Download Mapping of Lipid-‐Binding Proteins and Their Ligandability in Cells

Mapping of Lipid-­‐Binding Proteins and Their Ligandability in Cells Crava%, B. F. et. al. Cell 2015, 161, 1668 Hannah Haley Literature Seminar 5 September 2015 tely 10 Å from the intracellular mem233, located near the midpoint of the
xternal and internal negative clusters
channels, the amino acids forming the
conserved
ascan glutamate
or aspartate.
Lipids have structural (e.g. stabilizing membranes or proteins) or signaling roles e.g. F O (C
U Seicosanoids) ORNE LVIIPE IW
DS
gle most conserved amino acid in Kv
Role of Lipids in Physiology and Pathophysiology Structural: Signaling: Box 1 | From phospholipids to eicosanoid signalling
O
X O
O
P
O
O–
H
O
O
O
O
cPLA2
X O
Arachidonic acid (AA)
O
–O
8
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11
H
O
Lyso-PC
OH
1
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PGH2S
COX1 or COX2
Conventional
NSAIDs
O
O
O–
+
5
P
COX2
inhibitors
e.g. aspirin Prostaglandin, prostacyclin and
thromboxane synthases
Conversion
Inhibition
Indirect
action
PGs, TXs
Tissue
homeostasis
PGs, TXs
Leukotriene
synthases
Leukotrienes
PAF
Inflammation
The production of eicosanoids is initiated by the release of C20-polyunsaturated fatty
acids,
such |asMolecular
arachidonic
Reviews
Biology
Arachidonic acid derived molecules mediate both Nature
physiological and Cellacid
(AA, C20:4), from phospholipids (X stands for a phospholipid headgroup; see figure) or diacylglycerol (not shown; see
2+
pathophysiological ignaling athways cPLA2 lipase action is the rateBOX 2). This hydrolysis is catalysed by
cytosolic phospholipasesA2
(cPLA2).pCa
-induced
Unusually posiMoned lipids region
e. a, Side view
of the transmembrane
limiting step in eicosanoid formation, and cells lacking cPLA2 are generally devoid of eicosanoids. Liberated fatty acids
hypothesized to influence structure are then stereospecifically oxygenated either through the cyclic prostaglandin synthase pathway (prostaglandin H2
epresentation,
coloured
according
to atom
synthase (PGH2S), including cyclooxygenase (COX) activity) or through the linear lipoxygenase-dependent pathway
funcMon of KcsA n; magenta,and phosphorous)
andchannel
the channel
(5-lipoxygenase; 5-LO), and are thus converted into one of four families of eicosanoids: the prostaglandins (PGs),
rom the extracellular side of the membrane
prostacyclins, thromboxanes (TXs) and leukotrienes.
Eicosanoids
short
half-life,
ranging
from
R. et. al. Nature 2007, 4at
50, the
376; Wymann, M. P., Schneiter, R. Nhave
at. Raev. Mol. Cell Biol. 2008, 9, 1seconds
62 to minutes. Their prime mode of action is mediated by binding
n MacKinnon, sphere. The
lipid
observed
Chemical Proteomic Probes to Characterize Lipid-­‐Protein Interac>ons ental Figures
Probe design based on small molecule-­‐protein binding affinity and light-­‐induced crosslinking to capture protein Design elements: a)  Small molecule to be recognized by protein (“lipid element”) Lipid
b)  PhotoreacMve element that covalently links Probe
small molecule B
A
C
and protein upon UV irradiaMon c)  Alkyne to allow late-­‐stage conjugaMon to azide tag via Cu-­‐
1. CuAAC
catalyzed alkyne-­‐azide cycloaddiMon
b) photoreactive
group
lipid
c) latent
element
affinity handle
Aa)
arachidonoyl (20:4)
AA-DA
kDa
N
N 150–
100–
N
H
AEA-DA
AEA-DA
AA-DA
B
O
OH
oleoyl (18:1)
O
N
N
100–
O
probe targets
Fluorescent
imaging
N
H
C
N N
Me
OH
O
N Ngel
OEA-DA
O
H
N
OH
N N
arachidonoyl (20:4)
kDa
150–
A-DA
OH
Crosslinked
palmitoyl (16:0)
H
N
A-DA
O-DA
S-DA
Alkyne
affinity handle
arachidonoyl (20:4)
AEA-DA
AA-DA
Cells
2. SDS-PAGE
2. Cell lysis
Set of lipid probes:
Diazirine
photocroslinking group
rhodamine-azide
oleoyl (18:1)
kDa
150–
O
PEA-DA
H
N
Me
kDa
150–
stearoyl (18:0)
N N
O-DA
100–
A-DA
O-DA
S-DA
Lipid probes
Rh N3
1. UV Light
100–
H
N
O
Me
N N
S-DA
Crava%, B. F. et. al. Cell 2015, 161, 1668
mentalCharacteriza>on Figuresof Lipid Probe Targets A
photoreactive
group
lipid
element
Lipid Probe
latent
affinity handle
A B C
1. CuAAC
Rh N3
1. UV Light
rhodamine-azide
2. SDS-PAGE
2. Cell lysis
ve
latent
affinity handle
Crosslinked
probe targets
kDa
150–
100–
75–
1. UV Light
kDa
150–
100–azide Rhodamine 1.
