Articles in PresS. Am J Physiol Endocrinol Metab (October 14, 2014). doi:10.1152/ajpendo.00125.2014
1 2 3 4
Regulation of FSP27 protein stability by AMPK and HSC70
5 6 7
Xiaodong Zhang1,2, Bradlee L. Heckmann1,2,4, Xitao Xie1,2, Alicia M. Saarinen1,2
8
and Jun Liu1,2,3,#
9 10 11 12 13
From Department of Biochemistry and Molecular Biology1, HEALth Program2, Division of Endocrinology3, Mayo Clinic in Arizona, Scottsdale, Arizona 85259, USA; Mayo Graduate School4, Rochester, Minnesota 55905, USA.
14 15 16 17 18 19
#To whom correspondence should be addressed: Jun Liu, MCCRB 3-054, 13400 E. Shea Boulevard, Scottsdale, AZ 85259. Phone: 480-301-6745; Fax: 480-301-8387; E-mail: [email protected]
20 21 22 23 24 25
Copyright © 2014 by the American Physiological Society.
26 27
ABSTRACT
28 29
Fat specific protein 27 (FSP27) plays a pivotal role in controlling the formation of large
30
lipid droplet and energy metabolism. The cellular levels of FSP27 are tightly regulated
31
through the proteasomal ubiquitin-mediated degradation. However, the upstream signals
32
that trigger FSP27 degradation and the underlying mechanism(s) have yet to be identified.
33
Here we show that AMP-activated protein kinase (AMPK) activation by AICAR (5-
34
amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide) or phenformin induced the
35
ubiquitination of FSP27 and promoted its degradation in 3T3-L1 adipocytes. The levels
36
of FSP27 protein could be maintained by either knocking down AMPKα1 or blocking
37
proteasomal pathway. Moreover, AICAR treatment induced multilocularization of LDs in
38
3T3-L1 adipocytes, reminiscent of the morphological changes in cells depleted of FSP27.
39
Furthermore, mass spectrometry-based proteomic analysis identified heat shock cognate
40
70 (HSC70) as a novel binding protein of FSP27. The specific interaction was confirmed
41
by co-immunoprecipitation of both ectopically expressed and endogenous proteins.
42
Importantly, knockdown of HSC70 by small interference RNA resulted in increased half-
43
life of FSP27 in cells treated with a protein synthesis inhibitor cycloheximide (CHX) or
44
AICAR. However, silencing of the E3 ubiquitin ligase CHIP (C terminus of HSC70-
45
Interacting Protein) failed to alter the stability of FSP27 protein under both conditions.
46
Taken together, our data indicate that AMPK is a negative regulator of FSP27 stability
47
through the proteasomal ubiquitin-dependent protein catabolic process. Promotion of
48
FSP27 degradation may be an important factor responsible for the beneficial effect of
49
AMPK activators on energy metabolism. 2
50 51
INTRODUCTION
52 53
Adipose tissue plays a central role in the development of obesity, which occurs when
54
energy intake exceeds its expenditure. There are two types of adipose tissue, white
55
adipose tissue (WAT) for energy storage and brown adipose tissue (BAT) for energy
56
consumption to support heat production (9). White adipocytes, characterized by a large
57
unilocular lipid droplet (LD), are responsible for the intracellular storage of triglycerides
58
(TGs) in the fed state and provision of fatty acids as an energy substrate via lipolysis for
59
other tissues in response to fasting. Brown adipocytes, on the other hand, contain
60
numerous smaller LDs along with a large number of mitochondria and high oxidative
61
activity. Enlargement of LDs or adipocytes in WAT contributes to the development of
62
obesity and related insulin resistance (10, 13, 32). Thus, the induction of fat-burning
63
brown adipocyte-like capacity in white adipocytes may represent a potential therapeutic
64
strategy to combat obesity and its related disorders.
65 66
Recent studies have implicated AMPK as a potential target for inducing "BAT-like
67
phenotype" in WAT (6). For example, it was recently demonstrated that WAT
68
metabolism was remodeled toward energy dissipation through AICAR (5-amino-1-β-D-
69
ribofuranosyl-imidazole-4-carboxamide)-induced AMPK activation (7, 38). Specifically,
70
prolonged AMPK activation by AICAR caused increases in expression of PPAR-γ,
71
PPARα, and PPARδ in isolated rat epididymal adipocytes. Higher expression of PGC-1α
72
and carnitinepalmitoyl transferase-1b (CPT-1b) was accompanied by an increase in FA
3
73
oxidation in these cells. Moreover, a separate study indicated that mice injected with
74
AICAR showed increased UCP-1 expression and induced accumulation of brown
75
adipocytes within the WAT (40). However, the molecular mechanisms underlying the
76
effect of AMPK activation on the WAT plasticity remained incompletely defined.
