Imino sugars and perturbation of protein folding pathways in the ER Dom Alonzi.
Transcript of Imino sugars and perturbation of protein folding pathways in the ER Dom Alonzi.
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Imino sugars and perturbation of protein folding pathways
in the ER
Dom Alonzi
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Imino Sugars
N CH2OH
OH
OHHO
Therapeutic roles
Lysosomal storage disorders:Gaucher, Sandhoff
Antiviral: HCV, HBV
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ER
Golgi
Further GolgiProcessing toComplex-type
+
Cytoplasm
Proteosome
-Glc I & IICalnexin/
Calreticulin
+
Sec 61
Ceramideglucosyltransferase
Plasmamembrane
< 1 min
Chitobiase
N-alkyl-DNJs
N-alkyl-DNJs
N-alkyl-DNJs
N-alkyl-DNJs
N-alkyl-DNJs
Ceramide Glucosylceramide
Endoman’ase
GSLs
The effect of imino sugars on N-linked oligosaccharide processing in the cell
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Free oligosaccharide (FOS) generation - a normal cellular process
Unconjugated polymannose-type oligosaccharides are, in the main, produced as a by-product of the proteasomal degradation of misfolded glycoproteins.
A smaller proportion are produced during the dolichol pathway of N-linked glycosylation. These are segregated from their glycoprotein-linked counterparts and follow a non-vesicular trafficking pathway that leads to their degradation in the lysosome.
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-glucosidase I & II
ATPATP
-glucosidase II
Calnexin/calreticulin
Glucosyl transferase
Post ER modification
-SH
HS-
-s-s- -s-s-
HS-ERp57
-s-s-
NB-DNJ
NB-DNJ
Disruption of glycoprotein folding in the ER
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ER lumen
Cytosol PNGase
Chitobiase
Cytosolic Mannosidase
Proteasome
Misfolded glycoprotein
?
Sec61/Der-1
Free oligosaccharides produced by glycoprotein misfolding
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FOS is produced following treatment of HL60 cells with 1mM NB-DNJ for 24hrs
M4N
M5N
G1M5N
G3M5N
G3M6-9N1/2
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00
Control
1mM NB-DNJ 24hrs
G1M5NM5N
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Build up of Mono-glucosylated FOS is time dependent
0 5 10 15 200
10
20
30
40
50
1mM Butyl-DNJ
Time (hours)
[G1M
5N]
rela
tive
to
[M5N
]
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Build up of Tri-glucosylated FOS is time dependent
0 5 10 15 200
25
50
75
100
1mM Butyl-DNJ
Time (hours)
[G3M
5N]
rela
tive
to
[M5N
]
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G3M5N build up is Butyl-DNJ concentration dependent
0 250 500 750 10000
25
50
75
100Butyl-DNJ
Inhibitor concentration (µM)
[G3M
5N]
rela
tive
to
[M5N
]
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N CH2OH
OH
OHHO
N CH2OH
OH
OHHO
Butyl-DNJ Vs Nonyl-DNJ
Effect of chain length on glucosidase inhibition in the ER. Is it the result of differential inhibition of the enzymes or an access/localisation issue?
