Abstract Introduction Understanding the Role of Lipid Modifications in ER Protein Quality Control Melissa Roberts 1,2,3 , Clark Peterson 2 , Milton To 2 , James A.Olzmann 2 Methods and Results Figure 6. Identification of palmitoylated ERAD proteins. A-F) Affinity purified proteins from cells incubated in the presence or absence of 17-ODYA were reacted with a rhodamine moiety to enable detection of palmitoylation. Complexes were also incubated with hydroxylamine (HAM) to remove the thioester-linked palmitate group and confirm specificity of the signal. Proteins were separated by SDS-PAGE and gels scanned for fluorescence to detect the reporter or analyzed by Western blotting to detect the affinity purified protein. DFP is a non-fluorescent GFP variant used as a negative control, and calnexin is used as a positive control. Figure 9. The palmitoylation inhibitor 2-BP impairs CD147 degradation. A) Cells were pretreated for 16hr with 2-bromopalmitate (2-BP) and the degradation kinetics of CD147 analyzed by immunoblotting. B) CD147 levels were quantified using ImageJ and plotted. 1 Diablo Valley College, 2 Dept. of Nutritional Sciences & Toxicology, University of California, Berkeley 3 2015 Transfer-to-Excellence Research Experiences for Undergraduates Program (TTE REU Program) Anti-S 75 17-ODYA-rhodamine Hrd1-S - HAM + - + - 17-ODYA - + + A C D B 37 37 Anti-HA 100 75 100 75 17-ODYA-rhodamine Anti-S 17-ODYA-rhodamine Calnexin-HA - HAM + - + AUP1-S - 17-ODYA HAM 17-ODYA - + + - + - - + + Anti-S 25 25 17-ODYA-rhodamine DFP-S - HAM + - + - 17-ODYA - + + A B 75 0 vehicle 1 3 6 CD147 (mat.) CD147 (CG) tubulin 50 50 37 0 emetine (hr.) 2-BP 1 3 6 0 1 2 3 5 4 vehicle 2-BP 120 100 80 60 40 20 0 % CD147 (CG) remaining emetine chase (hr.) Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cellular process responsible for the identification and degradation of misfolded proteins. Defining the mechanisms underlying this process is vital to understanding the pathogenesis of numerous human diseases that result from impaired ER protein quality control. Recent reports indicate that the inhibition of acyl-CoA synthetases with small molecule, triacsin c disrupts ERAD. However, it remains unknown why acyl-CoA synthetases are required for ERAD. Palmitoylation, the covalent addition of the fatty acid palmitate to a protein, requires acyl-CoA synthetase activity and can significantly impact protein localization, structure, and physical interactions. Therefore, we hypothesized that palmitoylation of ERAD machinery regulates the identification and degradation of ERAD substrates. To test this hypothesis, we employed copper-catalyzed azide-alkyne cycloaddition to probe for palmitoylated ERAD proteins. Our results reveal palmitoylation of four prominent ERAD proteins: the E2-recruitment factor AUP1, the E3 ligase Hrd1, the rhomboid pseudoprotease Derlin1, and the mannosidase ERMan1, indicating that palmitoylation may regulate ERAD at multiple steps. Consistent with a functional role for palmitoylation in ERAD, we find that treatment the palmitoylation inhibitor 2-bromopalmitate significantly attenuated ERAD. Together, our results identify an unprecedented mechanism of ERAD regulation that will broadly impact our understanding of ERAD-associated diseases. Inhibition of long chain acyl-CoA synthetases impairs the glycan trimming step of ERAD ERMan1, AUP1, Hrd1, and Derlin1 are palmitoylated ERAD proteins Palmitoylation is required for efficient ERAD 