Investigation of the FAT10 conjugation pathway

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Zusammenfassung

Posttranslational modifications are important means to alter a proteins’ activity, function, stability or its intracellular localization. The post-translational conjugation of one or more molecules of Ub and ubiquitin-like proteins (UBLs) to selected substrates, namely ubiquitination, is one of the most important and multifaceted regulatory mechanisms in biology and requires the sequential interaction of a 3-step enzyme cascade. The initiating step for ubiquitin or UBL conjugation to its target proteins is the formation of an energy-rich thioester at its C-terminus. This activation takes places through an ubiquitin activating E1 enzyme. The activated ubiquitin is then transferred in a trans-thioesterification cascade to one of multiple E2 conjugating enzymes. The ubiquitin-charged E2 enzyme and a specific substrate protein are then both bound by a ubiquitin protein ligase (E3), which catalyzes the transfer of the activated ubiquitin between its carboxy-terminal hydroxyl-group onto the ε-amino-side chain of an internal lysine residue of the acceptor protein. Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, SUMO, NEDD8, and ISG15 are transferred by a similar E1-E2-E3 multi-enzyme cascade to its targets. For the ubiquitin-like modifier FAT10, the enzyme cascade has not yet been characterized completely.



The IFN-γ and TNF-α inducible modifier FAT10 is a young member of ubiquitin-like proteins, which can be conjugated to target proteins via its C-terminal diglycine motif. Moreover, it is to date the only identified ubiquitin-like protein, which can assign substrate proteins, in an ubiquitin-independent manner, for proteasomal degradation.



Recently, the FAT10 activating enzyme (E1) UBA6 and a FAT10 conjugating enzyme (E2), namely USE1 was identified, which interestingly, was at the same time the first known substrate for FAT10, as it was auto-FAT10ylated in cis.

The aim of this thesis was the characterization of the FAT10 conjugation pathway, starting with the identification of UBA6 interacting proteins in a yeast two-hybrid screen.

Given that at the beginning of the doctoral thesis no FAT10 conjugating E2 enzymes were known so far, the focus here was the identification of potential FAT10 E2 enzymes. In a yeast two-hybrid approach, a direct interaction of UBA6 and the C-terminal end of BRUCE, containing the entire highly conserved ubiquitin conjugating domain, could be shown. This interaction was further characterized with a construct encoding for full length BRUCE via co-immunoprecipitation experiments, especially in terms of exhibiting a putative FAT10 E2 function.



A further aim of this doctoral thesis was the identification of putative FAT10 substrates. In a yeast two-hybrid screen with the putative FAT10 E3 ligase TRIM11 and a cDNA-library from human thymus, a specific interaction between TRIM11 and JunB as well as with Ambra1 could be observed. The RING finger containing E3 ligase TRIM11 was previously identified in a yeast two-hybrid screen with FAT10 and a cDNA-library from human thymus. For both JunB and Ambra1, a specific interaction with TRIM11 could be verified in co-immunoprecipitation assays. Moreover, it could be demonstrated that JunB becomes covalently linked to either ubiquitin or FAT10, but not in the presence of a FAT10 mutant, lacking the di-glycine motif, which is required for isopeptide linkages to substrate proteins. This result points to a conjugate formation between JunB with the ubiquitin-like modifier ubiquitin and FAT10, which indicates, that JunB is a ubiquitin as well as FAT10 specific substrate. Moreover, a non-covalent linkage of JunB with either ubiquitin or FAT10 could be detected. Furthermore, cycloheximide experiments revealed evidence that not only the isopeptide linked form of JunB and FAT10 became assigned for proteasomal degradation but also JunB, which was non-covalently modified with FAT10. Proteasome inhibition with MG132 led to an accumulation of the JunB-FAT10 conjugate, which would be expected for a FAT10 substrate. The role of TRIM11 as a FAT10 specific E3 ligase could not be solved definitely, due to the fact that JunB, in presence of recombinant FAT10, UBA6 (E1), USE1 (E2) and TRIM11 (putative FAT10 E3) did not become FAT10ylated in vitro. TRIM11 overexpression resulted in a decreased protein level of ubiquitin, FAT10, JunB and also the JunB-ubiquitin and JunB-FAT10 conjugates.
A clear co-localization of FAT10 and JunB at the nuclear membrane could be detected by means of confocal microscopy. MG132 treatment caused the translocation of JunB into the cytosol. As a presumable functional consequence, JunB FAT10ylation led to a reduced JunB trans-activating capacity in reporter assays.



