94T) were expressed from the pET-19b plasmids. For
structural analysis, the gene encoding full-length PITPNA was cloned
into a modified pHIS2 vector containing an N-terminal His6x-tag
followed by a SUMO tag. E. coli BL21 Star cells were transformed by
these expression plasmids, and the cells were grown in 5052-ZYP
autoinduction media. The cells were harvested and lysed by sonication
in lysis buffer ((50mM Tris pH 8, 300mM NaCl, 20mM imidazole,
and 10% glycerol). Upon clearing the lysate by centrifugation, the
protein was isolated by affinity chromatography on Ni-NTA resin and
eluted in lysis buffer supplemented with 300 mM imidazole. The
proteins for functional assays were further subjected to size exclusion
chromatography (20 mM Tris 7.4, 300 mM NaCl, 10% glycerol, 3 mM
β-mercaptoethanol), concentrated at 3–12 mg/ml and stored at
−80 °C until needed.
His6x-SUMO tag was cleaved off from the protein for crystal-
lization trials by the Ulp1 protease (4 °C overnight), the protein was
bound on HiTrap Q HP column (Cytiva) in 20 mM Tris pH 8.0 and
eluted with a gradient of 0–500 mM NaCl. The protein was further
purified by size exclusion chromatography (SEC) on HiLoad 16/600
Superdex75 pg column (Cytiva) in SEC buffer (20 mM Tris pH 7.4).
The protein was concentrated to 12 mg/ml in the size exclusion buffer
(20 mM Tris pH 7.4), mixed with VT01454 at a 1:1.5 molar ratio
(protein:inhibitor), and the mixture was incubated at room tempera-
ture for ~60 min. Crystallization was performed using the sitting drop
vapor diffusion technique. Drops were created by mixing 150 nl of
protein solution and 150nl of reservoir solution of several commercial
crystallization screens using a Mosquito robot (SPT Labtech). Crystals
were obtained in 5 days in a condition with well solution of 100 mM
imidazole pH 8, 10% PEG 8000. The crystals were cryo-protected in
well solution supplemented with 20% glycerol and flash-frozen in
liquid nitrogen. The crystallographic dataset was collected from a
single crystal at the home source. The crystals diffracted to 2.3Å and
belonged to the orthorhombic P 2 2
1
2
1
space group. The data were
integrated and scaled using XDS (Kabsch, 2010). The structure was
solved by molecular replacement using the structure of PITP in
complex with PI (PDB ID: 1T27) as the search model and further
refined in Phenix (Afonine et al, 2012) and Coot (Emsley et al, 2010)to
good geometry and R-factors (Table 1). Figures were generated with
the PyMOL Molecular Graphics (Schrödinger, LLC). The atomic
coordinates and structural factors were deposited in the Protein Data
Bank (https://www.rcsb.org) under the PDB accession code 8PQO.
In vitro PITP lipid binding assay
HEK293-AT1 cells (5,000,000 cells/dish) were cultured for 2 days
in 100 mm culture dishes, and cells were labeled with 1.5 µCi/ml
[14C]acetic acid in 7 ml DMEM containing 2% dialyzed FBS for last
1 day. Cells were scraped and homogenized in 2 ml ice-cold buffer
(250 mM sucrose, 10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol) with ten times trituration up-and-down through a
25 G syringe needle. Cell homogenates were then centrifuged briefly
at 600 × gfor 5 min to remove nuclei and cell debris. The post-
nuclear fraction was collected by centrifugation at 100,000 × gfor
1 h to obtain the microsome pellet. Microsomes were resuspended
in 300 μl of assay buffer (10 mM Hepes-Na, 50 mM NaCl, 1 mM
EDTA, pH 7.4, protease cocktail, 0.1 mM dithiothreitol). The
recombinant PITPNA proteins (50 μg) and microsome (300 μg
proteins) were mixed in 1 ml assay buffer and tumbled in a 1.5 ml
Eppendorf tube for 30 min at room temperature. The reaction
mixture was centrifuged at 200,000 × gfor 1 h, and supernatant
(PITPNA protein with bound lipids) and pellet (unbound
membrane fractions) were processed to extract lipids by an acidic
chloroform/methanol extraction as described previously (Naka-
nishi et al, 1995). The lower phase was dried and developed by TLC
(chloroform/methanol/acetic acid/water 85:15:10:3, (v/v). The same
amounts of individual proteins were loaded on SDS-PAGE gel and
stained with Coomassie Blue.