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+
-glucosidase I
Potential Inhibitor
2AA 2AA2AA
+
100% 75% 25%
t= 30mins
In Vitro Glucosidase inhibition assay
40.00Minutes
30.00 31.00 32.00 33.00 34.00 35.00 36.00 37.00 38.00 39.00
G3M5N
G3M5N + -glucosidase I
G3M5N + -glucosidase I + 5M NN-DNJ
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In vitro IC50 determination
IC50 NN-DNJ= 0.54M and NB-DNJ = 0.83M
0 5 10 15 20 250
25
50
75
100
Butyl-DNJ
Nonyl-DNJ
Inhibitor concentration (µM)0.01 0.1 1 10 1000
25
50
75
100
Butyl-DNJ
Nonyl-DNJ
Log Inhibitor concentration (µM)
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FOS production: Nonyl-DNJ vs Butyl-DNJ
0 10 20 30 40 50 60 70 80 90 1000
10
20
30
40
50
60
70
80
90
Nonyl-DNJ
Butyl-DNJ
Inhibitor concentration (µM)
[G3M
5N]
rela
tive
to
[M5N
]
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NB-DNJ and NN-DNJ treatment of MDBK cells shows differential inhibition of glucosidases
Control
100µM NB-DNJ 72hrs
100µM NN-DNJ 72hrs
G3M5N G3M7N2
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00
Differential inhibition
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Minutes
20.00 24.00 28.00 32.00 36.00 40.00 44.00
Type of FOS produced on treatment with NB-DNJ is Endomannosidase dependent
Control
Functional Endomannosidase
Non-utilised Endomannosidase
G3M5N G3M7N2
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ER ERGIC
cis-Golgi
+
The Endomannosidase pathway
Bulk flow or ERGIC-53 mediated
Retrograde transport
Cytosolic Mannosidase
Endomannosidase
Golgi Mannosidase I
Further Golgi processing to Complex N-links
?
PNGase in the ER or Cytosol or both?
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Fate of FOS following long term 1mM NB-DNJ treatment of HL60 cells
Control
1mM NB-DNJ 24hrs
1mM NB-DNJ 72hrs
1mM NB-DNJ 48hrs
G3M5NG3M4N
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00
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G3M5N is converted to G3M4N in long term recovery experiments in HL60s
Control
1mM NB-DNJ 24hrs
1mM NB-DNJ 24hrs + 72hrs recovery
1mM NB-DNJ 24hrs + 96hrs recovery
1mM NB-DNJ 24hrs + 120hrs recovery
G3M5NG3M4N
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00
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Where?
Enzyme?
Fate of Glucosylated Man5 FOS?
Most likely theory: Cytosolic Mannosidase Glucosylated FOS cannot gain access to the lysosome {Saint-Pol A et al JBC (1999)}
lysosome
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Mouse: G1M4N in serum is dependent on NB-DNJ dose
Control
300mg/kg/day
1200mg/kg/day
G1M4N
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00
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G1M4N build up in serum following NB-DNJ treatment
0.0 2.5 5.0 7.5 10.0 12.5 15.00
25
50
75
100
2400mg/kg/day
days treated with 2400mg/kg/day
[G1M
4N]
rela
tive
to
[M3N
]
(Serum concentration ~ 40M)
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Minutes
22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.0020.00
Control
Treated
[Serum] = 40.8M
G1M4N is excreted in mouse urine following NB-DNJ treatment
G1-3M7N2
G1M4N
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FOS detected in serum from NPC patient treated with 100mg/day NB-DNJ
Untreated (day 0)
Treated (16 months)
G3M4N
G1M4N
Minutes
20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00
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Lysosome
ERGolgi
PNGaseDer-1/Sec61
Proteasome
Endomannosidase
Chitobiase
Cytosolic mannosidase
-glucosidase I & II
?
Retrograde transport
Pathway for FOS clearance
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ConclusionsCharacterised cellular FOS produced in the presence/absence of glucosidase inhibitors
Demonstrated differential inhibition of glucosidases I and II by imino sugars
FOS as biological markers - provide a simple cell based assay for glucosidase inhibitor efficacy and entry into the ER, and calnexin/calreticulin mediated protein folding
Formation of glucosylated Man4 species in cells, as a result of the cytosolic mannosidase, is the metabolic endpoint
Glucosylated FOS, in serum and urine of mouse and human following NB-DNJ treatment, reveals pathway of clearance
Detection of two PNGases in the cell: (i) ER lumen and (ii) cytosolic
Trans-renal clearance of glucosylated FOS prevents adverse effects on normal cellular processes
Endomannosidase influences the FOS pathway due to differential activities on protein folding states
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Acknowledgements
Dr David Neville
Neidys Sanchez-Hernandez
Dr Howard Mellor
Mark Hussey
Adam Pilling
Dr Terry Butters
Professor Raymond Dwek
1st floor