The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATPdependent ubiquitination and degradation by the proteasome. We have investigated the inf luence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP͞ubiquitin͞ proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin͞proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.
Research School of Chemistry Australian National University Canberra, ACT 0200, AustraliaThe PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains. Keywords: PYRIN domain; human ASC; NMR spectroscopy; threedimensional protein structure; death-domain superfamily *Corresponding author IntroductionThe death domain fold is the unifying structural motif of a superfamily of protein domains comprising the death domain (DD) itself, 1 the death effector domain (DED) 2 and the caspase recruitment domain (CARD).3 Their names express the prominent roles of these domains in programmed cell death. Domains from all three subfamilies occur as modules in diverse human apoptosis proteins in a variety of domain contexts. They all form a-helical bundles acting as adapters in signalling pathways and recruiting other proteins into signalling complexes. 4 Domains from the different death domain subfamilies tend to interact with each other, suggesting that their common fold was frequently reused as a module during the evolution of apoptotic adapter proteins, providing the structural backbone of the signalling pathways that control programmed cell death. Commensurate with their biological importance and despite often poor solubility due to self-association, several The PYRIN domain, also called DAPIN, PAAD or PYD, is a recently identified domain that has been suggested to present a new member of the DD superfamily. 15 -20 No experimentally determined structure of a PYRIN domain has been reported to date. An attempt to solve the structure of the PYRIN domain of CARD7 failed due to limited solubility. 17PYRIN domains are located at the N terminus of proteins that are linked intimately to a variety of human diseases, ranging from cancer to inflammatory syndromes. 15,16,18,21,22 The PYRIN domain was originally found in pyrin, the product of the fa...
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