77 78
Fat-specific protein 27 (FSP27), a member of the cell death-inducing DFF45-like effector
79
(CIDE) family, is a LD-associated protein abundantly expressed in the WAT (16, 17, 19).
80
FSP27 possesses a profound role in maintaining the integrity of large unilocular LDs and
81
the function of WAT as a TG storage organ. While overexpression of FSP27 in
82
nonadipocyte cells often results in enlarged LDs with increased capacity to store TGs,
83
knockdown of FSP27 in mature white adipocytes leads to the appearance of smaller
84
multilocular LDs as well as enhanced lipolysis (14-16, 30, 37, 42). In the liver, increased
85
FSP27 expression is also causally linked to the enhanced hepatic TG accumulation and
86
formation of LDs in ob/ob mice and fasted wild-type mice (24, 41). Mice with FSP27
87
deficiency exhibit multilocularization of LDs, upregulation of mitochondrial activity, and
88
eventual acquirement of brown adipose-like properties in the WAT. These animals are
89
also resistant to diet-induced obesity, hepatic steatosis and insulin resistance (29, 39). In
90
humans, treatment of obese patients with a very low calorie diet causes a reduction of
91
FSP27 expression in WAT (23). FSP27 protein is known to be rapidly degraded and has a
92
short half-life of less than 2 hours (28, 31), making the modulation of its stability a
93
potential therapeutic approach for the treatment of metabolic diseases.
94
4
95
In the present study, we have obtained evidence that AMPK activation plays a critical
96
role in the control of protein stability of FSP27. We show that prolonged AMPK
97
activation promotes FSP27 degradation through the proteasomal pathway and induces
98
multilocularization of LDs in adipocytes. We also provide evidence that HSC70 is a new
99
FSP27-interacting protein and is required for mediating the degradation of FSP27
100
downstream of AMPK activation.
101 102
RESULTS
103 104
AMPK activation decreases protein level of FSP27
105
Since AMPK activation and FSP27 ablation both led to brown-like phenotypes in white
106
adipocytes, we asked whether AMPK would be involved in the control of FSP27
107
expression. In differentiated 3T3-L1 white adipocytes, treatment with AICAR caused a
108
time-dependent decrease in the levels of endogenous (Figure 1A) as well as ectopically
109
expressed FSP27 (Figure 1B). The ectopic expression was achieved by infection with a
110
recombinant adenovirus (Ad FSP27-FLAG) that encodes mouse FSP27 with a C-terminal
111
FLAG epitope tag under the control of a CMV promoter. Similar effects were observed
112
when cells were treated with phenformin (Figure 1C and 1D), another pharmaceutical
113
activator of AMPK. By contrast, neither AICAR nor phenformin affected the protein
114
levels of Perilipin 1 and ABHD5 (α/β hydrolase domain-containing protein 5), two well-
115
known LD-associated proteins (Figure 1B and 1D). Both AICAR and phenformin
116
increased the phosphorylation of AMPKα and its target ACC (acetyl-CoA carboxylase)
117
(Figure 1E and 1F), confirming the successful activation of AMPK. In addition, energy
5
118
depletion by treatment of cells with an ATP synthase inhibitor, oligomycin, also activated
119
AMPK and decreased the protein levels of FSP27 (Figure 1G). Importantly, AICAR
120
exhibited no significant influence on the mRNA levels of FSP27 (Figure 1H). Thus,
121
AMPK activation induced by different pharmaceutical compounds all lead to decreased
122
protein level of FSP27 in 3T3-L1 adipocytes.
