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.


FAT10ylated UBE1 is degraded by the proteasome.HEK293 cells were transiently transfected with expression plasmids for HA-UBE1 with or without His-3xFLAG-FAT10 (FLAG-FAT10), as indicated. Cell lysates were used for immunoprecipitation with anti-HA-agarose followed by western blot analysis with antibodies reactive against HA or FLAG. Cells were additionally treated for the indicated time periods with cycloheximide (CHX) and/or the proteasome inhibitor MG132, as indicated. Proteins were separated under reducing conditions (4% 2-ME) on 4–12% NuPAGE gels. The upper panels show immunoprecipitated proteins, the lower panels show the protein expression in total cell lysates (load). β-actin was used as loading control. One representative experiment out of four experiments with similar outcomes is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120329.g005: FAT10ylated UBE1 is degraded by the proteasome.HEK293 cells were transiently transfected with expression plasmids for HA-UBE1 with or without His-3xFLAG-FAT10 (FLAG-FAT10), as indicated. Cell lysates were used for immunoprecipitation with anti-HA-agarose followed by western blot analysis with antibodies reactive against HA or FLAG. Cells were additionally treated for the indicated time periods with cycloheximide (CHX) and/or the proteasome inhibitor MG132, as indicated. Proteins were separated under reducing conditions (4% 2-ME) on 4–12% NuPAGE gels. The upper panels show immunoprecipitated proteins, the lower panels show the protein expression in total cell lysates (load). β-actin was used as loading control. One representative experiment out of four experiments with similar outcomes is shown.

Mentions: To obtain information about the influence of FAT10 modification on the activity of UBE1 we addressed the question whether FAT10ylation of UBE1 would have an influence on bulk ubiquitin conjugate formation in cells. To this aim, HEK293 cells were transiently transfected with an expression plasmid for FLAG-FAT10, or endogenous FAT10 expression was induced by treating the cells with IFNγ and TNFα for up to 72 hours. FLAG-FAT10 lacking the C-terminal diglycine motif (FLAG-FAT10ΔGG) served as negative control since it is not covalently conjugated to UBE1 and therefore should not have an influence on the activity of UBE1. Cells were harvested after 24, 48 and 72 hours and cell lysates were subjected to western blot analysis with a ubiquitin-specific antibody. As shown in Fig. 4, the presence of either overexpressed or induced FAT10 had no influence on the level of bulk ubiquitin conjugates. This result may be attributed to the fact that only a small amount of UBE1 becomes modified with FAT10 while the main portion of UBE1 remains unmodified. This might render it difficult to detect differences in bulk ubiquitin conjugate formation. In most cases, covalent modification of proteins with FAT10 leads to their proteasomal degradation as e.g. shown for the FAT10 substrates p62/SQSTM1 or USE1 [22,23]. Therefore it was pertinent to investigate whether FAT10ylation of UBE1 would also guide UBE1 to proteasomal degradation. To monitor the degradation of FAT10ylated UBE1, cells were treated for 2.5 or 5 hours with cycloheximide (CHX) to block de novo protein synthesis. As shown in Fig. 5 (upper right panel) and S3 Fig., the UBE1-FAT10 conjugate was degraded within five hours by about 40% (see quantification of ECL signals in S3 Fig.) and degradation was rescued when cells were treated at the same time with proteasome inhibitors MG132, bortezomib or lactacystin, confirming the results shown in Fig. 1A where the HA-UBE1-FLAG-FAT10 conjugate also accumulated upon MG132 treatment. At the same time, unconjugated UBE1 in the presence or absence of FAT10 remained stable and did not accumulate when the proteasome was inhibited (Fig. 5, load WB: HA and IP: HA, WB: HA). Moreover we have previously shown an accumulation of the HA-UBE1-FLAG-FAT10 conjugate upon specific knockdown of the FAT10 proteasome receptor Rpn10 [33]. Taken together, our data confirm that UBE1 is a novel substrate of FAT10 conjugation, and that a consequence of UBE1 FAT10ylation is to target UBE1 for proteasomal degradation.


