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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.


UBE1 is not a second E1 activating enzyme for FAT10, but a substrate of FAT10ylation in vitro.Recombinant proteins were incubated in reaction buffer at 30°C for 60 min and separated on 3–8% gradient Tris/Acetate NuPAGE SDS gels. Western blot analysis was performed under non-reducing (-2-mercaptoethanol (2-ME), left panels) or reducing (+ 2-ME (4%), right panels) conditions by using an antibody against FLAG to visualize FLAG-UBA6 or a polyclonal antibody reactive to UBE1. (A) Upper western blot panels show FLAG-UBA6 thioester-linked ubiquitin and FAT10, respectively (arrow heads in lane 3 and 5). Lower panels show ubiquitin activation by UBE1 (arrow head in lane 3), but no activation of FAT10 by UBE1. One experiment out of three experiments with similar outcomes is shown. (B) Western blot showing the in vitro conjugation of recombinant FAT10 onto UBE1 in presence of UBA6 and/or USE1 under reducing conditions (4% 2-ME). Arrow heads indicate the UBE1-FAT10 conjugate in lane 3 and 5. One representative experiment out of four experiments with similar outcomes is shown.
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pone.0120329.g003: UBE1 is not a second E1 activating enzyme for FAT10, but a substrate of FAT10ylation in vitro.Recombinant proteins were incubated in reaction buffer at 30°C for 60 min and separated on 3–8% gradient Tris/Acetate NuPAGE SDS gels. Western blot analysis was performed under non-reducing (-2-mercaptoethanol (2-ME), left panels) or reducing (+ 2-ME (4%), right panels) conditions by using an antibody against FLAG to visualize FLAG-UBA6 or a polyclonal antibody reactive to UBE1. (A) Upper western blot panels show FLAG-UBA6 thioester-linked ubiquitin and FAT10, respectively (arrow heads in lane 3 and 5). Lower panels show ubiquitin activation by UBE1 (arrow head in lane 3), but no activation of FAT10 by UBE1. One experiment out of three experiments with similar outcomes is shown. (B) Western blot showing the in vitro conjugation of recombinant FAT10 onto UBE1 in presence of UBA6 and/or USE1 under reducing conditions (4% 2-ME). Arrow heads indicate the UBE1-FAT10 conjugate in lane 3 and 5. One representative experiment out of four experiments with similar outcomes is shown.

Mentions: For a long time, UBE1 was thought to solely activate ubiquitin [26,27] but more recently it has emerged that UBE1 additionally activates and transfers NEDD8 under specific stress conditions [28,29]. Therefore we were interested to investigate whether UBE1 would in addition serve as a second E1 activating enzyme for FAT10. To address this question the active site cysteine of UBE1 was mutated to an alanine (HA-UBE1 C632A) and the mutant UBE1 was co-expressed together with FLAG-tagged FAT10 in HEK293 cells followed by co-immunoprecipitation and western blot analysis under reducing conditions. Independent of a functional active site cysteine the UBE1-FAT10 conjugate was still formed (Fig. 1A, lane 8), showing that the formation of the UBE1-FAT10 conjugate was not dependent on the preceding transfer of FAT10 onto the active site cysteine of UBE1, as it is e.g. shown in case of auto-FAT10ylation of USE1 [18]. To corroborate that UBE1 did not act as a second E1 activating enzyme of FAT10, UBE1 was recombinantly expressed and purified from E. coli, and subjected to in vitro FAT10- or ubiquitin-activation assays. The respective proteins were incubated at 30°C for 60 min and western blot analysis was performed under non-reducing and reducing (4% 2-mercaptoethanol, 2-ME) conditions to monitor thioester bond formation. Recombinant FLAG-UBA6 was used as positive control for FAT10 activation. As shown in Fig. 3A, both, ubiquitin and FAT10 were activated by FLAG-UBA6 and the thioester bonds were almost completely reducible in presence of 4% 2-mercaptoethanol (+2-ME) (Fig. 3A, upper panels, arrow heads in lanes 3 and 5). In comparison to this, recombinant UBE1 and ubiquitin formed a thioester bond which was completely absent under thioester-reducing conditions (Fig. 3A lower panels, arrow head in lane 3). In contrast, no activation of recombinant FAT10 by UBE1 was detectable under non-reducing conditions (Fig. 3A lower panels, lanes 5) showing that UBE1 does not act as a second E1 activating enzyme for FAT10.