Data availability
The atomic coordinates and structural factors were deposited in the
Protein Data Bank (https://www.rcsb.org) under the PDB accession
code 8PQO.
Expanded view data, supplementary information, appendices are
available for this paper at https://doi.org/10.1038/s44318-024-00096-3.
Peer review information
Apeerreviewfile is available at https://doi.org/10.1038/s44318-024-00096-3
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Acknowledgements
We would like to thank Dr. Radim Nencka (Institute of Organic Chemistry and
Biochemistry AS CR, Prague 6, Czech Republic) and Dr. Matthew Shair
(Department of Chemistry and Chemical Biology, Harvard University) for the MI-
14 and OSW1 inhibitors, respectively. We are also thankful to Dr. Peter Varnai
(Semmelweis University, Medical School, Budapest, Hungary), Dr.
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Antonietta (Antonella) De Matteis (Telethon Institute of Genetics and Medicine,
Naples, Italy), and Dr. Jurgen Wess (NIDDK, NIH, Bethesda, MD) for sharing
DNA constructs. We would also like to express our thanks to Dr. Shamshad
Cockcroft for the PITP antibodies. This work was partly funded by the Intramural
Research Program of the Eunice Kennedy Shriver National Institute of Child Health
and Human Development of the National Institutes of Health, Bethesda, MD,
USA. (HHS|NIH|NICHD - Z01:HD000196-25). Confocal imaging was performed
at the Microscopy & Imaging Core of the National Institute of Child Health and
Human Development, NIH, with the kind assistance of Drs. Vincent Schram and
Ling Yi. The research of A.E. and E.B. was funded by the project of the National
Institute Virology and Bacteriology (Program EXCELES, Project No.
LX22NPO5103) —Funded by the European Union—Next Generation Program.
The Academy of Sciences of the Czech Republic RVO: 61388963 for its support
of the Boura group is also acknowledged.
Author contributions
Yeun Ju Kim: Conceptualization; Data curation; Formal analysis; Investigation;
Methodology; Writing—review and editing. Joshua G Pemberton: Resources;
Formal analysis; Validation; Investigation; Methodology; Writing—review and
editing. Andrea Eisenreichova: Formal analysis; Investigation; Methodology. Amrita
Mandal: Investigation; Methodology. Alena Koukalova: Investigation; Methodology.
Pooja Rohilla: Investigation; Methodology. Mira Sohn: Resources; Data curation.
Andrei W Konradi: Resources. Tracy T Tang: Resources; Writing—review and
editing. Evzen Boura: Formal analysis; Funding acquisition; Writing—review and
editing. Tamas Balla: Conceptualization; Data curation; Supervision; Funding
acquisition; Visualization; Writing—original draft; Project administration.
Disclosure and competing interests statement
Drs. Tracy T Tang and Andrei W Konradi are employees and shareholders of
Vivace Therapeutics, Inc. The remaining authors declare no competing
interests.
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Expanded View Figures
Figure EV1. Ability of recombinant PITPNA proteins to extract PI and PC from prelabeled mem branes.
(A,B) Membranes prepared from HEK293-AT1 cells prelabeled with [14C]acetate overnight were incubated with purified recombinant PITPNA wild-type or mutated in the
indicated residues. After centrifugation, to pellet the membranes, the supernatant was subjected to lipid extraction and TLC analysis as detailed in the Methods.
Autoradiography films with different exposure times are shown. (B) PI and PC spots were quantified from two independent experiments using a Phosphor-Imager and
normalized to the values obtained in the wild-type proteins. (C) The same amounts of PITPs used in the above lipid binding assays were also run on SDS gels and visualized
with Coomassie staining. (D,E) Western Blot analysis of the various mutant mRFP-tagged PITPNA and PITPNB proteins expressed in the rescue experiments. Membranes
were probed with specific antibodies against PITPNA and PITPNB, which were generous gift from Dr. Shamshad Cockcroft, as indicated.