123 124
AMPKα1 is involved in the regulation of FSP27 protein level
125
AMPKα1 was reported to be the major isoform of AMPK catalytic α subunit in
126
adipocytes, accounting for approximately 90% of the total AMPK activity (4, 21). To
127
investigate whether AMPK mediates AICAR/phenformin-induced downregulation of
128
FSP27 expression, we performed siRNA-mediated gene silencing to deplete AMPKα1 in
129
3T3-L1 adipocytes. Compared with the transfection with control siRNA, only about 50%
130
of AMPKα1 remained in cells transfected with an AMPKα1-specific siRNA (Figure 2A
131
and C). Importantly, AMPKα1 knockdown markedly inhibited AICAR/phenformin-
132
induced phosphorylation of AMPKα subunit and a typical AMPK subunit ACC,
133
demonstrating an effective blockade of AMPK activation by AICAR/phenformin. In cells
134
treated with control siRNA, treatment with AICAR and phenformin reduced the protein
135
levels of FSP27-FLAG by about 80% and 40%, respectively (Figure B and D). However,
136
in cells treated with AMPKα1 siRNA, FSP27-FLAG only experienced a reduction by 32%
137
and 17%, respectively. These results suggest that AICAR and phenformin induce the
138
downregulation of FSP27 protein expression specifically via the activation of AMPK.
139 140
6
141
AMPK activation induces FSP27 degradation via ubiquitin-dependent proteasomal
142
pathway
143
Next, we determined whether the protein level of FSP27 could be maintained by
144
prevention of protein degradation. As shown in Figure 3A-D, addition of the proteasomal
145
inhibitor, MG-132 or epoxomicin, blocked the degradation of FSP27 in 3T3-L1
146
adipocytes treated with AICAR. By contrast, a lysosomal protease inhibitor chloroquine
147
was incapable of preventing such turnover (Figure 3E and F). To examine potential
148
ubiquitination of FSP27, we infected cells with either Ad FSP27-FLAG or a control null
149
virus (Ad-null). Following a 2-h treatment with AICAR, FSP27-FLAG was
150
immunoprecipitated and then subject to anti-ubiquitin immunoblotting analysis. As
151
expected, AICAR caused a decrease in the amount of FSP27 protein isolated from cells
152
infected with Ad FSP27 (Figure 3G). FSP27 ubiquitination, however, was increased in
153
AICAR-treated cells in comparison to control cells. Thus, our data suggest that AMPK
154
activation promotes FSP27 ubiquitination and degradation through the proteasomal
155
pathway.
156 157
HSC70 specifically interacts with FSP27
158
To uncover the mechanism by which the protein stability of FSP27 is regulated, we
159
searched for the potential interacting protein(s) of FSP27 by combining the usage of co-
160
immunoprecipitation and mass spectrometry-based protein identification methods. We
161
expressed FSP27-FLAG protein in 3T3-L1 adipocytes via infection with Ad FSP27-
162
FLAG. As control, cells were infected with Ad-null. Following anti-FLAG
163
immunoprecipitation and resolution on SDS-PAGE, SYPRO® Ruby staining revealed
7
164
the presence of a single specific band at approximately 73 kDa in FSP27-FLAG
165
precipitates (Figure 4A). Mass spectrometry analysis identified the corresponding protein
166
as HSC70, which was confirmed by anti-HSC70 immunoblotting (Figure 4B). Anti-
167
HSC70 immunoprecipitation followed by anti-FSP27 immunoblotting confirmed that a
168
specific interaction also existed between the endogenous proteins (Figure 4C). It is well
169
known that FSP27 is a lipid droplet-associated protein. Figure 4D showed that FSP27-
170
FLAG localized at the surface of LDs in 3T3-L1 adipocytes. Interestingly, while it was
171
mainly distributed scatterly or diffusely in the cytosol of control cells, HSC70 was
172
recruited to the surface of LDs in the presence of overexpressed FSP27-FLAG. Taking
173
together, these data indicate that HSC70 is a specific FSP27-binding protein.
174 175
HSC70 mediates AICAR-induced FSP27 degradation
176
HSC70 is a member of the heat shock protein 70 (HSP70) family, and serves as a
177
molecular chaperone to promote correct folding of nascent polypeptides or target proteins
178
for degradation (3, 26). To test whether HSC70 would mediate FSP27 degradation, we
179
silenced HSC70 expression via the HSC70-specific siRNA in 3T3-L1 adipocytes.