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)

UBE1 is not a second E1 activating enzyme for FAT10, but a substrate of FAT10ylation in vitro.Recombinant proteins were incubated in reaction buffer at 30°C for 60 min and separated on 3–8% gradient Tris/Acetate NuPAGE SDS gels. Western blot analysis was performed under non-reducing (-2-mercaptoethanol (2-ME), left panels) or reducing (+ 2-ME (4%), right panels) conditions by using an antibody against FLAG to visualize FLAG-UBA6 or a polyclonal antibody reactive to UBE1. (A) Upper western blot panels show FLAG-UBA6 thioester-linked ubiquitin and FAT10, respectively (arrow heads in lane 3 and 5). Lower panels show ubiquitin activation by UBE1 (arrow head in lane 3), but no activation of FAT10 by UBE1. One experiment out of three experiments with similar outcomes is shown. (B) Western blot showing the in vitro conjugation of recombinant FAT10 onto UBE1 in presence of UBA6 and/or USE1 under reducing conditions (4% 2-ME). Arrow heads indicate the UBE1-FAT10 conjugate in lane 3 and 5. One representative experiment out of four experiments with similar outcomes is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359146&req=5

pone.0120329.g003: UBE1 is not a second E1 activating enzyme for FAT10, but a substrate of FAT10ylation in vitro.Recombinant proteins were incubated in reaction buffer at 30°C for 60 min and separated on 3–8% gradient Tris/Acetate NuPAGE SDS gels. Western blot analysis was performed under non-reducing (-2-mercaptoethanol (2-ME), left panels) or reducing (+ 2-ME (4%), right panels) conditions by using an antibody against FLAG to visualize FLAG-UBA6 or a polyclonal antibody reactive to UBE1. (A) Upper western blot panels show FLAG-UBA6 thioester-linked ubiquitin and FAT10, respectively (arrow heads in lane 3 and 5). Lower panels show ubiquitin activation by UBE1 (arrow head in lane 3), but no activation of FAT10 by UBE1. One experiment out of three experiments with similar outcomes is shown. (B) Western blot showing the in vitro conjugation of recombinant FAT10 onto UBE1 in presence of UBA6 and/or USE1 under reducing conditions (4% 2-ME). Arrow heads indicate the UBE1-FAT10 conjugate in lane 3 and 5. One representative experiment out of four experiments with similar outcomes is shown.
Mentions: For a long time, UBE1 was thought to solely activate ubiquitin [26,27] but more recently it has emerged that UBE1 additionally activates and transfers NEDD8 under specific stress conditions [28,29]. Therefore we were interested to investigate whether UBE1 would in addition serve as a second E1 activating enzyme for FAT10. To address this question the active site cysteine of UBE1 was mutated to an alanine (HA-UBE1 C632A) and the mutant UBE1 was co-expressed together with FLAG-tagged FAT10 in HEK293 cells followed by co-immunoprecipitation and western blot analysis under reducing conditions. Independent of a functional active site cysteine the UBE1-FAT10 conjugate was still formed (Fig. 1A, lane 8), showing that the formation of the UBE1-FAT10 conjugate was not dependent on the preceding transfer of FAT10 onto the active site cysteine of UBE1, as it is e.g. shown in case of auto-FAT10ylation of USE1 [18]. To corroborate that UBE1 did not act as a second E1 activating enzyme of FAT10, UBE1 was recombinantly expressed and purified from E. coli, and subjected to in vitro FAT10- or ubiquitin-activation assays. The respective proteins were incubated at 30°C for 60 min and western blot analysis was performed under non-reducing and reducing (4% 2-mercaptoethanol, 2-ME) conditions to monitor thioester bond formation. Recombinant FLAG-UBA6 was used as positive control for FAT10 activation. As shown in Fig. 3A, both, ubiquitin and FAT10 were activated by FLAG-UBA6 and the thioester bonds were almost completely reducible in presence of 4% 2-mercaptoethanol (+2-ME) (Fig. 3A, upper panels, arrow heads in lanes 3 and 5). In comparison to this, recombinant UBE1 and ubiquitin formed a thioester bond which was completely absent under thioester-reducing conditions (Fig. 3A lower panels, arrow head in lane 3). In contrast, no activation of recombinant FAT10 by UBE1 was detectable under non-reducing conditions (Fig. 3A lower panels, lanes 5) showing that UBE1 does not act as a second E1 activating enzyme for FAT10.

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.