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)

FAT10ylated UBE1 is degraded by the proteasome.HEK293 cells were transiently transfected with expression plasmids for HA-UBE1 with or without His-3xFLAG-FAT10 (FLAG-FAT10), as indicated. Cell lysates were used for immunoprecipitation with anti-HA-agarose followed by western blot analysis with antibodies reactive against HA or FLAG. Cells were additionally treated for the indicated time periods with cycloheximide (CHX) and/or the proteasome inhibitor MG132, as indicated. Proteins were separated under reducing conditions (4% 2-ME) on 4–12% NuPAGE gels. The upper panels show immunoprecipitated proteins, the lower panels show the protein expression in total cell lysates (load). β-actin was used as loading control. One representative experiment out of four experiments with similar outcomes is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120329.g005: FAT10ylated UBE1 is degraded by the proteasome.HEK293 cells were transiently transfected with expression plasmids for HA-UBE1 with or without His-3xFLAG-FAT10 (FLAG-FAT10), as indicated. Cell lysates were used for immunoprecipitation with anti-HA-agarose followed by western blot analysis with antibodies reactive against HA or FLAG. Cells were additionally treated for the indicated time periods with cycloheximide (CHX) and/or the proteasome inhibitor MG132, as indicated. Proteins were separated under reducing conditions (4% 2-ME) on 4–12% NuPAGE gels. The upper panels show immunoprecipitated proteins, the lower panels show the protein expression in total cell lysates (load). β-actin was used as loading control. One representative experiment out of four experiments with similar outcomes is shown.
Mentions: To obtain information about the influence of FAT10 modification on the activity of UBE1 we addressed the question whether FAT10ylation of UBE1 would have an influence on bulk ubiquitin conjugate formation in cells. To this aim, HEK293 cells were transiently transfected with an expression plasmid for FLAG-FAT10, or endogenous FAT10 expression was induced by treating the cells with IFNγ and TNFα for up to 72 hours. FLAG-FAT10 lacking the C-terminal diglycine motif (FLAG-FAT10ΔGG) served as negative control since it is not covalently conjugated to UBE1 and therefore should not have an influence on the activity of UBE1. Cells were harvested after 24, 48 and 72 hours and cell lysates were subjected to western blot analysis with a ubiquitin-specific antibody. As shown in Fig. 4, the presence of either overexpressed or induced FAT10 had no influence on the level of bulk ubiquitin conjugates. This result may be attributed to the fact that only a small amount of UBE1 becomes modified with FAT10 while the main portion of UBE1 remains unmodified. This might render it difficult to detect differences in bulk ubiquitin conjugate formation. In most cases, covalent modification of proteins with FAT10 leads to their proteasomal degradation as e.g. shown for the FAT10 substrates p62/SQSTM1 or USE1 [22,23]. Therefore it was pertinent to investigate whether FAT10ylation of UBE1 would also guide UBE1 to proteasomal degradation. To monitor the degradation of FAT10ylated UBE1, cells were treated for 2.5 or 5 hours with cycloheximide (CHX) to block de novo protein synthesis. As shown in Fig. 5 (upper right panel) and S3 Fig., the UBE1-FAT10 conjugate was degraded within five hours by about 40% (see quantification of ECL signals in S3 Fig.) and degradation was rescued when cells were treated at the same time with proteasome inhibitors MG132, bortezomib or lactacystin, confirming the results shown in Fig. 1A where the HA-UBE1-FLAG-FAT10 conjugate also accumulated upon MG132 treatment. At the same time, unconjugated UBE1 in the presence or absence of FAT10 remained stable and did not accumulate when the proteasome was inhibited (Fig. 5, load WB: HA and IP: HA, WB: HA). Moreover we have previously shown an accumulation of the HA-UBE1-FLAG-FAT10 conjugate upon specific knockdown of the FAT10 proteasome receptor Rpn10 [33]. Taken together, our data confirm that UBE1 is a novel substrate of FAT10 conjugation, and that a consequence of UBE1 FAT10ylation is to target UBE1 for proteasomal degradation.

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.