In case of Ambra1, a non-covalent interaction with ubiquitin and FAT10 could be verified in co-immunoprecipitation experiments. Addition of MG132 led to an accumulation of over-expressed Ambra1, indicating that Ambra1 becomes degraded by the proteasome. However, no accumulation after proteasome inhibition was observable, when Ambra1 was ectopically co-expressed with FAT10, suggesting that the non-covalent interaction with FAT10 led to a different degradation mechanism other than the proteasome. Experiments with confocal microscopy illustrate an unambiguous co-localization of FAT10 and Ambra1 in punctuated structures, suggesting that the interaction of Ambra1 and FAT10 led to translocation of both proteins into punctuated structures.

Zusammenfassung in einer weiteren Sprache

Posttranslationale Modifikationen können die Aktivität, Funktion, Stabilität oder die intrazelluläre Lokalisation von Proteinen verändern. Posttranslationale Konjugation von einen oder mehreren Ubiquitin oder Ubiquitin-ähnlichen Proteinen zu ausgewählten Substraten, Ubiquitinierung genannt, ist eines der wichtigsten und vielfältigsten regulatorischen Mechanismen in der Biologie und erfordert das fortlaufende Zusammenspiel einer 3-Schritt Enzymkaskade. Initiierender Schritt zur Konjugation von Ubiquitin oder Ubiquitin-ähnlichen Proteinen an seine Zielproteine ist die Ausbildung eines energiereichen Thioesters an seinem C-Terminus. Diese Aktivierung erfolgt durch ein Ubiquitin-aktivierendes E1 Enzym. Das aktivierte Ubiquitin wird in einer Transesterifizierungskaskade auf eines von mehreren Ubiquitin konjugierenden E2-Enzymen übertragen. Das Ubiquitin-beladene E2 und ein spezifisches Substratprotein werden dann von einer Ubiquitin Proteinligase (E3) gebunden, welche den Transfer des aktivierten Ubiquitins zwischen seiner carboxy-terminalen Hydroxylgruppe auf die ε-Amino-Seitenkette eines internen Lysinrestes des Akzeptorproteins katalysiert. Ubiquitin-ähnliche Proteine (UBLs) wie beispielsweise SUMO, NEDD8 und ISG15 werden durch eine vergleichbare E1-E2-E3 Multienzym-Kaskade an ihre Zielproteine ligiert. Für das Ubiquitin-ähnliche Protein FAT10 ist der Konjugierungsmechanismus noch nicht vollständig erforscht.



Das IFN-γ und TNF-α induzierbare Protein FAT10 ist ein junges Mitglied der Ubiquitin-ähnlichen Proteine, welches über sein C-terminales Di-Glycin-Motiv kovalent an Zielproteine binden kann. Zudem ist es bisher das einzig identifizierte Ubiquitin-ähnliche Protein, welches Substratproteine Ubiquitin-unabhängig für den proteasomalen Abbau markieren kann. Erst vor Kurzem wurde ein FAT10 aktivierendes E1-Enzym, UBA6 und ein FAT10 konjugierendes E2 Enzym, USE1, identifiziert, welches interessanterweise zugleich das erste bekannte Substrat für FAT10 darstellt, da es in cis autoFAT10yliert wird.



Das Ziel dieser Doktorarbeit war die Charakterisierung des Konjugierungswegs von FAT10, beginnend mit der Identifizierung von UBA6 interagierenden Proteinen in einem „Yeast two-hybrid Screen“. Da zu Beginn der Doktorarbeit noch kein FAT10-spezifisches E2 Enzym bekannt war, lag der Fokus hier in der Identifikation von möglichen FAT10 E2 Enzymen.





In einem „Yeast two Hybrid Screen“ konnte eine direkte Interaktion von UBA6 und dem C-terminalen Ende von BRUCE gezeigt werden, welches eine vollständige hochkonservierte Ubiquitin-konjugierende Domäne enthält. Diese Interaktion wurde in dieser Arbeit mittels Co-Immunopräzipitations-Experimente mit einem Expressionskonstrukt, welches für das vollständige BRUCE-Protein kodiert, besonders hinsichtlich einer möglichen FAT10 E2 Funktion weiter charakterisiert.



