Exploring the activity and essentiality of the Δ-6 desaturase in Trypanosoma brucei Michela Cerone a and Terry K Smith a a School of Chemistry, BSRC, University of St Andrews, North Haugh, St Andrews, Fife, Scotland, UK Reference Nat. Rev. Mol. Cel. Bio., 1, 31-39 (2000); Nat.,459, 213-217(2009). Acknowledgments I would like to thank Prof Terry K Smith for his support and supervision and all members of TKS group; Prof. Keith Matthews from University of Edinburgh for providing anti-PAD1 antibody; and EPSRC and CRITICAT CDT for funding. N C Fe 2+ Fe 2+ Δ-6 T. brucei His His His Fe Fe Outer Membrane Inner Membrane TM TM TM TM H 3 C O S H 3 C S O 2 Ferrocytochrome b5 2 Ferricytochrome b5 2 NADH O 2 2 NAD + CoA CoA S O CoA H 3 C S O CoA H 3 C 2 H 2 O 1. How does Δ6-desaturase from T. brucei work? Fig 1. Schematic representation of Δ-6-desaturase trans-membrane enzyme and its reaction mechanism. Green indicates the ω-3 PUFAs biosynthetic pathway and orange the ω-6 PUFAs biosynthetic pathway. x Overexpression (OE) of Δ6-Desaturase Knock down (KD) of Δ6-Desaturase Δ6-DESATURASE Tet T7 HA tag p2T77 or pLEW100 vectors Growth Curve Immunofluorescence microscopy ESI-MS analysis of lipids GC-MS analysis of fatty acids T. brucei PCF and BSF OE and KD T. brucei PCF and BSF WT OE and KD Culture media rich in FBS High fat media +Tet +Tet -Tet -Tet Culture media low in FBS Low fat media +Tet +Tet -Tet -Tet 2. Our approach: looking for a phenotype x 4. Looking for a phenotype: polyunsaturated fatty acids production and growth curve PCF WT -TET PCF KD-D6 + TET PCF OE-D6 + TET High fat media Low fat media 20:4 20:4 20:3 20:3 20:2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20:2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20:2 22:6 22:5 22:4 0 1 2 5 10 15 FAME Relative Abundance (%) *** 20:4 20:4 20:3 20:3 20.2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20.2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20.2 22:6 22:5 22:4 0 1 5 10 15 FAME Relative Abundance (%) **** WT KD OE 0 20 40 60 80 100 22:4 --> 22:5 Conversion (%) 0 20 40 60 80 100 22:4 --> 22:5 Conversion (%) **** **** High fat media Low fat media 22:4 22:5 Δ6 High fat media Low fat media 20:4 20:4 20:3 20:3 20:2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20:2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20:2 22:6 22:5 22:4 0 1 2 5 10 15 FAME Relative Abundance (%) **** 20:4 20:4 20:3 20:3 20.2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20.2 22:6 22:5 22:4 20:4 20:4 20:3 20:3 20.2 22:6 22:5 22:4 0 1 2 5 10 15 FAME Relative Abundance (%) **** WT KD OE WT PCF -Tet KD-D6-PCF + TET OE-D6-PCF + TET High fat media Low fat media 0 20 40 60 80 100 22:4-->22:5- - >22:6 Conversion (%) * ** 0 20 40 60 80 100 22:4-->22:5- - >22:6 Conversion (%) ** **** 22:4 22:5 Δ6 22:6 Desaturase 0 20 40 60 0 1 × 10 6 2 × 10 6 3 × 10 6 4 × 10 6 TIME (h) Cell density (x10 4 ml - ) BSF WT +Tet BSF Δ6-KD +Tet BSF Δ6-OE +Tet BSF Δ6-KD + Tet + 22:6 BSF WT + Tet + 22:6 0 50 100 150 200 0 5 × 10 6 1 × 10 7 TIME (h) Cell density (x10 4 ml - ) PCF Δ6-KD + Tet PCF WT + Tet PCF WT + Tet + 22:6 PCF Δ6-KD + Tet + 22:6 PCF Δ6-OE + Tet back in SDM-79 with 10% FBS A B C Fig 3. Panel A and B show bar charts (top and middle) of the