75 Anti-S 75 17-ODYA-rhodamine ERMan1-S - HAM + - + - 17-ODYA - + + F 75 Conclusions Acknowledgements Figure 2. The long chain acyl-CoA synthetase inhibitor triacsin c impairs CD147 degradation. A) CD147 degradation kinetics were analyzed by immunoblotting following a 16-hr triacsin c pretreatment . B) CD147 levels in panel (A) were quantified using ImageJ and plotted. palmitate ACSL palmitoyl-acyl transferase palmitoyl-CoA CoA triacsin c 2-BP substrate ER LUMEN CYTOPLASM p97/ VCP ubiquitin AUP1 Hrd1 E2 ERMan1 ERMan1-trimmed glycan palmitate 26S Metabolic labeling 17-ODYA Lysis, click chemistry O HO O S O N N N protein S protein Reporter 120 75 0 vehicle 1 3 6 CD147 (mat.) CD147 (CG) tubulin 50 50 37 100 80 60 40 20 0 0 g n i n i a m e r ) G C ( 7 4 1 D C % 1 2 3 emetine chase (hr.) 6 5 4 vehicle triacsin c 0 emetine (hr.) triacsin c 1 3 6 tubulin 0 37 50 1 3 6 vehicle 0 1 3 6 triacsin c 0 1 3 6 kifunensine CD147 (CG,untrimmed) CD147 (Mat) CD147 (CG,trimmed) Figure 3. Triacsin c impairs glycan trimming. Immunoblot analysis of CD147 degradation in cells pretreated for 16 hr. with triacsin c or cotreated with the mannosidase inhibitor kifunensine. Fatty acid modification of ERAD factors regulates ER protein quality control. Thank you very much to my research mentor Clark Peterson, Principal Investigator Dr. James Olzmann, and the Olzmann Lab. Thank you to the Transfer-to-Excellence Research Experiences for Undergraduates program staff and Synberc. Support Information This work was funded by National Science Foundation Award ECCS-0939514 & ECCS-1157089 & ECCS-1461157 and National Institutes of Health Awards GM112948 and DK095921. A B Figure 1. Representation of Endoplasmic Reticulum-Associated Degradation (ERAD). The four major steps of ERAD are: recognition of misfolded proteins’ trimmed glycans by ER resident sugar-binding lectins and chaperones; substrate dislocation across the ER lipid bilayer presumably through a proteinaceous pore; polyubiq- uitination by E3 ubiquitin ligases; and degradation by the 26S proteasome in the cytoplasm. Future Directions Continue to probe for palmitoylation of ERAD proteins Elucidate the role of palmitoylation in ERAD Create mutated, palmitoylation-defective forms of ERMan1, AUP1, Hrd1, and Derlin1 and track degradation of CD147 Use fluorescence microscopy to localize palmitoylated and mutated, palmitoylation- defective ERAD proteins Analyze the protein complexes formed by palmitoylation-defective mutants Anti-S 17-ODYA-rhodamine 75 WT C4A + + + + C71A C96A ERMan1-S WT - + C556A + C582R 75 IB: anti-S vehicle triacsin c 2-BP ERMan1-S (palmitoylated) ERMan1-S + - - - - - - + + 75 75 ERMan1-S 17-ODYA-rhodamine 0 control triacsin c 2BP 20 40 80 60 100 120 Figure 7. ERMan1 is palmitoylated at cysteine-71. A) ERMan1 domain structure. Cysteine residues are indicated. B) ERMan1-S WT and cysteine mutants were affinity purified from cells incubated with 17-ODYA and palmitoylation was detected using click chemistry. Asterisk indicates nonspecific band. Figure 8. ERMan1 palmitoylation is impaired by triacsin c and 2-BP treatment. A) Illustration of our hypothesis that triacsin and 2-BP inhibit ERMan1 palmitoylation. B) ERMan1-S was affinity purified from cells incubated with 17-ODYA and the indicated inhibitors. Palmitoylation was detected by reaction wtih a rhodamine moiety. C) The levels of palmitoylated ERMan1 in panel (B) were quantified using ImageJ and plotted. Palmitoylated ERMan1 (% vehicle