Yeun Ju Kim et al The EMBO Journal
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1:PITPNAwt
12341234
2:PITPNAT58E
3:PITPNAC94S 4:PITPNAC94T
short exposure longer exposure
BSA
To ta l
labeled
membrane
12 3 4
BSA
0
1
2
3
4
1234
BSA
Relative intensity
(normalized to WT)
0.0
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Relative intensity
(normalized to WT)
1234
BSA
PI
PC
Total
labeled
membrane
A
B
C
PITPNAwt
PITPNA
C94A
PITPNA
C94T
PITPNA
C94S
PITPNBwt
mock
PITPNBwt
PITPNBC94S
T59A
T59E
'6
FV/AA
WW/AA
mock
mRFP-PITP
endogenous
PITP
anti-PITPNA anti-PITPNB
DE
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PS
PE
VT01454 -+-+
C
0
1000
2000
3000
4000
5000
0
200
400
600
parental
PITPNA KO
PITPNB KO
VT01454 -+-+-+-+-+-+
AngIIcontrol
parental
PITPNA KO
PITPNB KO
[3H]inositol in PI (cpm)
D
AngII 2 min control
mCherry-PITPNA-'5
EGFP-PKD-C1ab merge
AngII 2 min control
mCherry-PITPNB-'6
EGFP-PKD-C1ab
A
B
[3H]inositol in PI (cpm)
[3H]serine labeling
merge
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Figure EV2. Co-localization of C-terminally truncated PITPs and a DAG sensor in HEK293-AT1 cells and effects of VT01454 on synthetic rates of PS and PI
production.
(A,B) Representative confocal images showing HEK293-AT1 cells transfected with the indicated constructs before (top rows) and after (bottom rows) stimulation with
AngII (100 nM). Note the rapid increase in PM association of both the DAG sensor (PKD-C1ab) and the truncated PITPs. Scale bar 10 µm (note that all images in panels
(A) and (B), respectively, show the same cells in the various channels before and after stimulation). (C) Incorporation of [14C]-serine into cellular lipids in a 2 h incubation
period shown as two biological replicates (see Methods for details). Note the inhibition of PS synthesis by treatment with VT01454 (100nM), which was present
throughout the labeling period. (D) Effects of VT01454 on the rate of PI labeling with myo-[3H]inositol in a 1 h incorporation period either in the presence (right) or absence
(left) of AngII. The results of two independent experiments are plotted. Parental HEK293-AT1 (blue) or their PITPNA (green) or PITPNB (red/orange) K/O derivatives are
shown. Note the different scales of the two graphs.
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mol%
mol%
mol%
AB
CD
mol%
PC PE
SM PG
PC 30:0
PC 30:1
PC 32:0
PC 32:1
PC 32:2
PC 34:1
PC 34:2
PC 36:1
PC 36:2
PC 34:0
PC 36:3
PC 36:4
PC 38:3
PC 38:5
PC 38:4
PC 40:4
PC 40:5
0
5
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20
SM 30:0;4
SM 32:0;4
SM 32:1;2
SM 32:1;4
SM 34:0;2
SM 34:1;2
SM 34:1;4
SM 34:2;2
SM 36:0;3
SM 34:0;4
SM 36:0;4
SM 36:1;2
SM 40:1;2
SM 42:1;2
SM 40:2;2
SM 42:2;2
0.0
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PG 40:7
PG 42:8
PG 44:8
0.0
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PE 30:0
PE 30:1
PE 32:0
PE 32:1
PE 32:2
PE 34:1
PE 34:2
PE 36:1
PE 36:2
PE 34:0
PE 36:3
PE 36:4
PE 38:3
PE 38:5
PE 38:4
PE 40:4
PE 40:5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Figure EV3. Lipidomics analyses of HEK293-AT1 cells treated with VT01454.
HEK293-AT1 cells were treated with VT01454 (100 nM) or DMSO for 90 min and prepared for lipidomic analysis, which were performed by Lipotype as described in the
Methods. Means ± SEM and the individual data points are shown from biological triplicates from one experiment that was repeated with essentially the same results. Dark
columns show VT01454-treated cells. No significant changes were observed in either of the four lipid classes shown in panels (A–D).
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