CuAAC
fluorescent reporter tag
N3
Rh75–
C
kDa
150–
Lipid
Probe
Me
A B C 100–
Me
+N
O
75–
rhodamine-azide
Me
2. Cell lysis
50–
2. SDS-PAGE
50–
Fluorescent
gel imaging
A-DA
O-DA
S-DA
Cells incubated with probe for 30 min before UV
AEA-DA
AA-DA
B
Cells
AEA-DA
AA-DA
igures
N
50–
O
N
H
CO2
-
IdenHficaHon of target proteins
kDa
150–
N3
100–
75–
Me
50–
A-DA
O-DA
S-DA
Lipid probes
Lipid Probes Differen>ally Label Proteins 2. SDS-PAGE
2. SDS-PAGE
2. Cell
2. lysis
Cell lysis
AEA-DA
O
N
H
OH
N
N
AA-DA
A-DA
kDakDa
150–
150–
D
A-DA
O-DA
A-DA
S-DA
O-DA
S-DA
C
A-DA
O-DA
A-DA
S-DA
O-DA
S-DA
CC
Fluorescent
Fluorescent
gel imaging
gel imaging
kDakDa
150–
150–
kDakDa
150–
150–
100–
100–
100–
100–
75–75–
75–75–
100–
100–
100–
100–
75– 75–
75–75–
50– 50–
50–50–
50–50–
50–50–
37– 37–
37–37–
37–37–
37–37–
25– 25–
25–25–
25–25–
25–25–
O
OH
N
B
AA-DA
kDakDa
150–
150–
Crosslinked
Crosslinked
probe
targets
probe
targets
AEA-DA
AA-DA
AEA-DA
Lipid probes:
AA-DA
BB
AEA-DA
AA-DA
AEA-DA
Cells
Cells
N
O
N N
N
H
Me
membrane
membrane
HEK293T
HEK293T
soluble
soluble
HEK293T
HEK293T
membrane
membrane
HEK293T
HEK293T
soluble
soluble
HEK293T
HEK293T
•  AA-­‐DA probe labels aLipid-Binding
lmost exclusively Figure
Chemical
Proteomic
Probes
Mapping
Proteins
in Cells,
to
•  Related
LocaMon of Figure
diazirine Figure
S1. S1.
Chemical
Proteomic
Probes
for for
Mapping
Lipid-Binding Proteins
in Cells,
Related
to Figure
1 1 effects protein membrane proteins (A)
Experimental
workflow
for
gel-based
profiling
of
lipid-binding
proteins
in
mammalian
cells.
Cells
were
incubated
with
lipid
probes
labeling rofile (A) Experimental workflow for gel-based profiling
of lipid-binding
proteins
mammalian cells. Cells
were pincubated
with
lipid
probes
for f
•  May be incorporated into in
phospho/
small
molecules
in native biological
systems
(Lee
and
Bogy
!
!
crosslinking
UV light
(10 min,
C) and
subsequent
cell
lysis.
Probe-labeled
proteins
were
then conjugated
a rhodamine-azide
(Rhcrosslinking
withwith
UV light
(10 min,
4 C)4and
subsequent
cell
lysis.
proteins
were
then
to atoirhodamine-azide
(Rh-N
3
•  conjugated
Protein s UV dSome
ependent neutral lipids or Probe-labeled
lipidated proteins 2013;
Simon et al., 2013;
Su etlabeling al., 2013).
probes re
copper-catalyzed
azide-alkyne
cycloaddition
(CuAAC
or ‘‘click’’)
chemistry
to allow
for visualization
of probe-labeled
targets
by SDS-PAGE
copper-catalyzed
azide-alkyne
cycloaddition
(CuAAC
or ‘‘click’’)
chemistry
to allow
for visualization
of probe-labeled
targets
by SDS-PAGE
andanin
onprobes
innate chemical reactivity with protein residues, wherea
• 
Decide t
o u
se f
a%y a
cid a
mide scanning.
scanning.
others
exploit
andcells.
light-induced
crosslinking
(B) Membrane and soluble protein labeling
profiles for the AEA-DA and
AA-DA
probesbinding
(20 mM) affinity
in HEK293T
Note that the
AA-DA prober
1. UV(S-DA)
Light
acyl chains, as wellrhodamine-azide
as photoreactive (diazirine) and alkyne groups.
2. SDS-PAGE
2. Cell
(B)lysis
AEA-DA and A-DA probes
show overlapping
but distinct protein interaction profiles in HEK293T
cells. Cells were
treated with each probe (20 mM)
Crosslinked
targets
for 30 min probe
in situ
before photocrosslinkingFluorescent
and
gel imaging
analysis of probe-modified proteins as described
in Figure S1.
(C) Arachidonoyl probe labeling of membrane and
soluble proteins depends on UV irradiation of cells.
kDa
kDa(D) Comparative labeling
profiles of kDa
lipid probes
150–
150–
150–
(20 mM, 30 min) in HEK293T cells. Red and blue
arrows mark representative
proteins preferentially
100–
100–
100–
labeled by arachidonoyl and oleoyl/palmitoyl
Preferential
75–
75–
probes, respectively. See Figure S1C for profiles of
75–
labeling
by
A-DA, O-DA, and S-DA.
AEA-DAC
D
O
N
H
N
OH
kDa
150–
N
OEA-DA
H
N
B
100–
OH
75–
O
C
A-DA
O-DA
S-DA
Lipid probes:
A-DA
O-DA
S-DA
Cells
AEA-DA
AA-DA
Lipid Probes Differen>ally Label Proteins AEA-DA
AA-DA
B
Lipid probes
arachidonoyl
probes
N N
50–
50–
50–
50–
in human cells by gel-based profiling.
HEK293T cells were
OH
37–
37– probe
37– treated with
37–
Preferential
O
(AA-DA
labeling
by or AEA-DA; 20 mM, 30 min), irradiated with UV light (10 min, 4! C), and lysed,
oleoyl/
N N
and the cell proteomes were fractionated
palmitoyl
probes
into membrane and soluble components
O
N N
A-DA
25–
25–
25–
25–
by centrifugation prior to conjugation to
N
Me
a fluorescent reporter tag (Rh-N3) using
H
CuAAC (Figure S1A). Analysis of probe
targets by SDS-PAGE and in-gel fluomembrane
soluble
membrane
soluble
rescence
scanning
revealed distinct
H
O-DA
Me
N
HEK293T
HEK293T
HEK293T
protein-labeling profiles
for each HEK293T
probe
(Figure S1B). The AA-DA probe showed
N N
O
Figure S1. Chemical Proteomic Probes for Mapping Lipid-Binding
Proteins in
Cells, Related
to Figure 1
almost exclusive
labeling
of membrane
• 
Polyunsaturated a
rachidonoyl p
robes (
AEA-­‐DA, A
-­‐DA) d
emonstrate more (A) Experimental
gel-basedproteins,
profiling ofwhich
lipid-binding
proteins inwas
mammalian
cells. Cells of
were
incubated w
mall molecules in native biological
systems workflow
(Lee andforBogyo,
we suspected
a consequence
rapid
extensive p
rotein l
abeling t
han m
onounsaturated (
OEA-­‐DA, O
-­‐DA) o
r s
aturated !