treated) RECOGNITION CYTOPLASM DISLOCATION UBIQUITINATION DEGRADATION E3 E3 VCP/p97 VCP/p97 recruitment factor adaptor adaptor dislocon substrate ubiquitin lectin chaperone E3 E3 ER LUMEN glycan disulfide bond - + + Figure 5. Schematic of the click chemistry method used to detect palmitoylation. A) Cells were pretreated for 8 hr with 17-octadecynoic acid (17-ODYA), an alkyne-containing palmitate analog. B) Affinity purified S-tagged proteins were reacted with the fluorescent azide-rhodamine reporter via copper-catalyzed azide-alkyne cycloaddition. emetine (hr) ERMan1 is palmitoylated at cysteine-71 glycerol-3-P LPA PA fatty acid ACSL acyl CoA CoA DAG phospholipids TAG triacsin c lipid droplet formation protein acylation β-oxidation Figure 4. Schematic of the pathways affected by triacsin c. Triacsin c inhibits long chain acyl-CoA synthetases (ACSLs), which are required for the activation of fatty acids that are employed for protein acylation, β-oxidation, and lipid biosynthesis. E 25 25 Anti-S 17-ODYA-rhodamine Derlin1-S HAM 17-ODYA - + - - + - + + H + [CuL ] n + CuL n-1 N N N + - CuL n-2 - N N + N N N N CuL n-2 CuL n-2 N N N H + N N N A B palmitate ACSL palmitoyl-CoA CoA triacsin c palmitoyl-acyl transferase 2-BP palmitoylated ERMan1 ERMan1 Contact Information Melissa Roberts Email: [email protected] A B A B C 6 Create ERMan1, AUP1, Hrd1, and Derlin1 knockout cell lines using CRISPR/Cas9 to verify ERAD defect and for use in future degradation rescue studies protein protein Reporter Reporter Reporter Reporter Reporter protein protein protein protein Derlin-1 C4 C71 C96 C527 C556 C582 ERMan1 0 100 200 300 400 500 600 TM Cytosol Lumenal catalytic domain 700 * * 50 26S
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Abstract
Introduction
Understanding the Role of Lipid Modifications in ER Protein Quality Control
Melissa Roberts1,2,3, Clark Peterson2, Milton To2, James A.Olzmann2
Methods and Results
Figure 6. Identification of palmitoylated ERAD proteins. A-F) Affinity purified proteins from cells incubated in the presence or absence of 17-ODYA were reacted with a rhodamine moiety to enable detection of palmitoylation. Complexes were also incubated with hydroxylamine (HAM) to remove the thioester-linked palmitate group and confirm specificity of the signal. Proteins were separated by SDS-PAGE and gels scanned for fluorescence to detect the reporter or analyzed by Western blotting to detect the affinity purified protein. DFP is a non-fluorescent GFP variant used as a negative control, and calnexin is used as a positive control.
Figure 9. The palmitoylation inhibitor 2-BP impairs CD147 degradation. A) Cells were pretreated for 16hr with 2-bromopalmitate (2-BP) and the degradation kinetics of CD147 analyzed by immunoblotting. B) CD147 levels were quantified using ImageJ and plotted.
1Diablo Valley College, 2Dept. of Nutritional Sciences & Toxicology, University of California, Berkeley32015 Transfer-to-Excellence Research Experiences for Undergraduates Program (TTE REU Program)
Anti-S75
17-ODYA-rhodamine
Hrd1-S
-HAM + - +-17-ODYA - + +
A C
D
B
37
37
Anti-HA
10075
10075
17-ODYA-rhodamine
Anti-S
17-ODYA-rhodamine
Calnexin-HA
-HAM + - +
AUP1-S-17-ODYA
HAM17-ODYA- + +
- +- -
+
+
Anti-S
25
25
17-ODYA-rhodamine
DFP-S
-HAM + - +-17-ODYA - + +
A B
750
vehicle
1 3 6
CD147 (mat.)
CD147 (CG)
tubulin
50
50
37
0 emetine (hr.)
2-BP
1 3 6
0 1 2 3 54
vehicle2-BP
120
100
80
60
40
20
0
% C
D14
7 (C
G) r
emai
ning
emetine chase (hr.)
