Μελέτη της μεταγωγής σήματος από τους υποδοχείς του TNF και χαρακτηρισμός της πρωτεϊνης USP31
Creators: name: Τζήμας, Χρίστος Γ.
Publisher: name: Aristotle University of Thessaloniki
Year: 2006
Subjects: Ουβικιτίνη
Παράγοντας ογκονέκρωσης
Tumor necrosis factor receptors

description: Οι πρωτεϊνες TRAF συμμετέχουν στη μεταγωγή σήματος από τους υποδοχείς της οικογένειας TNFR. Με την τεχνική των δυο υβριδίων στο σακχαρομύκητα, απομονώθηκε από βιβλιοθήκες cDNA του ανθρώπου, μια πρωτεϊνη που παρουσιάζει ομολογία με τα ένζυμα αποουβικιτίνωσης(USP31, Ubiquitin Specific Protease 31) και αλληλεπιδρά με την πρωτεϊνη TRAF2. Με υβριδισμό τύπου Northern, αναγνωρίστηκαν 2 mRNA καθώς και 2 CDNA που σχετίζονται με την πρωτεϊνη USP31. Διακρίνονται 2 ισομορφές, η πρωτεϊνη USP31(μεγάλη ισομορφή) που περιέχει 1035 αμινοξέα και η πρωτεϊνη USP31S1(μικρή ισομορφή) που περιέχει 485 αμινοξέα. Αποδείχτηκε ότι μόνο η μεγάλη ισομορφή USP31 και όχι η μικρή ισομορφή USP31S1, διαθέτει ενζυμική δράση αποουβικιτινόσης. Επίσης, η πρωτεϊνη USP31 παρουσιάζει εξειδίκευση απέναντι σε αλυσίδες πολυουβικιτίνης που συνδέονται μέσω της λυσίνης-63 και όχι μέσω της λυσίνης 4Β. Η υπερέκφραση της πρωτεϊνης USP31 σε κύτταρα HEK293T παρεμπόδισε τηεν ενεργοποίηση του μεταγραφικού παράγοντα NF-kB που επάγεται από τον TNFo, τον υποδοχέα CD4D, την πρωτεϊνη LMP1, τις πρωτεϊνες TRAF-2 και TRAF-6 και τις κινάσες ΙΚΚα, ΙΚΚβ. Τέλος, οι δύο ισομορφές της πρωτεϊνης USP31 αλληλεπιδρούν με τις πρωτεϊνες TRAF2 και p65/RelA όπως αποδείχτηκε με πειράματα ανοσοκατακρήμνισης
description: The tumor necrosis factor superfamily of cytokines and receptors (TNF/TNFR) activate signalling pathways for cell proliferation, differentiation and apoptosis. CD40 Receptor is a member of the Tumor Necrosis Factor Receptors (TNFR) and is expressed mainly on the surface of B lymphocytes. CD40 interacts with CD40 Ligand (CD154) which is expressed mainly on T lymphocytes. LMP1 is a pseudoreceptor of the TNFR superfamily which mimics CD40 signalling and is constitutively activated. TRAFs constitute a family of proteins that participate in signal transduction by members of the TNF receptor superfamily. The amino terminal region of TRAFs is rich in cysteine and histidine residues, arranged in putative zinc-finger motifs and a more complex RING finger motif. TRAF2 and TRAF6 have a ubiquitin ligase activity which is dependent on the integrity of their RING finger domain and has been associated with the ability to activate the NF-κB and AP1 signalling pathways. Protein modification by ubiquitination has been primarily associated with degradation by the proteasome. However, protein ubiquitination has also been implicated in other cellular functions, including cell cycle regulation, signal transduction, transcription regulation, receptor endocytosis, ribosomal function etc. In previous studies of our research team, a yeast two-hybrid screening was performed in order to elucidate further the TRAF2 signalling mechanism. One of the isolated proteins demonstrated a strong positive interaction with wild type TRAF2, but very weak or no interaction with a mutant TRAF2 at the RING finger domain. This protein was studied further and turned out to be identical to a ubiquitin specific protease named USP31. Comparison of the USP31 cDNA sequence with human ESTs revealed that at least two USP31 cDNAs exist. One cDNA encodes for a 1035-amino-acid long polypeptide (USP31, long isoform) whereas the other encodes for a 485-amino-acid long polypeptide (short isoform, USP31S1). The two isoforms of USP31 share a common amino terminal region of 483 amino acids and contain all eight characteristic motifs of known deubiquitinases. Northern blot analysis of normal tissues and cancer cell lines revealed two major mRNA species of approximately 4.4 and 2.4 kb. The 4.4 kb mRNA was expressed in all normal tissues and cancer cell lines tested, while the 2.4 kb mRNA was expressed only in cancer cell lines. These mRNAs are likely to be derived from alternative splicing and may code for USP31 and USP31S1 respectively. USP31 was examined for its ability to act as a ubiquitin specific protease (USP/UBP). In addition to wild type USP31, a point mutant containing a serine instead of cysteine at