180
Compared with the endogenous protein levels in cells transfected with a GC-matched
181
control siRNA, about 50% of HSC70 was present in cells transfected with HSC70-
182
specific siRNA (Figure 5A). Upon treatment of cells with cycloheximide to block protein
183
synthesis, endogenous FSP27 experienced a rapid turnover in the control cells with a 1-h
184
half-life, whereas in HSC70 knockdown cells the protein was relatively stable without
185
significant degradation within 2 h (Figure 5A and 5B). Similarly, the half-life of ectopic
186
FSP27 was determined to be 1.5 h and 0.5 h, respectively, in control and HSC70
8
187
knockdown cells (Figure 5C and 5D). In addition, FSP27 was highly unstable in the
188
presence of AICAR and experienced an over 85% and 97% reduction after 3 h and 6 h,
189
respectively (Figure 5E and 5F). However, in HSC70 knockdown cells, FSP27 stability
190
was dramatically improved with a reduction of 25% and 63%, respectively, after the
191
AICAR treatment for 3 h and 6 h (Figure 5E and 5F). Thus, HSC70 plays a critical role in
192
the regulation of FSP27 protein stability and its degradation in response to the AMPK
193
activation.
194 195
The effect of CHIP on FSP27 degradation
196
CHIP (C terminus of Hsc70-interacting protein) is an E3 ubiquitin ligase, which
197
commonly forms a pre-ubiquitination complex with HSC70 and its client proteins
198
targeting for degradation (25, 27). To determine whether CHIP mediates the degradation
199
of FSP27, we overexpressed FSP27-FLAG in adipocytes where expression of
200
endogenous CHIP was reduced via siRNA-mediated knockdown. Interestingly, CHIP did
201
not affect FSP27 degradation in the presence of either CHX (Figure 6A) or AICAR
202
(Figure 6C). Thus, we conclude that CHIP is not involved in FSP27 degradation.
203 204
AICAR induces multilocularization of LDs
205
FSP27 plays an important role in maintaining the integrity of large unilocular LDs. Next,
206
we determined whether the degradation of FSP27 by AICAR would similarly affect the
207
LD morphology as the FSP27 depletion. Consistent with our previous report (42), FSP27
208
knockdown produced a plenty of small LDs and fewer large droplets in comparison to the
209
untreated control cells (Figure 7A and 7B). Interestingly, control cells treated with
9
210
AICAR exhibited a reduction in the size of large LDs (>20µM) along with a dramatic
211
increase in the number of small LDs (
1 2 3 4
Regulation of FSP27 protein stability by AMPK and HSC70
5 6 7
Xiaodong Zhang1,2, Bradlee L. Heckmann1,2,4, Xitao Xie1,2, Alicia M. Saarinen1,2
8
and Jun Liu1,2,3,#
9 10 11 12 13
From Department of Biochemistry and Molecular Biology1, HEALth Program2, Division of Endocrinology3, Mayo Clinic in Arizona, Scottsdale, Arizona 85259, USA; Mayo Graduate School4, Rochester, Minnesota 55905, USA.
14 15 16 17 18 19
#To whom correspondence should be addressed: Jun Liu, MCCRB 3-054, 13400 E. Shea Boulevard, Scottsdale, AZ 85259. Phone: 480-301-6745; Fax: 480-301-8387; E-mail: [email protected]
20 21 22 23 24 25
Copyright © 2014 by the American Physiological Society.
26 27
ABSTRACT
28 29
Fat specific protein 27 (FSP27) plays a pivotal role in controlling the formation of large
30
lipid droplet and energy metabolism. The cellular levels of FSP27 are tightly regulated
31
through the proteasomal ubiquitin-mediated degradation. However, the upstream signals
32
that trigger FSP27 degradation and the underlying mechanism(s) have yet to be identified.
33
Here we show that AMP-activated protein kinase (AMPK) activation by AICAR (5-
34
amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide) or phenformin induced the
35
ubiquitination of FSP27 and promoted its degradation in 3T3-L1 adipocytes. The levels
36
of FSP27 protein could be maintained by either knocking down AMPKα1 or blocking
37
proteasomal pathway. Moreover, AICAR treatment induced multilocularization of LDs in
38
3T3-L1 adipocytes, reminiscent of the morphological changes in cells depleted of FSP27.
39
Furthermore, mass spectrometry-based proteomic analysis identified heat shock cognate
40
70 (HSC70) as a novel binding protein of FSP27. The specific interaction was confirmed
41
by co-immunoprecipitation of both ectopically expressed and endogenous proteins.