Ein weiteres Ziel der Doktorarbeit lag in der Identifikation von möglichen FAT10 Substraten. In einem „Yeast Two-Hybrid Screen“ mit einer möglichen FAT10 E3 Ligase TRIM11 als „bait“-Protein und einer cDNA-Bank aus humanem Thymus, konnte eine spezifische Interaktion zwischen TRIM11 sowohl mit JunB, als auch mit Ambra1 nachgewiesen werden. Die Ring-Finger E3 Ligase TRIM11 wurde zuvor in einem „Yeast Two-Hybrid Screen“ mit FAT10 und einer cDNA-Bank aus humanem Thymus identifiziert. Sowohl für JunB als auch für Ambra1 konnte eine spezifische Interaktion in HEK293 Zellen mittels Co-Immunopräzipitations-Experimenten mit TRIM11 ermittelt werden. Zudem konnte gezeigt werden, dass JunB kovalent mit Ubiquitin oder FAT10 modifiziert wird, nicht jedoch in Anwesenheit einer FAT10 Mutante, der das C-terminale Di-Glycin Motiv fehlt, welches für die Isopeptidbindung an Substratproteine verantwortlich ist. Dieses Ergebnis weist auf die Bildung eines Konjugats zwischen JunB mit beiden Ubquitin-ähnlichen Proteinen hin, was vermuten lässt, dass JunB sowohl ein Ubiquitin, als auch ein FAT10 spezifisches Substrat darstellt. Zudem konnte auch eine nicht kovalente Bindung von JunB sowohl mit Ubiquitin als auch FAT10 detektiert werden. Ferner konnte mittels „Cycloheximid-Chase“ Experimenten verdeutlicht werden, dass nicht nur die Isopeptid-gebundene Form von JunB und FAT10 für den proteasomale Abbau markiert wird, sondern auch JunB, welches nicht kovalent mit FAT10 modifiziert wurde. Proteasomale Inhibition mittels der Zugabe von MG132 führte zu einer Akkumulation des JunB-FAT10 Konjugats, welches im Falle eines FAT10 Substrats erwartet werden würde. Die Rolle von TRIM11 als FAT10 spezifische E3 Ligase mit JunB als Substrat konnte nicht eindeutig geklärt werden, da JunB in vitro in Anwesenheit von rekombinantem FAT10, UBA6 (E1), USE1 (E2) und TRIM11 (mögliche E3-Ligase) nicht FAT10yliert wurde. Überexpression von TRIM11 führte zu einer Herunterregulierung von Ubiquitin, FAT10 und JunB, als auch den JunB-Ubiquitin oder JunB-FAT10 Konjugaten auf Proteinebene. Mittels Konfokalmikroskopie konnte eine klare Co-Lokalisation von FAT10 und JunB an der nukleären Membran detektiert werden. Die Zugabe von MG132 führte zu einer Translokation von JunB in das Cytoplasma. Als vermutlich funktionale Konsequenz hatte JunB FAT10ylierung in Reporterassays eine deutlich verminderte JunB Transaktivierungs-Leistung zur Folge.



Im Falle von Ambra1 konnte eine nicht-kovalente Bindung sowohl mit Ubiquitin, als auch mit FAT10 mittels Co-Immunopräzipitationsversuchen nachgewiesen werden. MG132 Zugabe führte zu einer Akkumulation von überexprimiertem Ambra1, welches darauf hinweist, dass Ambra1 proteasomal degradiert wird. Akkumulation nach proteasomaler Inhibition konnte allerdings nicht beobachtet werden, wenn Ambra1 und FAT10 co-exprimiert wurden, was darauf schließen lässt, dass die nicht-kovalente Interaktion mit FAT10 vermutlich zu einem anderen oder zusätzlichen Degradationsmechanismus führt. Experimente mit Konfokalmikroskopie verdeutlichen eine eindeutige Co-Lokalisation von FAT10 und Ambra1 in aggresomalen Strukturen, was vermuten lässt, dass die Interaktion von Ambra1 und FAT10 zu einer Translokalisation beider Proteine hin zu solchen Strukturen führt.