withSome
UV lightprobes
(10 min,rely
4 C) and
subsequent cell
lysis.probe
Probe-labeled
proteins were
then conjugated
S-DA
013; Simon et
al., 2013; H
Su crosslinking
et al.,Me
2013).
sequestration
of this
into membranes
through
its meta-to a rhoda
probes (PEA-­‐DA, -­‐DA) N copper-catalyzed
azide-alkyne cycloaddition
(CuAACSor
‘‘click’’) chemistry to allow for visualization of probe-labeled targets b
n innate chemical reactivity with protein residues, whereas bolic incorporation into phospho/neutral-lipids or into lipidated
scanning.
N
O
thers exploit binding affinity
andNlight-induced
crosslinking
proteins,
has been
noted for
other
fatty acidlipid probes
(Haber•  reChoose to map for
pas
roteins that interact with arachidonoyl probes (B) Membrane and soluble protein
labeling
profiles
the AEA-DA
and AA-DA
probes
(20 mM) in HEK293T
cells. Note that
ctions to capture proteins (Heal et al., 2011). The latter group kant et al., 2013; Tate et al., 2015). In contrast, the AEA-DA probe
PEA-DA
H
N
Iden>fica>on of Protein Targets with SILAC and LC-­‐MS/MS SILAC Ratio
15.0
light/heavy
A
10.0
ratio 20.0
UltraCentrifuge
Membrane
CuACC
Biotin-azide
Streptavidin
enrichment
MS1 intensity
LC/LC-MS/MS
Protein ID and quantification
AEA-DA vs AEA-DA
5.0
15.0
3.0
C
On-bead
trypsin digest
C
0.0 10.0
0
Soluble
No UV
A-DA
O-DA
S-DA
No UV
A-DA
O-DA
S-DA
Protein number
20.0
Mix
MS1 intensity
0
200
600
Protein number
5.0
3.0
0.0
400
800
0
200
400
600
800
Protein number
Protein number
D
A-DA vs A-DA
15.0
60
10.0
40
30
UV
Bas proteins labeled AEA-DA
A-DA
•  Lipid probe No
protein targets defined in UV A-DA vs A-DA
dependent manner (SILAC raMo ≥ 3.0 in 20probe-­‐versus-­‐no UV) and not enriched in probe vs. probe control (SILAC raMo < 2.0) 0
20
40
60
80
Light:
10 A-DA
Heavy: 1) A-DA 442
(No UV)
2) A-DA
H
**
**
20
5.0
3.0
0
0.0 Membrane Soluble
0
200 400 600 800
40
Protein number
AEA-DA
A-DA
OMIM Disease
Hematological
Renal
Developmental
20.0
Reproduction
Immune
Ratio of light/heavy Psychiatric
15.0
Neurological
peaks definesCardiovascular
SILAC ratio
Cancer
10.0
Metabolic
A-DA Selective
0.0
A
O-DA/S-DA Selective
3.0
Crosslinked probe targets
Light
Heavy
• 
SILAC Ratio
i) ± UV Light
ii) Cell Lysis
and 13C6, 15N4arginine enriched
5.0
SNX9
TMED2
IPO7
ACAT2
Comparison heavy cell groups: HMGCS1
a)  Same condiMons as light cells (probe-­‐versus-­‐probe control) NUCB1
20.0
b)  Same probe as light cells but no UV (probe-­‐versus-­‐no UV) No UV PITRM1
AEA-DAKIAA0664
vs AEA-D
c)  Other lipid probe (OEA-­‐DA, O-­‐DA, PEA-­‐DA, S
-­‐DA) NUCB2
15.0
200
400
600
800
ALDH1A2
FDFT1
HADHA
10.0
FDPS
RTN4IP1
PSMB6
O
5.0
AEA-DA No UV
O
N NDUFB9
N
A-DA
3.0
OH
NAMPT
A-DA vs
N A-DA
N
Me
PMPCA
H
0.0
H
DCTN2
0
200
400
600
800
N
SRPRB
N
SNX1
AEA-DA
A-DA
100
NDUFS2
ZADH2
No UV
20.0
No UV OCIAD1
80
% of Proteins
Heavy Cells
10.0
15N -lysine
6,
2
SILAC Ratio
Light Cells
•  Light cells are control and treated with arachidonoyl probe and UV •  Heavy cells are comparison A-DA Targets
X = OEA,PEA, O or S
13C
SILAC Ratio
A(EA)-DA
or X-DA
ILAC Ratio
A(EA)-DA
AEA-DA vs AEA-DA
15.0
% of Proteins
in situ treatment
(30 min)
SILAC Ratio
Stable-­‐isotope labeling bBy amino C-­‐tandem 20.0 acids in cell culture A-DAM
vs.S (LC-­‐MS/MS)
A-DA vs.