different polyunsaturated fatty acids (X axis) and the relative abundance (Y axis) found in T. brucei wild type (WT), overexpression (OE) and knockdown (KD) of ∆6-desaturase cultured in high fat (10% FBS )media (top) and low fat (5% FBS) media (middle) in presence of tetracycline. Panel A and B also show the conversion rate of the substrate (22:4) into product (22:5) for T. brucei PCF (panel A, bottom), and into product (22:6) for T. brucei BSF (panel B, bottom) by ∆6-desaturase. Panel C represents the growth rate of T. brucei wild type (WT), overexpression (OE) and knockdown (KD) of ∆6-desaturase cultured in low fat (5% FBS) media with (solid line) or without (dotted line) supplementation of 10 M of the product 22:6, respectively for PCF (top) and BSF (bottom). x 3. Looking for a phenotype: polyunsaturated fatty acids production and growth curve DIC DAPI HA tag MITOTRACKER DAPI/HA tag/-MITOTRACKER MERGED Fig 2. Immunofluorescence microscopy pictures of BSF T. brucei OE-Δ6. The HA- tag (green) and mitotracker (red) show that Δ6-desaturase (white arrows) is a mitochondrial associated (white arrows) enzyme. The same result was obtained in PCF T. brucei OE-Δ6 (not shown). 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm m/z (Da) 600 650 700 750 800 850 900 950 1000 m/z, Da 0.0 2.0e5 4.0e5 6.0e5 8.0e5 1.0e6 1.2e6 1.4e6 1.6e6 1.8e6 2.0e6 2.2e6 2.3e6 Intensity, cps 914.9 913.7 865.4 863.5 911.5 916.8 887.1 866.5 889.1 867.8 890.3 918.7 885.5 837.4 860.9 920.0 838.5 899.1 882.7 780.2 832.5 871.1 935.4 907.2 853.2 945.7 808.5 975.4 732.4 764.9 intensity (cps) 600 650 700 750 800 850 900 950 1000 m/z, Da 0.0 5.0e5 1.0e6 1.5e6 2.0e6 2.5e6 3.0e6 3.5e6 4.0e6 4.5e6 5.0e6 5.5e6 6.0e6 6.5e6 6.9e6 914.7 916.1 912.0 865.3 863.3 887.8 886.7 918.7 884.9 910.1 836.8 868.0 919.7 780.6 920.9 900.5 849.2 834.3 882.1 923.2 784.2 794.2 618.6 960.3 993.7 intensity (cps) m/z (Da) 5. Looking for a phenotype: inositolphosphoryl-ceramide (IPC) production in OE-∆6 in BSF T. brucei stumpy form control T. brucei OE-∆6 +Tet in 5%FBS T. brucei WT -Tet in 5%FBS DIC DIC DIC DAPI/PAD1 DAPI/PAD1 DAPI/PAD1 MERGED MERGED MERGED A B Fig 4. Panel A shows ESI-MS/MS spectra in negative mode for precursor of m/z 241 to detect IPC species (CE 60 eV) respectively in BSF T. brucei wild type (WT) (left) and overexpression (OE) (right) of ∆6-desaturase cultured in low fat media (5% FBS) for 48 h in presence of tetracycline. The red arrows highlight the surprisingly formation of IPC species when ∆6-desaturase is overexpressed, which is only normally observed in PCF or in the T. brucei stumpy form. Panel B shows immunofluorescence microscopy images of samples of the same cells, revealing the possible presence of the surface transporter for the differentiation signal, PAD1 (red, white arrows), in cells overexpressing ∆6-desaturase. 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm
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Exploring the activity and essentiality of theΔ-6 desaturase in Trypanosoma brucei
Michela Ceronea and Terry K Smitha
a School of Chemistry, BSRC, University of St Andrews, North Haugh, St Andrews, Fife, Scotland, UK
ReferenceNat. Rev. Mol. Cel. Bio., 1, 31-39 (2000);Nat.,459, 213-217(2009).