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cellular process responsible for the identification and degradation of misfolded proteins. Defining the mechanisms underlying this process is vital to understanding the pathogenesis of numerous human diseases that result from impaired ER protein quality control. Recent reports indicate that the inhibition of acyl-CoA synthetases with small molecule, triacsin c disrupts ERAD. However, it remains unknown why acyl-CoA synthetases are required for ERAD. Palmitoylation, the covalent addition of the fatty acid palmitate to a protein, requires acyl-CoA synthetase activity and can significantly impact protein localization, structure, and physical interactions. Therefore, we hypothesized that palmitoylation of ERAD machinery regulates the identification and degradation of ERAD substrates. To test this hypothesis, we employed copper-catalyzed azide-alkyne cycloaddition to probe for palmitoylated ERAD proteins. Our results reveal palmitoylation of four prominent ERAD proteins: the E2-recruitment factor AUP1, the E3 ligase Hrd1, the rhomboid pseudoprotease Derlin1, and the mannosidase ERMan1, indicating that palmitoylation may regulate ERAD at multiple steps. Consistent with a functional role for palmitoylation in ERAD, we find that treatment the palmitoylation inhibitor 2-bromopalmitate significantly attenuated ERAD. Together, our results identify an unprecedented mechanism of ERAD regulation that will broadly impact our understanding of ERAD-associated diseases.
Inhibition of long chain acyl-CoA synthetases impairs the glycan trimming step of ERAD
ERMan1, AUP1, Hrd1, and Derlin1 are palmitoylated ERAD proteins
Palmitoylation is required for efficient ERAD
75
Anti-S75
17-ODYA-rhodamine
ERMan1-S
-HAM + - +-17-ODYA - + +
F
75
Conclusions
Acknowledgements
Figure 2. The long chain acyl-CoA synthetase inhibitor triacsin c impairs CD147 degradation. A) CD147 degradation kinetics were analyzed by immunoblotting following a 16-hr triacsin c pretreatment . B) CD147 levels in panel (A) were quantified using ImageJ and plotted.
palmitateACSL
palmitoyl-acyltransferase
palmitoyl-CoACoA
triacsin c
2-BPsubstrate
ER LUMEN
CYTOPLASM
p97/VCP
ubiquitinAUP1
Hrd1
E2
ERMan1
ERMan1-trimmedglycan palmitate
26S
Metaboliclabeling
17-ODYA
Lysis,click chemistry
O
HO
O
S
O N
NN
prot
ein
S
prot
ein
Reporter
120
750
vehicle
1 3 6
CD147 (mat.)
CD147 (CG)
tubulin
50
50
37
100
80
60
40
20
00
gniniamer )
GC( 741
DC
%
1 2 3emetine chase (hr.)
654
vehicletriacsin c
0 emetine (hr.)
triacsin c
1 3 6
tubulin
0
37
50
1 3 6vehicle
0 1 3 6triacsin c
0 1 3 6kifunensine
CD147 (CG,untrimmed)
CD147 (Mat)
CD147 (CG,trimmed)
Figure 3. Triacsin c impairs glycan trimming. Immunoblot analysis of CD147 degradation in cells pretreated for 16 hr. with triacsin c or cotreated with the mannosidase inhibitor kifunensine.
Fatty acid modification of ERAD factors regulates ER protein quality control.
Thank you very much to my research mentor Clark Peterson, Principal Investigator Dr. James Olzmann, and the Olzmann Lab. Thank you to the Transfer-to-Excellence Research Experiences for Undergraduates program staff and Synberc.
Support InformationThis work was funded by National Science Foundation Award ECCS-0939514 & ECCS-1157089 & ECCS-1461157 and National Institutes of Health Awards GM112948 and DK095921.
A B
Figure 1. Representation of Endoplasmic Reticulum-AssociatedDegradation (ERAD). The four major steps of ERAD are: recognition of misfoldedproteins’ trimmed glycans by ER resident sugar-binding lectins and chaperones; substrate dislocation across the ER lipid bilayer presumably through a proteinaceous pore; polyubiq-uitination by E3 ubiquitin ligases; and degradation by the 26S proteasome in the cytoplasm.