No UV(SILAC) and LD
A
361-4.0 3170
LMNB1
SLC25A5
SLC25A4
ALG1
SLC25A3
SDHA
PDIA6
GANAB
SAR1B
UBA52
SLC25A6
1000
SLC1A5
RUVBL1
SLC25A11
FKBP4
GOLIM4
ATP5O
IRS4
4.0
AEA-DA A-DA
Classifica>on of Iden>fied Protein T0 argets
200 AEA-DA Ta
E
N
A
O
P
20 3.0
N
A
O
P
B
% of Pr
i) 80
± UV Light
**
ii) Cell Lysis
15.0
% of Proteins
SILAC Ra
AEA-DA vs AEA-DA
mRNA Pro
MED25
SLC25A44A
FECH
Electron T
HEATR3
40
0
PITRM1 In
5.0
Host-Virus
442
361
317
TNPO3
3.0
Lipid Me
OTUB1
20
COPS6
Protein T
0.0
SCARB2
0
200 400
600targets
800 1000
Crosslinked
probe
RRM2
0
NUDT1
803
678
Membrane Soluble
SNX2
NUCB2
40
AHSA1
AEA-DA
A-DA
mRNA Processing
ARF4
23 10
5
Protein Classes CACYBP
Apoptosis
44
AEA-DA
vs.
AEA-DA vs.
30 UltraNUCB1
Electron Transport
Other
NAMPT
Centrifuge
PSMB6
Enzymes
Host-Virus Interaction
20
TOMM22
263
Transporters FAH
Lipid Metabolism
420
Light:
Chaperones KIAA0664
Heavy:
Protein Transport
MED25
PFKL
10
Receptors
SLC25A44
0
20 40 60 80 100 120 PCK2
60
10
0.0
400
600
8
M
e
ito
ch us
on
dr
ia
C
el
lM
E
em R
br
an
e
G
o
Ly
l
so gi
so
AEA-DA Selective m
e
Protein number
sm
10.0
N N
N
H
40
Me
op
AEA-DA
A-DA
20
10
442
361
317
la
op
yt
C
803
678
C
uc
l
N
Cancer
LRRC59
cells).
For instances where
multiple
isoforms of a given protein are en
Heavy:
1) AEA-DA (No UV)
Metabolic
PTGR2
-4.0 values
0 for the4.0
Figure 2. Mapping Protein Targets of Lipid Probes
by
Quantitative
Proteomics
(H)SSR4
Heatmap showing the relative
protein enrichment
AE
2) AEA-DA
Protein ID and quantification
0
20
40
60
80
100
120
LOG
TRAM1
3)
OEA-DA
0
20
40
60prob
2R
(A) Heavy/light SILAC ratio plots for total proteins identified
in
experiments
comparing
the
labeling
profiles
of
lipid
probe
itself
with
(AEA-DA)
or
without
UV
irradiation
(no
UV)
as
controls,
CCDC47
4) PEA-DA
R =of
Number
of
Targets
S1HMGCS1
for complete
list oflight
lipidcells
probetreated
targets.with
probe without UV irradiation) or the equivalent
probe
(both
heavy and
20 mM
the same p
Number
ofLight/Heavy
Targets
s
N m
M ucl
e
ito
ch us
on
dr
ia
C
el
lM
E
em R
br
an
e
G
Ly olg
so
i
so
m
e
0
Protein Transport
A/PEA-DA Selective
% of Proteins
AEA-DA
A-DA
30
Adaptors
MS1 intensity
yt
Protein number
40
409
FECH
C
600 800 1000
Membrane Soluble
5.0
3.0
HEATR3
Number ofChannels
Targets
0.0
678Soluble
RPN1
Membrane0 803
PITRM1
LRRC59 8
0
200
400
600
TNPO3
Cell
PTGR2
OTUB1
CuACC
Proteins d
ifferenHally e
nriched b
y Mitochon
acetyl-CoA
F
G
membrane
SSR4
COPS6
Protein
number
Protein class distribuHon
10Biotin-azide
SCARB2
TRAM1
two 23
probes
(from HEK293T (human) FASN (6.6)
RRM2 2. Mapping Protein Targets of Lipid Probes by CCDC47
Figure
5
Protein Classes
CPT1A Quanti
(7.9)
ACSL
44 and N
FATP
NUDT1
Streptavidin
euro2a (mouse) cells)(A)SNX2
HMGCS1
FA-C
FA-CoA
FA
FA
Heavy/light
SILAC
ratio
plots
for
total
proteins
identified
in
experime
OMIM
Disease
enrichment
ALDH3A2
Other NUCB2
(18) GPAT
SLC25A20
SCARB1
COPS3and
probe
heavy
AHSA1 without UV irradiation) or the equivalent probe (both(20)
Biological Enzymes
Process
(20)
(20)
On-bead
POR
E
ARF4
Hematological
threshold
ratio
values
(R3-fold
in
no
UV
experiments)
for
designation
263 trypsin digest
MTDH
LPA
Transporters
CACYBP
420
ILVBL and
(B)
Venn diagram of shared Renal
and unique protein targets of AEA-DA
NUCB1
Chaperones
mRNA Processing
PAP
NAMPT
TBL2
Developmental
(C–F)
Analysis
of
lipid
probe
targets
based
on
(C)
presence
(membrane
PSMB6
LC/LC-MS/MS
SSR1
Receptors
Reproduction
Apoptosis
DAG
TOMM22
ELOVL2
subcellular
distribution;
(E)
involvement
in
specific
biological
process
Adaptors
Immune
409
FAH
SRPRB
PNPLA2
(20)
Electron Transport
DGAT
tations.
presented
as
the
mean
percentage
of
total
pro
KIAA0664Data in (C) are
Lipid
Probe Targets
Psychiatric
SPTLC1
Channels
ABHD5
(20)
PFKL
(Nolipid
UV probe
Ratio) targets (red) TAG
Host-Virus Interaction
(G)
Diagram highlighting
in major fattyLSS
acid m
Neurological
PCK2
Light
Cardiovascular
parentheses
next
to
gene
names
(data
shown
are
for
the
A-DA
probe
i
Heavy
Lipid Metabolism
RPN1
Light: AEA-DA
AEA-DA Targets
0400
200
317
la
20
361
OEA-DA/PEA-DA Selective
O
442
10.0
AEA-DA Targets
60 A-DA
MS1 intensity
% of Proteins
**
**
SILAC Ratio
N
80
H
No UV
AEA-DA
OEA-DA
PEA-DA
100
s
N m
u
M
c
ito leu
ch
s
on
dr
ia
C
el
AEA-DA Selective
lM
E
em R
br
an
e
G
Ly olg
so
i
so
m
e
OH
No UVN
AEA-DA H A-DA
A-DA vs A-DA
N
No UV
AEA-DA
OEA-DA
PEA-DA
O
AEA-DA
% of Proteins
C
yt
op
la
IdenMfied protein targets include many with known links to lipid biology (eg enzymes and lipid carriers involved in fa%y acid uptake, transport, biosynthesis, and catabolism) but also m20.0
any without prior links No UV
Protein number
A-DA vs A-DA
200
400
600
800
Biological Process
E
15.0
Protein number
BD
AEA-DA
F
G
Mix A-DA
ALDH3A2
threshold ratio values (R3-fold in no
for designation of lipid probe targets (also see Figure S2).