Acknowledgments I would like to thank Prof Terry K Smith for his support and supervision and all members of TKS group; Prof. Keith Matthews fromUniversity of Edinburgh for providing anti-PAD1 antibody; and EPSRC and CRITICAT CDT for funding.
x N"
C"
His"
His"
His"Fe2+"
Fe2+"
O
nΔ-6 T. brucei
His
His
HisFe
Fe
Outer Membrane
Inner Membrane
TM TM TM TM
H3CO
S
H3C S
O
2 Ferrocytochrome b5
2 Ferricytochrome b5
2 NADH
O2
2 NAD+
CoA
CoA
S
O
CoAH3C
S
O
CoAH3C
2 H2O
1. How does Δ6-desaturase from T. brucei work?
Fig 1. Schematic representation of Δ-6-desaturase trans-membrane enzyme and its reaction mechanism.Green indicates the ω-3 PUFAs biosynthetic pathway and orange the ω-6 PUFAs biosynthetic pathway.
x
Overexpression (OE) of Δ6-Desaturase
Knock down (KD) of Δ6-Desaturase
Δ6-DESATURASETetT7 HA tag
p2T77 or pLEW100vectors
Growth Curve
Immunofluorescence microscopy
ESI-MS analysis oflipids
GC-MS analysis of fatty acids
T. brucei PCF and BSFOE and KD
T. brucei PCF and BSF WT
OE and KD
Culture media rich in FBS
High fat media
+Tet
+Tet -Tet
-Tet
Culture media low in FBS
Low fat media
+Tet
+Tet -Tet
-Tet
2. Our approach: looking for a phenotype
x
4. Looking for a phenotype: polyunsaturated fatty acids production and growth curve
PCF WT -TET
PCF KD-D6 + TET
PCF OE-D6 + TET
High fat media
Low fat media
20:4
20:4
20:3
20:3
20:2
22:6
22:5
22:4
20:4
20:4
20:3
20:3
20:2
22:6
22:5
22:4
20:4
20:4
20:3
20:3
20:2
22:6
22:5
22:4
0
1
2
5
10
15
FAME
Rel
ativ
e A
bund
ance
(%)
***
20:4
20:4
20:3
20:3
20.2
22:6
22:5
22:4
20:4
20:4
20:3
20:3
20.2
22:6
22:5
22:4
20:4
20:4
20:3
20:3
20.2
22:6
22:5
22:4
0
1
5
10
15
FAME
Rel
ativ
e A
bund
ance
(%)
****
WT KD OE
0
20
40
60
80
100
22:4 --> 22:5
Con
vers
ion
(%)
0
20
40
60
80
100
22:4 --> 22:5
Con
vers
ion
(%)
****
**** PCF WT -TET
PCF KD-D6 + TET
PCF OE-D6 + TET
High fat media Low fat media
22:4 22:5 Δ6
High fat media
Low fat media20
:420
:420
:320
:320
:222
:622
:522
:420
:420
:420
:320
:320
:222
:622
:522
:420
:420
:420
:320
:320
:222
:622
:522
:40
1
2
5
10
15
FAME
Rela
tive
Abun
danc
e (%
) ****
20:4
20:4
20:3
20:3
20.2
22:6
22:5
22:4
20:4
20:4
20:3
20:3
20.2
22:6
22:5
22:4
20:4
20:4
20:3
20:3
20.2
22:6
22:5
22:4
0
1
2
5
10
15
FAME
Rela
tive
Abun
danc
e (%
)
****WT KD OE
WT PCF -TetKD-D6-PCF + TET OE-D6-PCF + TET
High fat media Low fat media
0
20
40
60
80
100
22:4-->22:5- - >22:6
Conv
ersi
on (%
)
*
**
0
20
40
60
80
100
22:4-->22:5- - >22:6
Conv
ersi
on (%
)
BSF WT -Tet Control
BSF KD-D6 + TET
BSF OE-D6 + TET
**
****
22:4 22:5 Δ6 22:6Desaturase
0 20 40 600
1×106
2×106
3×106
4×106
TIME (h)
Cell
dens
ity (x
104
ml- )
BSF WT +Tet BSF Δ6-KD +Tet BSF Δ6-OE +Tet BSF Δ6-KD + Tet + 22:6 BSF WT + Tet + 22:6
0 50 100 150 2000
5×106
1×107
TIME (h)
Cell
dens
ity (x
104
ml- )
PCF Δ6-KD + Tet PCF WT + Tet
PCF WT + Tet + 22:6PCF Δ6-KD + Tet + 22:6
PCF Δ6-OE + Tet
back in SDM-79with 10% FBS
A B C
Fig 3. Panel A and B show bar charts (top and middle) of the different polyunsaturated fatty acids (X axis) and the relative abundance (Y axis) found in T. brucei wild type (WT), overexpression (OE) and knockdown (KD) of ∆6-desaturasecultured in high fat (10% FBS )media (top) and low fat (5% FBS) media (middle) in presence of tetracycline. Panel A and B also show the conversion rate of the substrate (22:4) into product (22:5) for T. brucei PCF (panel A, bottom), andinto product (22:6) for T. brucei BSF (panel B, bottom) by ∆6-desaturase. Panel C represents the growth rate of T. brucei wild type (WT), overexpression (OE) and knockdown (KD) of ∆6-desaturase cultured in low fat (5% FBS) mediawith (solid line) or without (dotted line) supplementation of 10 𝝁M of the product 22:6, respectively for PCF (top) and BSF (bottom).
