Limits...
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 a substrate of FAT10ylation.(A) HEK293 cells were transiently transfected with expression plasmids for HA-UBE1, the active site cysteine mutant of HA-UBE1 (HA-UBE1 C632A), His-3xFLAG-FAT10 (FLAG-FAT10), His-3xFLAG-FAT10 with a mutated diglycine motif at the C terminus (FLAG-FAT10 ΔGG), or a lysine-less mutant of His-3xFLAG-FAT10 (FLAG-FAT10 K0). Where indicated, cells were additionally treated with 10 μM of the proteasome inhibitor MG132 for six hours prior to harvesting. Cell lysates were subjected to immunoprecipitation with anti-HA antibody coupled to agarose. Proteins were separated on 4–12% Bis/Tris NuPAGE gels, and western blot analysis was performed with antibodies reactive against HA or FLAG. β-actin was used as loading control. The upper panels show the immunoprecipitated proteins and the lower western blot panels show the total protein expression in the cell lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were transfected with the expression constructs indicated, harvested and lysed as described in (A). Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody coupled to agarose and subsequent western blots were performed as described in (A). An asterisk in lane 6 marks an unspecific background band in the WB against FLAG. Cartoons describe the covalent and non-covalent interactions of UBE1 and FAT10. One representative experiment out of three experiments with similar outcomes is shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359146&req=5

pone.0120329.g001: UBE1 is a substrate of FAT10ylation.(A) HEK293 cells were transiently transfected with expression plasmids for HA-UBE1, the active site cysteine mutant of HA-UBE1 (HA-UBE1 C632A), His-3xFLAG-FAT10 (FLAG-FAT10), His-3xFLAG-FAT10 with a mutated diglycine motif at the C terminus (FLAG-FAT10 ΔGG), or a lysine-less mutant of His-3xFLAG-FAT10 (FLAG-FAT10 K0). Where indicated, cells were additionally treated with 10 μM of the proteasome inhibitor MG132 for six hours prior to harvesting. Cell lysates were subjected to immunoprecipitation with anti-HA antibody coupled to agarose. Proteins were separated on 4–12% Bis/Tris NuPAGE gels, and western blot analysis was performed with antibodies reactive against HA or FLAG. β-actin was used as loading control. The upper panels show the immunoprecipitated proteins and the lower western blot panels show the total protein expression in the cell lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were transfected with the expression constructs indicated, harvested and lysed as described in (A). Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody coupled to agarose and subsequent western blots were performed as described in (A). An asterisk in lane 6 marks an unspecific background band in the WB against FLAG. Cartoons describe the covalent and non-covalent interactions of UBE1 and FAT10. One representative experiment out of three experiments with similar outcomes is shown.

Mentions: Recently we published proteomic data to identify new interaction partners and substrates of endogenous FAT10. In this analysis, the ubiquitin-activating enzyme UBE1 was identified and classified as a novel putative substrate of FAT10 [23]. In order to confirm this classification and to show that UBE1 and FAT10 indeed interact and form a stable conjugate, co-immunoprecipitations of HA-tagged UBE1 (HA-UBE1) and different versions of FLAG-tagged FAT10 (FLAG-FAT10) transiently overexpressed in HEK293 cells were performed and analyzed under reducing conditions by western blot analysis (Fig. 1A). Upon expression of HA-UBE1 and FLAG-FAT10 a non-reducible conjugate with an apparent molecular mass of approximately 150 kDa was formed (Fig. 1A, upper panel, lane 4) and the amount of the conjugate increased upon proteasome inhibition with MG132 (Fig. 1A, lane 5), suggesting that FAT10ylated UBE1 may be guided to proteasomal degradation. When HA-UBE1 was co-expressed with a FLAG-FAT10 mutant lacking the C-terminal diglycine motif (FLAG-FAT1ΔGG) only a minor portion of FLAG-FAT10ΔGG formed a conjugate with HA-UBE1, most probably due to the high protein concentrations under overexpression conditions (Fig. 1A, lane 6), further supporting the conception of a covalent attachment of FAT10 to UBE1 via its C-terminal diglycine motif. Additionally, the overexpression of a lysine-less FAT10 mutant (FLAG-FAT10 K0) revealed a conjugate with the same size as the wildtype HA-UBE1-FLAG-FAT10 conjugate, indicating that UBE1 becomes modified by one single FAT10 molecule (Fig. 1A, lane 7). Monomeric FAT10 was also immunoprecipitated together with HA-UBE1, pointing to an additional non-covalent interaction of the two proteins (Fig. 1A, lanes 4–8). To further characterize the non-covalent interaction of FAT10 and UBE1, an immunoprecipitation of FLAG-tagged FAT10 or its diglycine mutant FLAG-FAT10ΔGG was performed and the non-covalent interaction of HA-UBE1 was investigated by a subsequent western blot analysis using an HA-reactive antibody (Fig. 1B). As shown in Fig. 1B, both, the HA-UBE1-FLAG-FAT10 conjugate as well as unconjugated HA-UBE1 were immunoprecipitated with FLAG-FAT10 (Fig. 1B, lane 3). HA-UBE1 was also immunoprecipitated with the FAT10 diglycine mutant FLAG-FAT10ΔGG (Fig. 1B, lane 5), indicating a non-covalent interaction of UBE1 and FAT10 which might be independent of the FAT10 diglycine motif. To verify the formation of a stable non-reducible isopeptide linkage between UBE1 and FAT10, an immunoprecipitation of overexpressed proteins under denaturing conditions was performed. A subsequent western blot analysis under reducing conditions revealed that the UBE1-FAT10 conjugate was still detectable confirming that UBE1 and FAT10 formed a stable isopeptide linkage (S1 Fig.).