42
Importantly, knockdown of HSC70 by small interference RNA resulted in increased half-
43
life of FSP27 in cells treated with a protein synthesis inhibitor cycloheximide (CHX) or
44
AICAR. However, silencing of the E3 ubiquitin ligase CHIP (C terminus of HSC70-
45
Interacting Protein) failed to alter the stability of FSP27 protein under both conditions.
46
Taken together, our data indicate that AMPK is a negative regulator of FSP27 stability
47
through the proteasomal ubiquitin-dependent protein catabolic process. Promotion of
48
FSP27 degradation may be an important factor responsible for the beneficial effect of
49
AMPK activators on energy metabolism. 2
50 51
INTRODUCTION
52 53
Adipose tissue plays a central role in the development of obesity, which occurs when
54
energy intake exceeds its expenditure. There are two types of adipose tissue, white
55
adipose tissue (WAT) for energy storage and brown adipose tissue (BAT) for energy
56
consumption to support heat production (9). White adipocytes, characterized by a large
57
unilocular lipid droplet (LD), are responsible for the intracellular storage of triglycerides
58
(TGs) in the fed state and provision of fatty acids as an energy substrate via lipolysis for
59
other tissues in response to fasting. Brown adipocytes, on the other hand, contain
60
numerous smaller LDs along with a large number of mitochondria and high oxidative
61
activity. Enlargement of LDs or adipocytes in WAT contributes to the development of
62
obesity and related insulin resistance (10, 13, 32). Thus, the induction of fat-burning
63
brown adipocyte-like capacity in white adipocytes may represent a potential therapeutic
64
strategy to combat obesity and its related disorders.
65 66
Recent studies have implicated AMPK as a potential target for inducing "BAT-like
67
phenotype" in WAT (6). For example, it was recently demonstrated that WAT
68
metabolism was remodeled toward energy dissipation through AICAR (5-amino-1-β-D-
69
ribofuranosyl-imidazole-4-carboxamide)-induced AMPK activation (7, 38). Specifically,
70
prolonged AMPK activation by AICAR caused increases in expression of PPAR-γ,
71
PPARα, and PPARδ in isolated rat epididymal adipocytes. Higher expression of PGC-1α
72
and carnitinepalmitoyl transferase-1b (CPT-1b) was accompanied by an increase in FA
3
73
oxidation in these cells. Moreover, a separate study indicated that mice injected with
74
AICAR showed increased UCP-1 expression and induced accumulation of brown
75
adipocytes within the WAT (40). However, the molecular mechanisms underlying the
76
effect of AMPK activation on the WAT plasticity remained incompletely defined.
77 78
Fat-specific protein 27 (FSP27), a member of the cell death-inducing DFF45-like effector
79
(CIDE) family, is a LD-associated protein abundantly expressed in the WAT (16, 17, 19).
80
FSP27 possesses a profound role in maintaining the integrity of large unilocular LDs and
81
the function of WAT as a TG storage organ. While overexpression of FSP27 in
82
nonadipocyte cells often results in enlarged LDs with increased capacity to store TGs,
83
knockdown of FSP27 in mature white adipocytes leads to the appearance of smaller
84
multilocular LDs as well as enhanced lipolysis (14-16, 30, 37, 42). In the liver, increased
85
FSP27 expression is also causally linked to the enhanced hepatic TG accumulation and
86
formation of LDs in ob/ob mice and fasted wild-type mice (24, 41). Mice with FSP27
87
deficiency exhibit multilocularization of LDs, upregulation of mitochondrial activity, and
88
eventual acquirement of brown adipose-like properties in the WAT. These animals are
89
also resistant to diet-induced obesity, hepatic steatosis and insulin resistance (29, 39). In
90
humans, treatment of obese patients with a very low calorie diet causes a reduction of
91
FSP27 expression in WAT (23). FSP27 protein is known to be rapidly degraded and has a
92
short half-life of less than 2 hours (28, 31), making the modulation of its stability a
93
potential therapeutic approach for the treatment of metabolic diseases.
94
4
95
In the present study, we have obtained evidence that AMPK activation plays a critical
96
role in the control of protein stability of FSP27. We show that prolonged AMPK
97
activation promotes FSP27 degradation through the proteasomal pathway and induces
98
multilocularization of LDs in adipocytes. We also provide evidence that HSC70 is a new
99
FSP27-interacting protein and is required for mediating the degradation of FSP27
100
downstream of AMPK activation.
