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The balance of ubiquitin conjugation and deconjugation

Final Report Summary - UBIQUITIN BALANCE (The balance of ubiquitin conjugation and deconjugation)

Ubiquitin conjugation is an important system to convey information in the cell. A protein that is modified with one or more ubiquitins can be recognized, relocalized or broken down by the proteasome, dependent on the type of ubiquitin chains and sometimes on the site of the modification. The important role of ubiquitin in crucial cellular pathways make it attractive as a drug target, but for effective drug design it is important that the regulation of the enzymes involved is well understood.

In this project we have studied the regulation of the process of ubiquitination and deubiquitination in chromatin regulation. One focus in our studies was the specifiity of E3 ligases. In these studies we have resolved how RNF168 produces H2A ubiquitination at K13/15, a novel chromatin mark, that is specific for DNA damage and the regulation of its use. Intriguingly we could show that the target plays critical catalytic roles in the reaction, both for H2A and for PCNA.

Regulation of ubiquitination levels is also carefully controled by control of the enzymatic activity of deubiquitinating enzymes (DUBs). In this project we have analyzed the mechanisms of different DUBs. We have found different regulatory mechanisms in different ubiquitin specific proteases (USPs), that nevertheless make use of the same switching loop in the catalytic domain. Interestingly, the catalytic domains of USP4 and USP7 are not very active, but they can be activated by interaction with additional domains. In the case of USP4 this promotes release of the product ubiquitin, whereas in the case of USP7 this promotes ubiquitin binding and rearrangement of the catalytic triad. In both cases the inhibition of the catalytic domain is controlled by the same switching loop. In the UCH family of DUBs the regulation is purely at the level of ubiquitin binding by DEUBAD domain proteins. Structural studies showed how these related domains could either activate UCH-L5 (by RPN13) or inactivate its catalytic activity (by INO80G) by large conformational changes and allosteric activation. We have shown biochemically how this activation mechanism is conserved in BAP1. Intriguingly the BAP1 activity is regulated at the level of the substrate, as it is selectively active against the K119 site on H2A in nucleosomes. Our biochemical studies are starting to show how this H2A selectivity is organized.

In conclusion we can state that the balance of ubiquitin conjugation is divided over a large number of individual regulatory mechanisms, both at the level of the conjugation machinery and at the level of the DUBs that together promote the correct and timely modification for cellular signalling.