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Conjugation of the ubiquitin activating enzyme UBE1 with the ubiquitin-like modifier FAT10 targets it for proteasomal degradation.

Bialas J, Groettrup M, Aichem A - PLoS ONE (2015)

Bottom Line: Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines.By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway.Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Institute Thurgau at the University of Konstanz, Unterseestrasse 47, CH-8280, Kreuzlingen, Switzerland; Division of Immunology, Department of Biology, University of Konstanz, D-78457, Konstanz, Germany.

ABSTRACT
The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directly targets its substrates for proteasomal degradation by becoming covalently attached via its C-terminal diglycine motif to internal lysine residues of its substrate proteins. The conjugation machinery consists of the bispecific E1 activating enzyme Ubiquitin-like modifier activating enzyme 6 (UBA6), the likewise bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By mass spectrometry analysis the ubiquitin E1 activating enzyme ubiquitin-activating enzyme 1 (UBE1) was identified as putative substrate of FAT10. Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines. FAT10ylation of UBE1 depends on the diglycine motif of FAT10. By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway. Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway.

No MeSH data available.


Endogenous UBE1-FAT10 conjugate formation is dependent on UBA6 and USE1.(A) Total cell extracts from IFNγ and TNFα-stimulated HEK293 cells were used to immunoprecipitate endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1) or an unspecific IgG-agarose as control, followed by western blot analysis using polyclonal antibodies against FAT10 and UBE1. Before harvesting, cells were treated with proteasome inhibitor MG132 for 6 hours, where indicated. Proteins were separated under reducing conditions (4% 2-mercaptoethanol) on 4–12% Bis/Tris NuPAGE gels. The upper panel shows the immunoprecipitated UBE1-FAT10 conjugate, the middle panel the immunoprecipitated FAT10 conjugates, the lower western blot panels show protein expression levels in total cell lysates (load). β-actin was used as loading control. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were treated with IFNγ/TNFα to stimulate endogenous FAT10 expression as described in (A). Additionally, cells were treated on two days either with control siRNA or FAT10-specific siRNA to downregulate endogenous FAT10 expression at the same time as it was induced. Cells were harvested on day three, and cell lysates were subjected to immunoprecipitation of endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1). Proteins were separated on 4–12% NuPAGE gels and western blot analysis was performed under reducing conditions (4% 2-mercaptoethanol) with polyclonal antibodies against FAT10 and UBE1. β-actin was used as loading control. The upper western blot panel shows the disappearance of the UBE1-FAT10 conjugate after siRNA treatment, the middle panel shows immunoprecipitated FAT10 conjugates, and the lower panels show protein expression levels in total protein lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (C) Same experimental setup as in (B) only that UBA6 and USE1 were specifically knocked down by treating HEK293 cells with specific siRNAs against UBA6 and USE1, respectively. Control cells were either untreated or treated with unspecific control siRNA. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown.
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pone.0120329.g002: Endogenous UBE1-FAT10 conjugate formation is dependent on UBA6 and USE1.(A) Total cell extracts from IFNγ and TNFα-stimulated HEK293 cells were used to immunoprecipitate endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1) or an unspecific IgG-agarose as control, followed by western blot analysis using polyclonal antibodies against FAT10 and UBE1. Before harvesting, cells were treated with proteasome inhibitor MG132 for 6 hours, where indicated. Proteins were separated under reducing conditions (4% 2-mercaptoethanol) on 4–12% Bis/Tris NuPAGE gels. The upper panel shows the immunoprecipitated UBE1-FAT10 conjugate, the middle panel the immunoprecipitated FAT10 conjugates, the lower western blot panels show protein expression levels in total cell lysates (load). β-actin was used as loading control. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were treated with IFNγ/TNFα to stimulate endogenous FAT10 expression as described in (A). Additionally, cells were treated on two days either with control siRNA or FAT10-specific siRNA to downregulate endogenous FAT10 expression at the same time as it was induced. Cells were harvested on day three, and cell lysates were subjected to immunoprecipitation of endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1). Proteins were separated on 4–12% NuPAGE gels and western blot analysis was performed under reducing conditions (4% 2-mercaptoethanol) with polyclonal antibodies against FAT10 and UBE1. β-actin was used as loading control. The upper western blot panel shows the disappearance of the UBE1-FAT10 conjugate after siRNA treatment, the middle panel shows immunoprecipitated FAT10 conjugates, and the lower panels show protein expression levels in total protein lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (C) Same experimental setup as in (B) only that UBA6 and USE1 were specifically knocked down by treating HEK293 cells with specific siRNAs against UBA6 and USE1, respectively. Control cells were either untreated or treated with unspecific control siRNA. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown.

