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Function and regulation of promyelocytic leukemia (PML) nuclear bodies (NB) and their role in innate immunity and antiviral resistance

Final Activity Report Summary - PML NUCLEAR BODIES (Function and regulation of promyelocytic leukemia (PML) nuclear bodies (NB) and their role in innate immunity and antiviral resistance)

The nucleus of a eukaryotic cell is the organelle where the genetic information is stored in the Deoxyribonucleic acid (DNA) and transcribed into Ribonucleic acid (RNA). Within the nucleus several discrete structures or sub-nuclear organelles exist and are involved in the regulation of gene expression, such as nucleoli, Cajal bodies, nuclear speckles and Promyelocytic leukemia (PML) bodies. Various characteristics were attributed to PML-bodies, including an active role in transcription, DNA repair, DNA replication, RNA, or a role as intranuclear protein depots. They were identified by the presence of the PML protein, which was essential for their formation because of its multimerisation and the subsequent recruitment of other proteins.

In a special form of leukaemia, the PML-protein was fused to a transcription factor, leading to uncontrolled cells' proliferation and PML-bodies' dispersal. The PML protein also played a role in a number of human infectious diseases by viruses which needed the nuclear machinery of the host for their own replication. Infection with these viruses also led to dispersal of PML-bodies. The expression of the PML gene was highly up-regulated by interferons which pointed towards a function in innate immunity against infectious diseases. The assembly of PML-bodies was regulated by the covalent attachment of the Small ubiquitin-like modifier (SUMO) protein to PML. The SUMO conjugated form of PML then displayed binding sites for other proteins which were localised to PML bodies.

Over 30 other proteins were found in PML-bodies so far, at least under certain conditions. The members of the Sp100 protein family were the best characterised out of these. Like PML, Sp100 was induced by interferons and could be modified by SUMO which regulated the binding to the heterochromatin binding proten 1 (HP1). Sp100 and the related proteins Sp110 and Sp140 contained DNA-binding domains and were found to function as transcriptional regulators of various genes, including certain viruses. Apart from these similarities in function and regulation, there was no evidence for a direct interaction between PML and Sp100. For most proteins, the mechanism of their recruitment into PML-bodies was unclear.

In order to understand the assembly and regulation of PML-bodies and their antiviral activity, we studied the molecular interactions of the PML and Sp100 proteins. The complementary Deoxyribonucleic acids (cDNAs) of several PML-localised proteins and the enzymes involved in SUMO modification were cloned into bacterial expression vectors. We designed, based on these, a large number of deletion constructs around the previously characterised domains. The recombinant proteins were produced in bacteria and most of them could be isolated and purified in high yields. It turned out that all proteins comprising the N-terminal regions of the PML or Sp100 proteins had a strong tendency to form large complexes. These homotypic interactions of PML and Sp100 could also be shown by using the yeast-two hybrid system.

Furthermore, we could analyse the binding of Sp100 to HP1 and the oligomerisation properties of Sp110 and Sp140. The interactions were verified by affinity purification of proteins from cell extracts with our recombinately produced proteins. In order to obtain these proteins in their SUMO conjugated form, we cloned the components of the SUMO modification enzyme cascade into commercially available co-expression vectors. The production of SUMO conjugated PML in bacteria could be demonstrated. With these modified proteins at hand we were then able to analyse the importance of the SUMO conjugation for the assembly of PML-bodies and their antiviral function.