position 98 (USP31C98S) was constructed and tested for its enzymatic activity. Cysteine-98 corresponds to a highly conserved cysteine of the catalytic domain of deubiquitinating enzymes (Cys box). USP31, USP31C98S and USP31S1 were tested for their ability to cleave tetraubiquitin in vitro. Wild type full length USP31 readily cleaved the tetrameric substrate to its monomer, dimer and trimer. However, the point mutant USP31C98S and the short isoform USP31S1 were enzymatically inactive. Furthermore, USP31 was tested for its deubiquitinating specificity towards lysine-48 or lysine-63-linked polyubiquitin chains. Lysine-48-linked polyubiquitin chains usually mark proteins for proteasomal degradation whereas lysine-63-linked polyubiquitin chains have been associated primarily with non-proteolytic regulatory processes. Wild type USP31 cleaved almost completely lysine-63-linked polyubiquitin. Moreover, it cleaved partially lysine-48-linked polyubiquitin. The point mutant USP31C98S or the short isoform USP31S1 did not cleave any of the two substrates. These results demonstrate that USP31 is a deubiquitinating enzyme that cleaves preferentially lysine-63-linked polyubiquitin chains. USP31 can also cleave lysine-48-linked polyubiquitin chains albeit to a lesser extent. The ability of USP31 to interact with TRAF2 in mammalian cells was tested by co-immunoprecipitation experiments in HEK 293T cells. These experiments confirmed the specific interaction of USP31 and TRAF2 which requires an intact RING finger domain of TRAF2. Indirect immunofluorescense showed that USP31 is localized predominantly in the nucleus and to some extent in the cytoplasm. The nuclear localization of USP31 prompted us to investigate potential target molecules in the nucleus. p65/RelA was considered as a possible target of USP31, since it is a major component of NF-κΒ and it is ubiquitinated and degraded by the proteasome in the nucleus. The ability of p65/RelA to interact with USP31 was confirmed by co-immunoprecipitation experiments in HEK 293T cells. To determine whether USP31 is implicated in NF-κΒ activation, transient cotransfection experiments were performed in HEK 293T cells. USP31 overexpression inhibited NF-κB activation mediated by TNFα, CD40 and LMP1. USP31 also inhibited NF-κΒ activation mediated by TRAF2 and TRAF6, as well as by the IKKα and IKKβ kinases. Overexpression of USP31 did not affect Smad mediated transcription activation, indicating that USP31 is not a general regulator of transcription. These results reinforce the notion that USP31 has a specific role in NF-κB activation induced by TRAFs and members of the TNFR family. Additionally, some of its targets are located downstream of IKKs, and possibly in the nucleus. The latter is consistent with the nuclear localization of USP31. Another object of this thesis was the design of inhibitory peptides towards TNFR mediated signal transduction. The carboxyterminal activation regions (CTARs) CTAR1 and CTAR2 on the cytoplasmic tail of LMP1 are responsible for activation of NF-κΒ and binding of signalling molecules like TRAFs. Recombinant proteins which mimic the structure of CTARs could inhibit TNFR signalling, due to competition for binding signalling molecules. For this purpose CTAR1 and CTAR2 were cloned as fusion proteins with GST and the membrane translocating sequence, MTS. The recombinant proteins were expressed in bacteria and were purified by affinity chromatography. In concentration and time course experiments the recombinant proteins were detected in HEK 293T cell extracts. Proteins were detected at concentrations larger than 5 μΜ and even after one hour of incubation. The inhibitory effect of the recombinant proteins on signal transduction was studied by transient cotransfection experiments. The ability of the recombinant proteins to inhibit LMP1 mediated NF-κΒ activation was tested in HEK 293T cells. The recombinant proteins CTAR1 and CTAR2 inhibit LMP1 mediated NF-κΒ activation, at concentrations over 5 μΜ. However, it was rather a non specific effect since it was also observed to the control experiment. This effect could be attributed to a non specific inhibition mediated by high protein concentrations that are required by this system