Mentions: To verify that the UBE1-FAT10 conjugate formation was not due to overexpression of the two proteins but was formed also under endogenous conditions, HEK293 cells were treated for 24 hours with the proinflammatory cytokines interferon (IFN)γ and tumor necrosis factor (TNF)α to induce endogenous FAT10 expression. To visualize the endogenous UBE1-FAT10 conjugate, endogenous FAT10 was immunoprecipitated with a FAT10-reactive monoclonal antibody followed by western blot analysis with an antibody specific for endogenous UBE1. As shown in Fig. 2A, the UBE1-FAT10 conjugate was formed also under endogenous conditions, it appeared as a double band and was absent when the unspecific isotype control instead of the FAT10-reactive antibody was used for immunoprecipitation (Fig. 2A, lane 4). Mass spectrometry analysis of the two bands revealed that the upper band contained the UBE1-FAT10 conjugate because both proteins were identified at a molecular mass, corresponding to the UBE1-FAT10 conjugate (S1 Table, sample S2). As a control, by analysis of corresponding gel slices deriving from unstimulated cells and therefore from cells, not expressing FAT10, no UBE1 or FAT10 was identified (S1 Table, sample C2). UBE1 and FAT10 were both also identified in the lower band deriving from cytokine stimulated cells (S1 Table, sample S1), but UBE1 was also identified in the respective unstimulated control (S1 Table, sample C1). Therefore we suggest that a portion of UBE1 unspecifically interacted with the protein A sepharose used for immunoprecipitation of FAT10. However, since in most cases the amount of UBE1 in the lower band increased when FAT10 was expressed and immunoprecipitated and since at the same time the amount of UBE1 in the lysate (load) remained stable, it might also represent UBE1, non-covalently interacting with FAT10 as already shown in Fig. 1B in case of HA-UBE1 and FLAG-FAT10. As shown for the conjugate formed under overexpression conditions, the endogenous UBE1-FAT10 conjugate accumulated when cells were treated for six hours with the proteasome inhibitor MG132 (Fig. 2A, lanes 2 and 3). Modification of substrate proteins with FAT10 has previously been shown to depend on the activation of FAT10 by the E1 activating enzyme UBA6, and its transfer by the E2 conjugating enzyme USE1 [18]. To investigate whether this is also necessary for the formation of the UBE1-FAT10 conjugate, we used siRNAs to down-regulate FAT10, UBA6 or USE1 upon induction of FAT10 expression with cytokines. As expected, upon knockdown of endogenous FAT10 no monomeric FAT10 and no FAT10 conjugates were detectable anymore. In addition, the amount of the UBE1-FAT10 conjugate was clearly reduced, further supporting the UBE1-FAT10 conjugate formation under endogenous conditions (Fig. 2B, lane 4). A similar result was obtained after knockdown of UBA6 and USE1 mRNAs by about 85% which also strongly abrogated the formation of the endogenous UBE1-FAT10 conjugate (Fig. 2C, lanes 4 and 5). Taken together, the results obtained so far show that UBE1 is a novel substrate of FAT10 and that UBE1-FAT10ylation is dependent on a functional FAT10 conjugation pathway.


Conjugation of the ubiquitin activating enzyme UBE1 with the ubiquitin-like modifier FAT10 targets it for proteasomal degradation.