Future DirectionsContinue to probe for palmitoylation of ERAD proteins
Elucidate the role of palmitoylation in ERAD
Create mutated, palmitoylation-defective forms of ERMan1, AUP1, Hrd1, and Derlin1and track degradation of CD147
Use fluorescence microscopy to localize palmitoylated and mutated, palmitoylation-defective ERAD proteinsAnalyze the protein complexes formed by palmitoylation-defective mutants
Anti-S
17-ODYA-rhodamine
75
WT
C4A
+ + + +
C71A
C96A
ERMan1-S
WT
- +
C556
A
+
C582
R
75
IB: anti-S
vehicletriacsin c
2-BP
ERMan1-S(palmitoylated)
ERMan1-S
+ -
-
---
-+
+
75
75
ERMan1-S
17-ODYA-rhodamine 0
contr
oltria
csin
c2B
P
20
40
80
60
100
120
Figure 7. ERMan1 is palmitoylated at cysteine-71. A) ERMan1 domain structure. Cysteine residues are indicated. B) ERMan1-S WT and cysteine mutants were affinity purified from cells incubated with 17-ODYA and palmitoylation was detected using click chemistry. Asterisk indicates nonspecific band.
Figure 8. ERMan1 palmitoylation is impaired by triacsin c and 2-BP treatment. A) Illustration of our hypothesis that triacsin and 2-BP inhibit ERMan1 palmitoylation. B) ERMan1-S was affinity purified from cells incubated with 17-ODYA and the indicated inhibitors. Palmitoylation was detected by reaction wtih a rhodamine moiety. C) The levels of palmitoylated ERMan1 in panel (B) were quantified using ImageJ and plotted.
Pal
mito
ylat
ed E
RM
an1
(% v
ehic
le tr
eate
d)
RECOGNITION
CYTOPLASM
DISLOCATION UBIQUITINATION DEGRADATION
E3E3
VCP/p97 VCP/p97recruitment
factor
adaptor
adaptor
dislocon
substrate
ubiquitin
lectin
chaperone
E3 E3
ER LUMEN
glycan
disulfide bond
-+ +
Figure 5. Schematic of the click chemistry method used to detect palmitoylation. A) Cells werepretreated for 8 hr with 17-octadecynoic acid (17-ODYA), an alkyne-containing palmitate analog. B) Affinity purified S-taggedproteins were reacted with the fluorescent azide-rhodamine reporter via copper-catalyzed azide-alkyne cycloaddition.
emetine (hr)
ERMan1 is palmitoylated at cysteine-71
glycerol-3-P
LPA
PAfatty acidACSL
acyl CoACoA
DAG phospholipids
TAG
triacsin c
lipid dropletformation
proteinacylation
β-oxidation
Figure 4. Schematic of the pathways affected by triacsin c. Triacsin c inhibits long chain acyl-CoA synthetases (ACSLs), which are required for the activation of fatty acids that are employed for protein acylation, β-oxidation, and lipid biosynthesis.
E
25
25
Anti-S
17-ODYA-rhodamine
Derlin1-S
HAM17-ODYA
- +- -
+-+ +
H+
[CuL ]n+
CuL n-1
NN N+ -
CuL n-2
-
NN
+NNN
NCuL n-2
CuL n-2
NN
N
H+
NN
N
A B
palmitateACSL
palmitoyl-CoACoA
triacsin c
palmitoyl-acyltransferase
2-BPpalmitoylated
ERMan1
ERMan1
Contact InformationMelissa RobertsEmail: [email protected]
A
B
A
B C
6
Create ERMan1, AUP1, Hrd1, and Derlin1 knockout cell lines using CRISPR/Cas9 to verify ERAD defect and for use in future degradation rescue studies
protein
protein
Reporter
Reporter
Reporter
Reporter
Reporter
protein
protein
protein
protein
Derlin-1
C4 C71
C96
C527
C556
C582
ERMan10 100 200 300 400 500 600
TM
Cytosol Lumenalcatalytic domain
700
* *
50
26S
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