CellUV experiments)COPS3
Known or predicted Biological
Process
POR
acetyl-CoA
links and
to dNeuro2a
isease cells
G
(B) Venn diagram Protein of shared
and
unique
protein
targets
AEA-DA andMitochondria
A-DA probesProtein in HEK293T
FA
membranein biological involvement pofrocesses
MTDH
subcellular distribuHon
ILVBL
10
InFASN
Situ (6.6)
Drug Profiling with Lipid Probes
(C–F) Analysis of lipid probe targets based on (C) presence
23
mRNA Processing
TBL2 (membrane) or absence (soluble) of known/predicted tran
Lipid-­‐Interac>on Proteome Enriched in Known Drug Targets A
Non-DrugBank Targets
Lipid Probe Targets
Proton pump (ATP4A)
Cathepsin D (CTSD)
Dihydrofolate reductase (DHFR)
Epoxide hydrolase 2 (EPHX2)
Lanosterol synthase (LSS)
Leukotriene A-4 hydrolase (LTA4H)
Monocarboxylate transporter 1 (SLC16A1)
Multidrug resistance protein 1 (ABCB1)
Nicotinamide phosphoribosyltransferase (NAMPT)
Prostaglandin-endoperoxide synthase 1 (PTGS1)
Progesterone receptor component 1 (PGRMC1)
Sterol O-acyltransferase 1 (SOAT1)
Chaperones (4%)
Transporters
Receptors (2%)
22%
280
(25%)
Classes
considered
ligandable
71%
29%
800
Others
45%
Enzymes
27%
DrugBank Targets
Non-DrugBank Targets
Number of Proteins
A
840
(75%)
Aladin (AAAS)
B-cell receptor-associated protein 31 (BCAP31)
Cleft lip and palate transmembrane protein 1 (CLPTM1)
ER lumen protein-retaining receptor 1 (KDELR1)
HEAT repeat-contain protein 3 (HEATR3)
Mitochondrial carrier homolog 1/2 (MTCH1/2)
Nuclear pore complex protein (NUP205)
Nucleobindin 1/2 (NUCB1/2)
OCIA domain-containing protein 1 (OCIAD1)
Protein QIL1 (QIL1)
Transmembrane protein 97 (TMEM97)
Transportin-1 (TNPO1)
Considered
not
Neuro2a cells
ligandable
Light
A-DA
A-DA
PTGS1
(Neuro2a)
Heavy A-DA (NoUV) A-DA
B
600 82%
18%
PTGS2 (A549)
DMSO DMSO DMSO
Light
DMSO DMSO DMSO
A(EA)-DA
DrugBank
MeO 2S
MS1 intensity
C
• B25% DMSO
of the idenHfied Competitor
lipid interacHon proteome enriched in Non-DrugBank
drug targets, while DMSO
12% of Flurbi
total hRofe
uman pDMSO
roteome is dRofe
rugged 400
Flurbi
Heavy
MS1 intensity
Me
•  Suggests lipid probes may preferenHally interact with proteins that can bind other small molecule ligands 200
PTGS1
CO 2H
•  Hypothesize that lipid probes can provide methods to determine drug target engagement and selecHvity 0
e
O
bl
e
F
lu
Competed
target
br
i) UV Light
ii) Cell Lysis
O
So
Heavy Cells
an
Light Cells
Rofecoxib
SILAC Ratio
15
1.4PTGS1 and PTGS2 Inhibitors for M
em
(±)-Flurbiprofen
Prostaglandin biosyntheMc enzymes PTGS1 and PTGS2 are inhibititor)
(PTGS2
(PTGS1/2 inhibitor)
lipid probe targets:
Digest
LC-MS/MS Analysis
MS1 intensity
Non-competed
target
Light
SILAC Ratio
Heavy
Competed
target
15
1.4
A-DA
(±)-Flurbiprofen
Heavy A-DA (NoUV)
Neuro2a cells
5.0
4.0 PTGS1
3.0PTGS2
2.0
8.0
6.0
Neuro2a cells
3.0
2.0
1.0
0.0
A-DA
A-DA Rofecoxib
AKR1B8
PTGS1
1.0
0
SILAC Ratio
100
200
3.2
300
0.0
0
1.1
100
are known:
1.3
1.1
3.5
4.6
A549 cells
SILAC Ratio
Enrich
Light
D
SILAC Ratio
(DMSO/Competitor)
PTGS1
“Click”
MS1 intensity
ank
MS1 intensity
Light
A-DA
A-DA
Mix
Heavy A-DA
(NoUV) A-DA
D
A549 cells
MS1 intensity
B
C
Neuro2a
Lipid probe
targetscells
3.9
1.4
SILAC Ratio
200
20.0
Me No UV
(±)-Flurbiprofen
A-DA vs A-DA
A549 cells
CO H
15.0
10.0
5.0
3.0
0.0
2
5.0
4.0 PTGS2
F
3.0
4.0
2.0
2.0
(±)-­‐Flurbiprofin 0.0
0
O
5.0
0 1.0
200
400
600
PTGS1/2 inhibitor
300
MeO 2S
Rofecoxib
A549 cells
3.0
1.0
800
Protein number
100 200 300 400
0.0
0
PTGS2
O
Rofecoxib PTGS2 inhibitor 100 200 300 400
Prostaglandin-endoperoxide
synthase 1 (PTGS1)
Monocarboxylate transporter 1 (SLC16A1)
Progesterone receptor
component
(PGRMC1)
Multidrug
resistance1protein
1 (ABCB1)
Nicotinamide
phosphoribosyltransferase
(NAMPT)
Sterol O-acyltransferase
1 (SOAT1)
Others
45%
Protein QIL1 (QIL1)
Nuclear pore complex protein (NUP205)
Enzymes
Nucleobindin 1/2 (NUCB1/2) Transmembrane protein
OCIA domain-containing protein
1 (OCIAD1) (TNPO1)
27%
Transportin-1
Non-DrugBank Targets
Protein QIL1 (QIL1)
Chaperones (4%)
Aladin (AAAS)protein 97 (TMEM97)