Bialas J, Groettrup M, Aichem A - PLoS ONE (2015)

Endogenous UBE1-FAT10 conjugate formation is dependent on UBA6 and USE1.(A) Total cell extracts from IFNγ and TNFα-stimulated HEK293 cells were used to immunoprecipitate endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1) or an unspecific IgG-agarose as control, followed by western blot analysis using polyclonal antibodies against FAT10 and UBE1. Before harvesting, cells were treated with proteasome inhibitor MG132 for 6 hours, where indicated. Proteins were separated under reducing conditions (4% 2-mercaptoethanol) on 4–12% Bis/Tris NuPAGE gels. The upper panel shows the immunoprecipitated UBE1-FAT10 conjugate, the middle panel the immunoprecipitated FAT10 conjugates, the lower western blot panels show protein expression levels in total cell lysates (load). β-actin was used as loading control. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were treated with IFNγ/TNFα to stimulate endogenous FAT10 expression as described in (A). Additionally, cells were treated on two days either with control siRNA or FAT10-specific siRNA to downregulate endogenous FAT10 expression at the same time as it was induced. Cells were harvested on day three, and cell lysates were subjected to immunoprecipitation of endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1). Proteins were separated on 4–12% NuPAGE gels and western blot analysis was performed under reducing conditions (4% 2-mercaptoethanol) with polyclonal antibodies against FAT10 and UBE1. β-actin was used as loading control. The upper western blot panel shows the disappearance of the UBE1-FAT10 conjugate after siRNA treatment, the middle panel shows immunoprecipitated FAT10 conjugates, and the lower panels show protein expression levels in total protein lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (C) Same experimental setup as in (B) only that UBA6 and USE1 were specifically knocked down by treating HEK293 cells with specific siRNAs against UBA6 and USE1, respectively. Control cells were either untreated or treated with unspecific control siRNA. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown.
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pone.0120329.g002: Endogenous UBE1-FAT10 conjugate formation is dependent on UBA6 and USE1.(A) Total cell extracts from IFNγ and TNFα-stimulated HEK293 cells were used to immunoprecipitate endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1) or an unspecific IgG-agarose as control, followed by western blot analysis using polyclonal antibodies against FAT10 and UBE1. Before harvesting, cells were treated with proteasome inhibitor MG132 for 6 hours, where indicated. Proteins were separated under reducing conditions (4% 2-mercaptoethanol) on 4–12% Bis/Tris NuPAGE gels. The upper panel shows the immunoprecipitated UBE1-FAT10 conjugate, the middle panel the immunoprecipitated FAT10 conjugates, the lower western blot panels show protein expression levels in total cell lysates (load). β-actin was used as loading control. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were treated with IFNγ/TNFα to stimulate endogenous FAT10 expression as described in (A). Additionally, cells were treated on two days either with control siRNA or FAT10-specific siRNA to downregulate endogenous FAT10 expression at the same time as it was induced. Cells were harvested on day three, and cell lysates were subjected to immunoprecipitation of endogenous FAT10 and the UBE1-FAT10 conjugate with a mAb against FAT10 (4F1). Proteins were separated on 4–12% NuPAGE gels and western blot analysis was performed under reducing conditions (4% 2-mercaptoethanol) with polyclonal antibodies against FAT10 and UBE1. β-actin was used as loading control. The upper western blot panel shows the disappearance of the UBE1-FAT10 conjugate after siRNA treatment, the middle panel shows immunoprecipitated FAT10 conjugates, and the lower panels show protein expression levels in total protein lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (C) Same experimental setup as in (B) only that UBA6 and USE1 were specifically knocked down by treating HEK293 cells with specific siRNAs against UBA6 and USE1, respectively. Control cells were either untreated or treated with unspecific control siRNA. An asterisk indicates the heavy chain of the antibody used for immunoprecipitation. One representative experiment out of three experiments with similar outcomes is shown.