101 102
RESULTS
103 104
AMPK activation decreases protein level of FSP27
105
Since AMPK activation and FSP27 ablation both led to brown-like phenotypes in white
106
adipocytes, we asked whether AMPK would be involved in the control of FSP27
107
expression. In differentiated 3T3-L1 white adipocytes, treatment with AICAR caused a
108
time-dependent decrease in the levels of endogenous (Figure 1A) as well as ectopically
109
expressed FSP27 (Figure 1B). The ectopic expression was achieved by infection with a
110
recombinant adenovirus (Ad FSP27-FLAG) that encodes mouse FSP27 with a C-terminal
111
FLAG epitope tag under the control of a CMV promoter. Similar effects were observed
112
when cells were treated with phenformin (Figure 1C and 1D), another pharmaceutical
113
activator of AMPK. By contrast, neither AICAR nor phenformin affected the protein
114
levels of Perilipin 1 and ABHD5 (α/β hydrolase domain-containing protein 5), two well-
115
known LD-associated proteins (Figure 1B and 1D). Both AICAR and phenformin
116
increased the phosphorylation of AMPKα and its target ACC (acetyl-CoA carboxylase)
117
(Figure 1E and 1F), confirming the successful activation of AMPK. In addition, energy
5
118
depletion by treatment of cells with an ATP synthase inhibitor, oligomycin, also activated
119
AMPK and decreased the protein levels of FSP27 (Figure 1G). Importantly, AICAR
120
exhibited no significant influence on the mRNA levels of FSP27 (Figure 1H). Thus,
121
AMPK activation induced by different pharmaceutical compounds all lead to decreased
122
protein level of FSP27 in 3T3-L1 adipocytes.
123 124
AMPKα1 is involved in the regulation of FSP27 protein level
125
AMPKα1 was reported to be the major isoform of AMPK catalytic α subunit in
126
adipocytes, accounting for approximately 90% of the total AMPK activity (4, 21). To
127
investigate whether AMPK mediates AICAR/phenformin-induced downregulation of
128
FSP27 expression, we performed siRNA-mediated gene silencing to deplete AMPKα1 in
129
3T3-L1 adipocytes. Compared with the transfection with control siRNA, only about 50%
130
of AMPKα1 remained in cells transfected with an AMPKα1-specific siRNA (Figure 2A
131
and C). Importantly, AMPKα1 knockdown markedly inhibited AICAR/phenformin-
132
induced phosphorylation of AMPKα subunit and a typical AMPK subunit ACC,
133
demonstrating an effective blockade of AMPK activation by AICAR/phenformin. In cells
134
treated with control siRNA, treatment with AICAR and phenformin reduced the protein
135
levels of FSP27-FLAG by about 80% and 40%, respectively (Figure B and D). However,
136
in cells treated with AMPKα1 siRNA, FSP27-FLAG only experienced a reduction by 32%
137
and 17%, respectively. These results suggest that AICAR and phenformin induce the
138
downregulation of FSP27 protein expression specifically via the activation of AMPK.
139 140
6
141
AMPK activation induces FSP27 degradation via ubiquitin-dependent proteasomal
142
pathway
143
Next, we determined whether the protein level of FSP27 could be maintained by
144
prevention of protein degradation. As shown in Figure 3A-D, addition of the proteasomal
145
inhibitor, MG-132 or epoxomicin, blocked the degradation of FSP27 in 3T3-L1
146
adipocytes treated with AICAR. By contrast, a lysosomal protease inhibitor chloroquine
147
was incapable of preventing such turnover (Figure 3E and F). To examine potential
148
ubiquitination of FSP27, we infected cells with either Ad FSP27-FLAG or a control null
149
virus (Ad-null). Following a 2-h treatment with AICAR, FSP27-FLAG was
150
immunoprecipitated and then subject to anti-ubiquitin immunoblotting analysis. As
151
expected, AICAR caused a decrease in the amount of FSP27 protein isolated from cells
152
infected with Ad FSP27 (Figure 3G). FSP27 ubiquitination, however, was increased in
153
AICAR-treated cells in comparison to control cells. Thus, our data suggest that AMPK
154
activation promotes FSP27 ubiquitination and degradation through the proteasomal
155
pathway.