Receptors (2%) Transmembrane
Transporters
B-cell receptor-associated
protein 31 (BCAP31)
Transportin-1
(TNPO1)
Cleft lip and palate transmembrane protein 1 (CLPTM1)
ER lumen protein-retaining receptor 1 (KDELR1)
22%
HEAT repeat-contain protein 3 (HEATR3)
carrier homolog 1/2 (MTCH1/2)
Others PTGS1Mitochondrial
PTGS2 (A549)
(Neuro2a)
Nuclear pore complex protein (NUP205)
45%
Enzymes Light
Nucleobindin
1/2
(NUCB1/2)
DMSO DMSO DMSO
DMSO DMSO DMSO
OCIA domain-containing protein 1 (OCIAD1)
27%
DMSO Flurbi Rofe
DMSO Flurbi Rofe
Heavy
Protein QIL1 (QIL1)
MeO 2S
Transmembrane protein 97 (TMEM97)
Transportin-1 (TNPO1)
Compe>>on Between Lipid Probe and Drug For Protein A
DrugBank Targets
Non-DrugBank Targets
Engagement 280
840
CompeMtors :
(25%)
C
C
B
Light Cells
Light Cells
B
(±)-­‐Flurbiprofin (±)-Flurbiprofen
F
PTGS1/2 (PTGS1/2
inhibitor)inhibitor C
A(EA)-DA
Enrich
Mix
Digest
LC-MS/MS Analysis
Enrich
MS1 intensity
Light
Heavy
MS1 intensity
Non-competed
Competed
target
target
Lipid
probe targets
“Click”
CO 2H
D
Competed
target
(±)-Flurbiprofen
Neuro2a cells
5.0
SILAC Ratio SILAC Ratio
SILAC
Ratio
(DMSO/Competitor)
(DMSO/Competitor)
(DMSO/Competitor)
ii) Cell“Click”
Lysis
(±)-Flurbiprofen
MeO 2S inhibitor)
(PTGS1/2
Me
Heavy Cells
Mix
i)
UV
Light
Lipid probe targets
2
F
Lipid probe targets
Light Cells
CO
H
MeO2 S
CO H
Competed
target
Competed
Competitor
target
DMSO
Me
2
DrugBank
CO 2H
F Targets
Non-DrugBank Targets
Heavy
i) UV
Light Cells
i) UV Light
ii) Cell Lysis
Me
Me
Heavy Cells
ii) Cell Lysis
(75%)
D
F
O
O 8.0
O
O
2.0
5.0
1.0
O
O
Rofecoxib
Neuro2a
cells
O
Rofecoxib Neuro2a
cells
PTGS2 inhibitor
3.0
2.0
1.0
D0.0
0.0
4.0 PTGS1
(±)-Flurbiprofen
0
0
100
200
300
O
PTGS1
8.0
5.0
lipid probe
DM
DM
PTGS2 (A549)
PTGS1 (Neuro2a)
DMSO DMSO DMSO
Light
Rofecoxib
Flurbi 1.3
DMSO 3.9
Rofe
Heavy
SILAC
Ratio 1.4
(PTGS2
inhibititor)
Rofecoxib
(±)-Flurbiprofen
AKR1B8
6.0(PTGS2 inhibititor)
4.0(PTGS1/2
PTGS1
inhibitor)
(±)-Flurbiprofen
3.0
DMSO DMSO DMSO
DMSO Flurbi Rofe
Light
Heavy
Rofecoxib
(PTGS2 inhibititor)
MeO 2S
97
PTGS1 (Neuro2a)
MS1 intensity
CompeMMon experiment:
MS1 intensity
DrugBank Targets
Non-DrugBank Targets
MS1 intensity
Lipid Probe Targets
Prostaglandin-endoperoxide synthase 1 (PTGS1)
Proton1pump
(ATP4A)
Progesterone receptor component
(PGRMC1)
Cathepsin
D (CTSD)
Sterol O-acyltransferase
1 (SOAT1)
Dihydrofolate reductase (DHFR)
Epoxide hydrolase 2 (EPHX2)
Lanosterol synthase (LSS)
Leukotriene A-4 hydrolase (LTA4H)
Monocarboxylate transporter 1 (SLC16A1)
Multidrug resistance
protein 1 (ABCB1)
Competitor
Competitor(NAMPT)
DMSO Nicotinamide
DMSO phosphoribosyltransferase
Prostaglandin-endoperoxide
synthase 1 (PTGS1)
A(EA)-DA A(EA)-DA
Progesterone receptor component 1 (PGRMC1)
Sterol O-acyltransferase 1 (SOAT1)
lipid probe
SILAC
RatioNOT1.4
(±)-Flurbiprofen
competed
byA549
flurbicellscompeted
by rofe
5.0
DMSO DMSO DMSO
DMSO 3.5
Rofe
Flurbi 4.6
1.1
lipid probe
3.9
1.3
Rofecoxib
competed
1
flurbi
A549bycells
and rofe
CompeHHon ith known inhibitor 4.0 PTGS2 consistent w
4.0
(±)-Flurbiprofen
Rofecoxib
PTGS2
selecHvity
3.0
3.0
A549
Neuro2a
3.9
1.4
1.3
1.1 cells
3.5
4.6
SILAC
Ratio cells
AKR1B8
2.0
1.0
5.0
2.0
1.0
5.0
6.0
(±)-Flurbiprofen
Rofecoxib
Rofecoxib
0.0
0.0
4.0 PTGS2
4.0
100
200
300
0
100 A549
200 cells
300 400
0
100 A549
200 cells
300 400
Neuro2a cells
Neuro2a cells
Protein
3.0 Protein number
Protein number
Protein number
ProteinAnalysis
ID and quantification
3.0
3.0
LC-MS/MS
3.0 number
“Click”
5.0
8.0
5.0
5.0
Enrich
AKR1B8
Non-competed
Competed
2.0
6.0
4.0 PTGS1
2.0
2.0
2.0
4.0 PTGS2
4.0
target
targetDigest
PTGS1
PTGS2
Figure 4. The Lipid-Interaction Proteome Is3.0
Rich in Drug Targets
3.0
3.0
LC-MS/MS Analysis
3.0
1.0
1.0
1.0
1.0
(A) CategorizationNon-competed
of lipid probe
targets based on
distribution in DrugBank (left pie chart) and further analysis of non-DrugBank
targets by protein classe
Competed
Light
2.0
2.0
2.0
2.0
target
target
PTGS1
0.0
considered ligandable (e.g., enzymes, receptors,0.0
transporters) or not (others).
