KOPS - The Institutional Repository of the University of Konstanz

Characterization of novel E1 and E2 enzymes and their role in ubiquitin and FAT10 conjugation

Characterization of novel E1 and E2 enzymes and their role in ubiquitin and FAT10 conjugation

Cite This

Files in this item

Checksum: MD5:147959222e25016adcbda4a3467b49db

PELZER, Christiane, 2009. Characterization of novel E1 and E2 enzymes and their role in ubiquitin and FAT10 conjugation [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Pelzer2009Chara-7414, title={Characterization of novel E1 and E2 enzymes and their role in ubiquitin and FAT10 conjugation}, year={2009}, author={Pelzer, Christiane}, address={Konstanz}, school={Universität Konstanz} }

application/pdf 2009 A three-step enzyme cascade carries out the covalent conjugation of ubiquitin to target proteins. The formation of an isopeptide bond between ubiquitin and a lysine residue in a substrate requires the activation of the C-terminal glycine residue of ubiquitin by an activating enzyme (E1), which first adenylates ubiquitin and then transfers it onto its active site cysteine to form a thioester bond. The activated ubiquitin is subsequently passed onto a cysteine in the active site of a ubiquitin-conjugating enzyme (E2). 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 onto the substrate protein. This system was thought to be arranged in a hierarchal way with one E1, dozens of E2 enzymes and hundreds of E3 enzymes. The mechanism of conjugation not only applies to ubiquitin but also to the family of ubiquitin-like modifiers like ISG15, NEDD8 and SUMO. For the IFN-γ and TNF-α inducible modifier FAT10 no enzyme cascade has been characterized, even though it is known that FAT10 and its conjugates are rapidly degraded by the proteasome, which can be enhanced by the interaction of NUB1L.<br />The aim of this thesis was the characterization of the FAT10 conjugation pathway, starting with the identification of novel activating enzymes. The identification of the so far elusive FAT10 conjugate in a murine fibroblast cell line was another aim of this study.<br />During the course of experiments for the search of a new activating enzyme, UBA6 (UBE1L2) was identified as a second ubiquitin-activating enzyme, which can substitute for UBE1 in ubiquitylation assays with UBCH5B as E2 enzyme and E6-AP and HECTH9 as E3 enzymes. Furthermore, the MDM2-mediated ubiquitylation of p53 can occur, when UBE1 is replaced by UBA6. UBA6 mRNA is up-regulated in testes of mice, indicating an organ-specific function. Other well-known E1 enzymes were tested negative for the activation of the ubiquitin-like modifier FAT10. UBA6 turned out to interact with FAT10 in a yeast two-hybrid screen, which encouraged us to exchange the tags on HA-UBA6 and GST-FAT10 to GST-UBA6 and HA-FAT10 in GST pulldown assays. Under these conditions UBA6 could be shown to activate not only ubiquitin but also FAT10.<br />The UBA6-specific E2 enzyme USE1 could interact with FAT10 by a yeast two-hybrid screening. UBA6-activated FAT10 can be transferred onto USE1 in vitro and in vivo, leading to the formation of covalent conjugates. This was achieved by auto-FAT10ylation of USE1 in cis but not in trans. siRNA mediated down-regulation of USE1 mRNA resulted in a reduction of FAT10 conjugate formation suggesting that USE1 is an important E2 enzyme in the FAT10 conjugation cascade. The co-expression of FAT10 and NUB1L led to the degradation of the USE1-FAT10 conjugate by the proteasome pointing to a negative self-regulation by the auto-modification of USE1 with FAT10.<br />This thesis further showed that the protein linked to FAT10 in a stably transfected murine fibroblast cell line was identified as a fragment of the SV40 large T antigen. Peptides from the mass-spectrometric analysis revealed that the fragment linked to FAT10 mainly consists of the DNA binding domain of the large T antigen. A monoclonal antibody against the N-terminal 59 amino acids of large T did not recognize the conjugate indicating that also an N-terminal part is missing. The large T fragment did not bind to the FAT10 mutant, which lacks the diglycine motif.<br />In conclusion, the results presented in this thesis identify a second ubiquitin-activating enzyme and provide insight into the conjugation pathway of FAT10. Furthermore, they strengthen the role of FAT10 in substrate degradation and reveal an auto-regulatory process for FAT10 attachment with the help of NUB1L. The findings provide useful tools to identify FAT10 substrates and to elucidate the physiological role of FAT10 in the immune response in the future. Characterization of novel E1 and E2 enzymes and their role in ubiquitin and FAT10 conjugation eng 2011-05-04T10:02:08Z Charakterisierung neuer E1- und E2-Enzyme und ihre Rolle in der Ubiquitin- und FAT10-Konjugierung 2011-03-24T17:34:14Z terms-of-use Pelzer, Christiane Pelzer, Christiane

Downloads since Oct 1, 2014 (Information about access statistics)

Diss_Pelzer.pdf 351

This item appears in the following Collection(s)

Search KOPS


My Account