Mentions: To verify that the UBE1-FAT10 conjugate formation was not due to overexpression of the two proteins but was formed also under endogenous conditions, HEK293 cells were treated for 24 hours with the proinflammatory cytokines interferon (IFN)γ and tumor necrosis factor (TNF)α to induce endogenous FAT10 expression. To visualize the endogenous UBE1-FAT10 conjugate, endogenous FAT10 was immunoprecipitated with a FAT10-reactive monoclonal antibody followed by western blot analysis with an antibody specific for endogenous UBE1. As shown in Fig. 2A, the UBE1-FAT10 conjugate was formed also under endogenous conditions, it appeared as a double band and was absent when the unspecific isotype control instead of the FAT10-reactive antibody was used for immunoprecipitation (Fig. 2A, lane 4). Mass spectrometry analysis of the two bands revealed that the upper band contained the UBE1-FAT10 conjugate because both proteins were identified at a molecular mass, corresponding to the UBE1-FAT10 conjugate (S1 Table, sample S2). As a control, by analysis of corresponding gel slices deriving from unstimulated cells and therefore from cells, not expressing FAT10, no UBE1 or FAT10 was identified (S1 Table, sample C2). UBE1 and FAT10 were both also identified in the lower band deriving from cytokine stimulated cells (S1 Table, sample S1), but UBE1 was also identified in the respective unstimulated control (S1 Table, sample C1). Therefore we suggest that a portion of UBE1 unspecifically interacted with the protein A sepharose used for immunoprecipitation of FAT10. However, since in most cases the amount of UBE1 in the lower band increased when FAT10 was expressed and immunoprecipitated and since at the same time the amount of UBE1 in the lysate (load) remained stable, it might also represent UBE1, non-covalently interacting with FAT10 as already shown in Fig. 1B in case of HA-UBE1 and FLAG-FAT10. As shown for the conjugate formed under overexpression conditions, the endogenous UBE1-FAT10 conjugate accumulated when cells were treated for six hours with the proteasome inhibitor MG132 (Fig. 2A, lanes 2 and 3). Modification of substrate proteins with FAT10 has previously been shown to depend on the activation of FAT10 by the E1 activating enzyme UBA6, and its transfer by the E2 conjugating enzyme USE1 [18]. To investigate whether this is also necessary for the formation of the UBE1-FAT10 conjugate, we used siRNAs to down-regulate FAT10, UBA6 or USE1 upon induction of FAT10 expression with cytokines. As expected, upon knockdown of endogenous FAT10 no monomeric FAT10 and no FAT10 conjugates were detectable anymore. In addition, the amount of the UBE1-FAT10 conjugate was clearly reduced, further supporting the UBE1-FAT10 conjugate formation under endogenous conditions (Fig. 2B, lane 4). A similar result was obtained after knockdown of UBA6 and USE1 mRNAs by about 85% which also strongly abrogated the formation of the endogenous UBE1-FAT10 conjugate (Fig. 2C, lanes 4 and 5). Taken together, the results obtained so far show that UBE1 is a novel substrate of FAT10 and that UBE1-FAT10ylation is dependent on a functional FAT10 conjugation pathway.

Bottom Line: Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines.By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway.Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Institute Thurgau at the University of Konstanz, Unterseestrasse 47, CH-8280, Kreuzlingen, Switzerland; Division of Immunology, Department of Biology, University of Konstanz, D-78457, Konstanz, Germany.

ABSTRACT
The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directly targets its substrates for proteasomal degradation by becoming covalently attached via its C-terminal diglycine motif to internal lysine residues of its substrate proteins. The conjugation machinery consists of the bispecific E1 activating enzyme Ubiquitin-like modifier activating enzyme 6 (UBA6), the likewise bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By mass spectrometry analysis the ubiquitin E1 activating enzyme ubiquitin-activating enzyme 1 (UBE1) was identified as putative substrate of FAT10. Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines. FAT10ylation of UBE1 depends on the diglycine motif of FAT10. By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway. Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway.

No MeSH data available.