Characterisation of the Interaction between FAT10 and its Substrate Protein p62

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2014
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The ubiquitin-like modifier FAT10 has a C-terminal diglycine motif which is required for the conjugation to lysines (K) in its substrate proteins via isopeptide bonds. The biological function of FAT10, besides the proteasomal degradation of substrate proteins remains obscure. Sequestosome 1 (SQSTM1/p62) was found to be mono-FAT10ylated at several lysines and a non-covalent interaction between FAT10 and p62 was detectable too. The FAT10ylation of p62 leads to its proteasomal degradation. p62 can interact with a large number of proteins and changes its face by altering the binding partner(s). It is required for the formation of ubiquitylated protein aggregates and was found to act as a shuttling factor which links those aggregates to the autophagy machinery.


The aim of this study was to further characterise the covalent and non-covalent interaction between FAT10 and p62. Therefore, in vitro interaction studies with either recombinant proteins or proteins which were expressed in HEK293T cells were performed. By using a lysineless p62 mutant, the FAT10ylation was not always completely abolished. p62 deletion proteins were used in order to identify the covalent and non-covalent interaction domains. The deletion of the PB1, the NPI, the TRAF or the N-terminal PEST domain of p62 were found to impede the FAT10ylation. By the mutation of single lysines, it was shown that the lysines of p62 which become FAT10ylated seem to be redundant. For the non-covalent interaction with FAT10, the ZZ, the LIR, the CPI and the C-terminus of the C-terminal PEST domain of HA-p62 seem to be dispensable. By using non-phosphorylation, phospho-mimicking and non-oligomerisation p62 mutants it was shown that neither the phosphorylation status at seine 403, nor the oligomerisation capability of p62 seem to be prerequisites for the interaction with FAT10. An isolated PB1 domain of p62 did not interact with Flag-FAT10, neither covalently or non-covalently. According to the cycloheximide chase, proteasomal degradation rather than autophagosomal degradation is involved in the degradation of the FAT10-p62 conjugate. There was no interaction detectable between FAT10 and other autophagic adaptor proteins such as NBR1, NDP52 and OPTN.

Zusammenfassung in einer weiteren Sprache

Das Ubiquitin verwandte Protein FAT10 bildet über ein C-terminales Glycin-Glycin Motiv Isopeptide Brücken mit den Lysinen in seinen Substratproteinen. Die biologische Funktion von FAT10, neben dem proteasomalen Abbau der modifizierten Proteine, ist noch weitestgehend ungeklärt. Das Protein Sequestosome 1 (SQSTM1/p62) wird an verschiedenen Lysinen mono-FAT10yliert und interagiert auch nicht-kovalent mit FAT10. Die FAT10ylierung von p62 führt zu dessen proteasomalen Abbau. Durch die Interaktion mit einer großen Anzahl von Proteinen, übt p62 viele verschiedene Funktionen aus. Es ist zum Beispiel an der Bildung von Ubiquitin positiven Protein Aggregaten beteiligt und führt diese zum autophagosomalen Abbau.


Das Ziel dieser Studie war es, die Interaktionen zwischen FAT10 und p62 zu charakterisieren. Hierzu wurden in vitro Interaktionsstudien mit rekombinanten Proteinen und mit in HEK293T Zellen exprimierten Proteinen durchgeführt. Bei einer Lysin-freien p62 Mutante war die FAT10ylierung nicht immer vollständig verhindert. p62 Mutanten mit fehlenden Domänen wurden für die Identifizierung der kovalenten und nicht-kovalenten Interaktionsdomänen verwendet. Bei den p62 Mutanten ohne PB1, NPI, TRAF oder N-terminalen PEST Domäne waren keine FAT10-p62 Konjugate detektierbar. Die Analyse einzelner p62 Lysin Mutanten ergab, dass die für die FAT10ylierung verwendeten Lysine vermutlich redundant sind. Die ZZ, die LIR, die CPI Domäne und der C-Terminus der C-terminalen PEST Domäne von p62 sind scheinbar für die nicht-kovalente Interaktion mit FAT10 verzichtbar. Anhand von nicht phosphorylierbaren, „phospho-mimicking“ und nicht oligomerisierenden p62 Mutanten wurde gezeigt, dass weder der Phosphorylierungsstatus an Serin 403 noch die Fähigkeit von p62 Oligomere zu bilden Voraussetzungen für die Interaktion mit FAT10 sind. Mit einer isolierten p62 PB1 Domäne wurde gezeigt, dass diese weder kovalent noch nicht-kovalent mit p62 interagiert. Ein Cycloheximide „Chase“ ergab, dass das Proteasom der bevorzugte Abbauweg für das FAT10-p62 Konjugat ist. Die Untersuchung der autophagosomalen Adapterproteinen NBR1, NDP52 und OPTN auf ihre Interaktionsfähigkeit mit FAT10 blieb ohne weitere Treffer.

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
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FAT10, p62, NBR1, OPTN, NDP52, Autophagy, Proteasome
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ISO 690KLUGE, Kathrin, 2014. Characterisation of the Interaction between FAT10 and its Substrate Protein p62 [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Kluge2014Chara-27947,
  year={2014},
  title={Characterisation of the Interaction between FAT10 and its Substrate Protein p62},
  author={Kluge, Kathrin},
  address={Konstanz},
  school={Universität Konstanz}
}
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