Heavy
0.0
0.0
1.0
0
1.0300
1.0200
1.0
100 cells
300 with0vehicle
0
100
200
100
200or300
400 ligand
(B) Scheme for
in situ competitive profiling of ligands using lipid probes. Isotopically light and heavy
are treated
(DMSO)
competitor
0
Light
0.0
0.0
Heavy
0.0
0.0 enriched, and digested fo
Protein
number
Protein
number
0
respectively,
along
with
a
lipid
probe
for
30
min.
Cells
are
then
UV
irradiated
and
lysed,
and
light
and
heavy
lysates
are
combined,
Protein ID and quantification
100
200
300
0
100
200
300
0 100 200 300 400 Protein
0 100number
200 300 400
LC-MS/MS analysis.
Ligand
are designated as proteins
show light/heavy
ratios
of R3.0.
Protein that
number
Protein
number
Protein number
Protein number
Protein
ID and targets
quantification
MS1 intensity
MS1 intensity
Mix
Digest
MS1 intensity
MS1 intensity
B
840
(75%)
(25%)
(C) Chemical structures of the dual PTGS1/2-inhibitor (±)-flurbiprofen and PTGS2-selective inhibitor rofecoxib and representative peptide MS1 chromatogram
•  cells,
PTGS enzymes are among that
the (±)-flurbiprofen
most competed -­‐DA competes
target proteins, ndicaHng good selecHvity for PTGS1
and PTGS2 in Neuro2a
and A549
respectively,
showing
(25AmM)
A-DA (5imM)
labeling
of both
PTGS1 and PTGS2
. The
Lipid-Interaction
Proteome
IsIs
Rich
Drug
Targets
Figure
4. The Lipid-Interaction
Proteome
Rich
inin
Drug
Targets
• 
AKR1B8 i
s m
ouse o
rtholog o
f h
uman a
ldo-­‐keto r
eductase w
hich i
s m
odified/inhibited by prostaglandins whereas rofecoxib (25 mM) selectively competes PTGS2 labeling.
(A) Categorization
of lipid probe
targets
basedon
on distribution
in DrugBank
(left pie chart)
and further
of non-DrugBank
by protein class
orization
of lipid probe
targets
based
distribution
in DrugBank
(left pie
chart)analysis
and further
analysistargets
of non-DrugBan
Lipid Probes as Screening Tool for Discovery of New Ligands •  Nucleobindin protein NUCB1 known to interact with PTGS1 and PTGS2 and enhance PTGS2-­‐mediated prostaglandin synthesis, but not before known to bind small molecule ligands •  Hypothesized to play role in cellular lipid metabolism A
B
A
O
Me 2N
A
B
FI-­‐AEA FluoPol Probe + NMe
C
B
2
C
C
CO2-
O
N
H
N
H
O
Me
D
D
D
O 2N
O
med chem
N HN NH 2
H
initial HTS hit
G
16,000 compounds screened for compeHHve binding to NUCB1 relaHve to FI-­‐AEA probe G
G
Fluorescence polarizaHon by arachidonoyl but Fdecreased F
not palmitoyl compeHtor lipids
Increase in fluorescence polarizaHon upon FI-­‐AEA E bEinding Eto purified recombinant NFUCB1 H
R1
O 2N
R2
O
N
R3
21 compounds
R4
R1
O
N
R2
N
N
Me
MJN228 (R1 = Ph; R 2 = H)
KML110 (R1 = Ph; R 2 = Me)
KML181 (R1 = H; R 2 = H), inactive control
OpHmizaHon of iniHal screen hit to generate more potent NUCB1 binding ligands
H
H
MJN228 Competes Arachidonoyl Probe for NUCB1 Binding G
H
NUC1B ligands:
O 2N
R1
O
N
R2
A
N
N
Me
MJN228 (R1 = Ph; R 2 = H)
KML110 (R1 = Ph; R 2 = Me)
KML181 (R1 = H; R 2 = H), inactive control
Adapting Lipid Probes for HTS to Discover NUCB1 Ligands
CompeHHve binding of opHmized ligands to purified NUCB1 relaHve to FI-­‐AEA probe e of Fl-AEA probe.
on of the Fl-AEA probe (0.5 mM) with recombinant human NUCB1 (1.0 mM) produced a strong FluoPol signal that was significantly suppressed by the
lipid AA (20 mM; Z0 = 0.54).