x
3. Looking for a phenotype: polyunsaturated fatty acids production and growth curve DIC DAPI HA tag MITOTRACKER DAPI/HA tag/-MITOTRACKER MERGED
Fig 2. Immunofluorescence microscopypictures of BSF T. brucei OE-Δ6. The HA-tag (green) and mitotracker (red) showthat Δ6-desaturase (white arrows) is amitochondrial associated (white arrows)enzyme. The same result was obtained inPCF T. brucei OE-Δ6 (not shown).
10 μm10 μm10 μm10 μm10 μm10 μm
x
m/z (Da)
-Prec (241.00): 296 MCA scans from Sample 1 (MICHELA_090321) of BSF-WT-5FBS-241.wiff (Nanospray), Smoothed Max. 2.3e6 cps.
600 650 700 750 800 850 900 950 1000m/z, Da
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e62.3e6
Intensity, cps
914.9
913.7
865.4
863.5
911.5916.8887.1
866.5
889.1
867.8 890.3
918.7885.5837.4860.9 920.0838.5 899.1882.7780.2 832.5 871.1 935.4907.2853.2 945.7808.5 975.4732.4 764.9
inte
nsity
(cps
)
-Prec (241.00): 296 MCA scans from Sample 103 (OE-5FBS-TET-PICTURE-241) of MC-MAY21.wiff (Nanospray) Max. 6.9e6 cps.
600 650 700 750 800 850 900 950 1000m/z, Da
0.0
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e6
6.0e6
6.5e66.9e6
Intensity, cps
914.7
916.1
912.0
865.3
863.3887.8
886.7
918.7884.9 910.1836.8 868.0 919.7780.6
920.9900.5849.2834.3 882.1 923.2784.2 794.2618.6 960.3 993.7
inte
nsity
(cps
)
m/z (Da)
5. Looking for a phenotype: inositolphosphoryl-ceramide (IPC) production in OE-∆6 in BSF
T. b
ruce
ist
umpy
form
cont
rol
T. b
ruce
iO
E-∆6
+Te
tin
5%
FBS
T. b
ruce
iW
T -T
etin
5%
FBS
DIC
DIC
DIC
DAPI/PAD1
DAPI/PAD1
DAPI/PAD1
MERGED
MERGED
MERGED
A B
Fig 4. Panel A shows ESI-MS/MS spectra in negative mode for precursor of m/z 241 to detect IPC species (CE 60 eV) respectively in BSF T. brucei wild type (WT) (left) and overexpression (OE) (right) of ∆6-desaturase cultured in low fatmedia (5% FBS) for 48 h in presence of tetracycline. The red arrows highlight the surprisingly formation of IPC species when ∆6-desaturase is overexpressed, which is only normally observed in PCF or in the T. brucei stumpy form. PanelB shows immunofluorescence microscopy images of samples of the same cells, revealing the possible presence of the surface transporter for the differentiation signal, PAD1 (red, white arrows), in cells overexpressing ∆6-desaturase.
10 μm 10 μm 10 μm
10 μm 10 μm10 μm
10 μm10 μm10 μm
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