156 157
HSC70 specifically interacts with FSP27
158
To uncover the mechanism by which the protein stability of FSP27 is regulated, we
159
searched for the potential interacting protein(s) of FSP27 by combining the usage of co-
160
immunoprecipitation and mass spectrometry-based protein identification methods. We
161
expressed FSP27-FLAG protein in 3T3-L1 adipocytes via infection with Ad FSP27-
162
FLAG. As control, cells were infected with Ad-null. Following anti-FLAG
163
immunoprecipitation and resolution on SDS-PAGE, SYPRO® Ruby staining revealed
7
164
the presence of a single specific band at approximately 73 kDa in FSP27-FLAG
165
precipitates (Figure 4A). Mass spectrometry analysis identified the corresponding protein
166
as HSC70, which was confirmed by anti-HSC70 immunoblotting (Figure 4B). Anti-
167
HSC70 immunoprecipitation followed by anti-FSP27 immunoblotting confirmed that a
168
specific interaction also existed between the endogenous proteins (Figure 4C). It is well
169
known that FSP27 is a lipid droplet-associated protein. Figure 4D showed that FSP27-
170
FLAG localized at the surface of LDs in 3T3-L1 adipocytes. Interestingly, while it was
171
mainly distributed scatterly or diffusely in the cytosol of control cells, HSC70 was
172
recruited to the surface of LDs in the presence of overexpressed FSP27-FLAG. Taking
173
together, these data indicate that HSC70 is a specific FSP27-binding protein.
174 175
HSC70 mediates AICAR-induced FSP27 degradation
176
HSC70 is a member of the heat shock protein 70 (HSP70) family, and serves as a
177
molecular chaperone to promote correct folding of nascent polypeptides or target proteins
178
for degradation (3, 26). To test whether HSC70 would mediate FSP27 degradation, we
179
silenced HSC70 expression via the HSC70-specific siRNA in 3T3-L1 adipocytes.
180
Compared with the endogenous protein levels in cells transfected with a GC-matched
181
control siRNA, about 50% of HSC70 was present in cells transfected with HSC70-
182
specific siRNA (Figure 5A). Upon treatment of cells with cycloheximide to block protein
183
synthesis, endogenous FSP27 experienced a rapid turnover in the control cells with a 1-h
184
half-life, whereas in HSC70 knockdown cells the protein was relatively stable without
185
significant degradation within 2 h (Figure 5A and 5B). Similarly, the half-life of ectopic
186
FSP27 was determined to be 1.5 h and 0.5 h, respectively, in control and HSC70
8
187
knockdown cells (Figure 5C and 5D). In addition, FSP27 was highly unstable in the
188
presence of AICAR and experienced an over 85% and 97% reduction after 3 h and 6 h,
189
respectively (Figure 5E and 5F). However, in HSC70 knockdown cells, FSP27 stability
190
was dramatically improved with a reduction of 25% and 63%, respectively, after the
191
AICAR treatment for 3 h and 6 h (Figure 5E and 5F). Thus, HSC70 plays a critical role in
192
the regulation of FSP27 protein stability and its degradation in response to the AMPK
193
activation.
194 195
The effect of CHIP on FSP27 degradation
196
CHIP (C terminus of Hsc70-interacting protein) is an E3 ubiquitin ligase, which
197
commonly forms a pre-ubiquitination complex with HSC70 and its client proteins
198
targeting for degradation (25, 27). To determine whether CHIP mediates the degradation
199
of FSP27, we overexpressed FSP27-FLAG in adipocytes where expression of
200
endogenous CHIP was reduced via siRNA-mediated knockdown. Interestingly, CHIP did
201
not affect FSP27 degradation in the presence of either CHX (Figure 6A) or AICAR
202
(Figure 6C). Thus, we conclude that CHIP is not involved in FSP27 degradation.
203 204
AICAR induces multilocularization of LDs
205
FSP27 plays an important role in maintaining the integrity of large unilocular LDs. Next,
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we determined whether the degradation of FSP27 by AICAR would similarly affect the
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LD morphology as the FSP27 depletion. Consistent with our previous report (42), FSP27
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knockdown produced a plenty of small LDs and fewer large droplets in comparison to the
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untreated control cells (Figure 7A and 7B). Interestingly, control cells treated with
9
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AICAR exhibited a reduction in the size of large LDs (>20µM) along with a dramatic
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increase in the number of small LDs (