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 a substrate of FAT10ylation.(A) HEK293 cells were transiently transfected with expression plasmids for HA-UBE1, the active site cysteine mutant of HA-UBE1 (HA-UBE1 C632A), His-3xFLAG-FAT10 (FLAG-FAT10), His-3xFLAG-FAT10 with a mutated diglycine motif at the C terminus (FLAG-FAT10 ΔGG), or a lysine-less mutant of His-3xFLAG-FAT10 (FLAG-FAT10 K0). Where indicated, cells were additionally treated with 10 μM of the proteasome inhibitor MG132 for six hours prior to harvesting. Cell lysates were subjected to immunoprecipitation with anti-HA antibody coupled to agarose. Proteins were separated on 4–12% Bis/Tris NuPAGE gels, and western blot analysis was performed with antibodies reactive against HA or FLAG. β-actin was used as loading control. The upper panels show the immunoprecipitated proteins and the lower western blot panels show the total protein expression in the cell lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were transfected with the expression constructs indicated, harvested and lysed as described in (A). Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody coupled to agarose and subsequent western blots were performed as described in (A). An asterisk in lane 6 marks an unspecific background band in the WB against FLAG. Cartoons describe the covalent and non-covalent interactions of UBE1 and FAT10. One representative experiment out of three experiments with similar outcomes is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359146&req=5

pone.0120329.g001: UBE1 is a substrate of FAT10ylation.(A) HEK293 cells were transiently transfected with expression plasmids for HA-UBE1, the active site cysteine mutant of HA-UBE1 (HA-UBE1 C632A), His-3xFLAG-FAT10 (FLAG-FAT10), His-3xFLAG-FAT10 with a mutated diglycine motif at the C terminus (FLAG-FAT10 ΔGG), or a lysine-less mutant of His-3xFLAG-FAT10 (FLAG-FAT10 K0). Where indicated, cells were additionally treated with 10 μM of the proteasome inhibitor MG132 for six hours prior to harvesting. Cell lysates were subjected to immunoprecipitation with anti-HA antibody coupled to agarose. Proteins were separated on 4–12% Bis/Tris NuPAGE gels, and western blot analysis was performed with antibodies reactive against HA or FLAG. β-actin was used as loading control. The upper panels show the immunoprecipitated proteins and the lower western blot panels show the total protein expression in the cell lysates (load). One representative experiment out of three experiments with similar outcomes is shown. (B) HEK293 cells were transfected with the expression constructs indicated, harvested and lysed as described in (A). Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody coupled to agarose and subsequent western blots were performed as described in (A). An asterisk in lane 6 marks an unspecific background band in the WB against FLAG. Cartoons describe the covalent and non-covalent interactions of UBE1 and FAT10. One representative experiment out of three experiments with similar outcomes is shown.
Mentions: Recently we published proteomic data to identify new interaction partners and substrates of endogenous FAT10. In this analysis, the ubiquitin-activating enzyme UBE1 was identified and classified as a novel putative substrate of FAT10 [23]. In order to confirm this classification and to show that UBE1 and FAT10 indeed interact and form a stable conjugate, co-immunoprecipitations of HA-tagged UBE1 (HA-UBE1) and different versions of FLAG-tagged FAT10 (FLAG-FAT10) transiently overexpressed in HEK293 cells were performed and analyzed under reducing conditions by western blot analysis (Fig. 1A). Upon expression of HA-UBE1 and FLAG-FAT10 a non-reducible conjugate with an apparent molecular mass of approximately 150 kDa was formed (Fig. 1A, upper panel, lane 4) and the amount of the conjugate increased upon proteasome inhibition with MG132 (Fig. 1A, lane 5), suggesting that FAT10ylated UBE1 may be guided to proteasomal degradation. When HA-UBE1 was co-expressed with a FLAG-FAT10 mutant lacking the C-terminal diglycine motif (FLAG-FAT1ΔGG) only a minor portion of FLAG-FAT10ΔGG formed a conjugate with HA-UBE1, most probably due to the high protein concentrations under overexpression conditions (Fig. 1A, lane 6), further supporting the conception of a covalent attachment of FAT10 to UBE1 via its C-terminal diglycine motif. Additionally, the overexpression of a lysine-less FAT10 mutant (FLAG-FAT10 K0) revealed a conjugate with the same size as the wildtype HA-UBE1-FLAG-FAT10 conjugate, indicating that UBE1 becomes modified by one single FAT10 molecule (Fig. 1A, lane 7). Monomeric FAT10 was also immunoprecipitated together with HA-UBE1, pointing to an additional non-covalent interaction of the two proteins (Fig. 1A, lanes 4–8). To further characterize the non-covalent interaction of FAT10 and UBE1, an immunoprecipitation of FLAG-tagged FAT10 or its diglycine mutant FLAG-FAT10ΔGG was performed and the non-covalent interaction of HA-UBE1 was investigated by a subsequent western blot analysis using an HA-reactive antibody (Fig. 1B). As shown in Fig. 1B, both, the HA-UBE1-FLAG-FAT10 conjugate as well as unconjugated HA-UBE1 were immunoprecipitated with FLAG-FAT10 (Fig. 1B, lane 3). HA-UBE1 was also immunoprecipitated with the FAT10 diglycine mutant FLAG-FAT10ΔGG (Fig. 1B, lane 5), indicating a non-covalent interaction of UBE1 and FAT10 which might be independent of the FAT10 diglycine motif. To verify the formation of a stable non-reducible isopeptide linkage between UBE1 and FAT10, an immunoprecipitation of overexpressed proteins under denaturing conditions was performed. A subsequent western blot analysis under reducing conditions revealed that the UBE1-FAT10 conjugate was still detectable confirming that UBE1 and FAT10 formed a stable isopeptide linkage (S1 Fig.).

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.