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570 Biowissenschaften, Biologie
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ISO 690RYU, Stella, 2012. Investigation of the FAT10 conjugation pathway [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Ryu2012Inves-19379,
  year={2012},
  title={Investigation of the FAT10 conjugation pathway},
  author={Ryu, Stella},
  address={Konstanz},
  school={Universität Konstanz}
}
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    <dcterms:abstract xml:lang="eng">Posttranslational modifications are important means to alter a proteins’ activity, function, stability or its intracellular localization. The post-translational conjugation of one or more molecules of Ub and ubiquitin-like proteins (UBLs) to selected substrates, namely ubiquitination, is one of the most important and multifaceted regulatory mechanisms in biology and requires the sequential interaction of a 3-step enzyme cascade. The initiating step for ubiquitin or UBL conjugation to its target proteins is the formation of an energy-rich thioester at its C-terminus. This activation takes places through an ubiquitin activating E1 enzyme. The activated ubiquitin is then transferred in a trans-thioesterification cascade to one of multiple E2 conjugating enzymes. The ubiquitin-charged E2 enzyme and a specific substrate protein are then both bound by a ubiquitin protein ligase (E3), which catalyzes the transfer of the activated ubiquitin between its carboxy-terminal hydroxyl-group onto the ε-amino-side chain of an internal lysine residue of the acceptor protein. Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, SUMO, NEDD8, and ISG15 are transferred by a similar E1-E2-E3 multi-enzyme cascade to its targets. For the ubiquitin-like modifier FAT10, the enzyme cascade has not yet been characterized completely.&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;The IFN-γ and TNF-α inducible modifier FAT10 is a young member of ubiquitin-like proteins, which can be conjugated to target proteins via its C-terminal diglycine motif. Moreover, it is to date the only identified ubiquitin-like protein, which can assign substrate proteins, in an ubiquitin-independent manner, for proteasomal degradation.&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;Recently, the FAT10 activating enzyme (E1) UBA6 and a FAT10 conjugating enzyme (E2), namely USE1 was identified, which interestingly, was at the same time the first known substrate for FAT10, as it was auto-FAT10ylated in cis.&lt;br /&gt;&lt;br /&gt;The aim of this thesis was the characterization of the FAT10 conjugation pathway, starting with the identification of UBA6 interacting proteins in a yeast two-hybrid screen.&lt;br /&gt;&lt;br /&gt;Given that at the beginning of the doctoral thesis no FAT10 conjugating E2 enzymes were known so far, the focus here was the identification of potential FAT10 E2 enzymes. In a yeast two-hybrid approach, a direct interaction of UBA6 and the C-terminal end of BRUCE, containing the entire highly conserved ubiquitin conjugating domain, could be shown. This interaction was further characterized with a construct encoding for full length BRUCE via co-immunoprecipitation experiments, especially in terms of exhibiting a putative FAT10 E2 function.&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;A further aim of this doctoral thesis was the identification of putative FAT10 substrates. In a yeast two-hybrid screen with the putative FAT10 E3 ligase TRIM11 and a cDNA-library from human thymus, a specific interaction between TRIM11 and JunB as well as with Ambra1 could be observed. The RING finger containing E3 ligase TRIM11 was previously identified in a yeast two-hybrid screen with FAT10 and a cDNA-library from human thymus. For both JunB and Ambra1, a specific interaction with TRIM11 could be verified in co-immunoprecipitation assays. Moreover, it could be demonstrated that JunB becomes covalently linked to either ubiquitin or FAT10, but not in the presence of a FAT10 mutant, lacking the di-glycine motif, which is required for isopeptide linkages to substrate proteins. This result points to a conjugate formation between JunB with the ubiquitin-like modifier ubiquitin and FAT10, which indicates, that JunB is a ubiquitin as well as FAT10 specific substrate. Moreover, a non-covalent linkage of JunB with either ubiquitin or FAT10 could be detected. Furthermore, cycloheximide experiments revealed evidence that not only the isopeptide linked form of JunB and FAT10 became assigned for proteasomal degradation but also JunB, which was non-covalently modified with FAT10. Proteasome inhibition with MG132 led to an accumulation of the JunB-FAT10 conjugate, which would be expected for a FAT10 substrate. The role of TRIM11 as a FAT10 specific E3 ligase could not be solved definitely, due to the fact that JunB, in presence of recombinant FAT10, UBA6 (E1), USE1 (E2) and TRIM11 (putative FAT10 E3) did not become FAT10ylated in vitro. TRIM11 overexpression resulted in a decreased protein level of ubiquitin, FAT10, JunB and also the JunB-ubiquitin and JunB-FAT10 conjugates.&lt;br /&gt;A clear co-localization of FAT10 and JunB at the nuclear membrane could be detected by means of confocal microscopy. MG132 treatment caused the translocation of JunB into the cytosol. As a presumable functional consequence, JunB FAT10ylation led to a reduced JunB trans-activating capacity in reporter assays.&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;In case of Ambra1, a non-covalent interaction with ubiquitin and FAT10 could be verified in co-immunoprecipitation experiments. Addition of MG132 led to an accumulation of over-expressed Ambra1, indicating that Ambra1 becomes degraded by the proteasome. However, no accumulation after proteasome inhibition was observable, when Ambra1 was ectopically co-expressed with FAT10, suggesting that the non-covalent interaction with FAT10 led to a different degradation mechanism other than the proteasome. Experiments with confocal microscopy illustrate an unambiguous co-localization of FAT10 and Ambra1 in punctuated structures, suggesting that the interaction of Ambra1 and FAT10 led to translocation of both proteins into punctuated structures.</dcterms:abstract>
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