A
C lipids AEA, 2-AG, and AA, but not palmitoyl lipids PEA, 2-palmitoyl
tration-dependent
suppression of the NUCB1-FluoPol signal by arachidonoyl
D
B
PG), or palmitic acid (PA). Error bars represent SD (n = 5). See Figure S6A for profiling
of additional lipids.
of 16,000 compounds identified small molecules that inhibited the NUCB1-FluoPol signal by 20% or greater (dotted black line).
e of confirmed HTS hit 1 and positions modified for medicinal chemistry optimization. See Figures S6B–S6D for summary of medicinal chemistry
MJN228 selecHvely n of NUCB1 ligands.
inhibits AEA-­‐DA ration-dependent blockade of AEA-DA (5 mM) labeling of purified, recombinant NUCB1 (0.25 mM) doped into HEK293T lysates (0.75 mg/ml) by HTS
probe binding to 00 mM).
NUCB1 over ~400 Structures and competition profiling results (G) and IC50 curves and values (H) for NUCB1 ligands MJN228 and KML110 and the inactive control
other probe targets KML181.
and (H) represent mean values ± SD from at least three independent experiments.
InhibiHon of AEA-­‐DA probe labeling of NUCB1 in D 6H) and did not
new ligands
1 withBan IC50 value ofcells 3.3by mM
(Figure
o disrupt other arachidonoyl probe-protein interactions
E
(25 mM) produced
substantial (!3- to 5-fold) reductions in lipid
probe enrichment of NUCB1, whereas KML181 had no effect
Metabolic Effects of NUCB1-­‐ligand Interac>on A NUC1B ligands:
O 2N
C
R1
C
O
N
R2
N
N
Me
MJN228 (R1 = Ph; R 2 = H)
KML110 (R1 = Ph; R 2 = Me)
KML181 (R1 = H; R 2 = H), inactive control
•  Site on NUCB1 of both ADA-­‐DA probe and ligand MJN228 Ebinding mapped to PTGS1/2 binding domain D
B
•  Suggests common region for NUC1B lipid-­‐protein and protein-­‐protein interacHons
IdenMfying metabolic consequences of NUCB1-­‐MJN228 interacMon:
D
E
G
F
Cell treatment with MJN228 leads to elevated levels of N-­‐acyl ethanolamines (NAEs) and N-­‐acyl taurines (NATs), two classes of fady acid amides
NAEs and NATs are both metabolized by the enzyme fady acid amide hydrolase (FAAH), but neither MJN228 or KML110 Ginhibit FAAH (PF-­‐7845 is known FAAH inhibitor) Metabolic Effects of NUCB1-­‐ligand Interac>on D NUC1B ligands:
O 2N
D
D
E
E
R1
O
N
R2
E
N
N
Me
MJN228 (R1 = Ph; R 2 = H)
KML110 (R1 = Ph; R 2 = Me)
KML181 (R1 = H; R 2 = H), inactive control
NUCB1
G
ligands
G
control
compounds
FAAH
inhibitor
Cell treatment with MJN228 and KML110 leads to elevated levels of N-­‐acyl ethanolamines
G
NUCB1 ligands also inhibit the oxidaHve metabolism of exogenously added arachidonylethanolamine control
NUCB1
Engagement
and Effects
Lipid Metabolism
Effects of NUCB1 Ligands
shRNA
knockdown
Lipid Metabolism
of NUCB1 Ligands
eptide MS1
chromatograms
of AEA-DA
probe labeling
in Neuro2a
cells by MJN228 and KML110,
tograms
showing
blockade
of showing
AEA-DA
labeling
of endogenous
NUCB1ofinendogenous
Neuro2a cellsNUCB1
by MJN228
and KML110,
Knocking down expression of probe
Nblockade
UC1B leads to elevated levels of fady acid amides NUCB1
ligands
control PTGS2
inhibitor
on
performed
with MJN228
(10 mM) with
and the
AEA-DA
(5 mM).
forexperiment
in situ competition
experiment
performed
MJN228
(10probe
mM) and
the AEA-DA probe (5 mM).
nd
Lipid
Metabolism
Effects
of
NUCB1
Ligands
nent
MJN228-sensitive,
AEA-DA-modified
NUCB1
peptide
(aa
53–68)
in
Neuro2a
tification of a prominent MJN228-sensitive, AEA-DA-modified NUCB1 peptide cells.
(aa 53–68) in Neuro2a cells.
Data collecMvely uggests that Nshow
UCB1 pendogenous
lays i(10
ndirect role iinn Neuro2a
facilitaMng fby
a%y acid a(NAEs
mide metabolism, e.g. matograms
showing
blockade
of sAEA-DA
labeling
of
NUCB1
cells
MJN228
and
KML110,
als
that profiling
Neuro2a
cells
treated
with
MJN228
(10
mM)
elevated
fatty
acid
amides
(NAEsfatty
and
NATs)
compared
to
cells
bolite
reveals
that Neuro2a
cellsprobe
treated
with MJN228
mM)
show
elevated
acid
amides
and
NATs) compared to cells
serving See
s i5also
ntracellular to dTable
eliver S5.
lipids to fa%y acid amide hydrolase (FAAH)
(p
0.0001, (10
n = mM)
5 per(pcondition).
S5.carrier or <KML181
< 0.0001,
na=
perTable
condition).
See also
A Global Map of Lipid-Binding Proteins and T
Ligandability
in Cells Proteins and Their Globally M
apping Lipid-­‐Binding Ligandability Graphical Abstract
Authors
Micah J. Niphakis, Kenn
Armand B. Cognetta III,
Fabiana Piscitelli, Hugh
Benjamin F. Cravatt
Correspondence
[email protected] (M
[email protected] (B.
In Brief
A chemical proteomics a
a global lipid-protein inte
provides evidence for th
druggability of lipid-bind
cells.
Highlights
Proteomic probes allow mapping of cellular targets of small molecules and discovery of new small molecule d Chemical probes provide
a global
protein ligands map of lipid-binding