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Cambridge, United Kingdom

Wilson M.D.,Cancer Research UK Research Institute | Harreman M.,Cancer Research UK Research Institute | Taschner M.,Cancer Research UK Research Institute | Taschner M.,Max Planck Institute of Biochemistry | And 6 more authors.
Cell | Year: 2013

DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a "mechanism of last resort" employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function. © 2013 The Authors.


Cherblanc F.L.,Imperial College London | Chapman K.L.,Imperial College London | Reid J.,Domainex | Borg A.J.,Imperial College London | And 8 more authors.
Journal of Medicinal Chemistry | Year: 2013

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally related analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is time-dependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between 1 and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by 1 in vitro and has implications for the activity of ETPs against these important epigenetic targets. © 2013 American Chemical Society.


Littler E.,Domainex
Sp2 | Year: 2010

Combinatorial Domain Hunting (CDH) is a new technology developed by Domainex that generates large amounts of high-quality protein from genes that are traditionally difficult to express in recombinant systems.


Grant
Agency: GTR | Branch: Innovate UK | Program: | Phase: Feasibility Study | Award Amount: 150.00K | Year: 2013

Our aim is to establish the feasibility of a new treatment for cancer that works by targeting the enzyme SMYD3. Recent academic research has shown that SMYD3 is not present in normal cells, but in many cancer cells it switches on the production of other proteins that help tumours grow and spread throughout the body. So this work suggests that a drug that inactivates SMYD3 will switch off these mechanisms, and have powerful anti-cancer properties, perhaps with few side-effects. This new mechanism would be very different than those of existing drugs, and of many other experimental treatments. This project will test this idea by identifying SMYD3-blocking compounds, and finding out whether they have the expected effects on tumour cells, and are safe to normal cells. If successful, this project will provide the starting-points for a larger programme to find a drug candidate that works in this way.


Methods for producing and identifying fragments of proteins, and more particularly to methods for generating and identifying soluble protein domains are disclosed based on a method for generating a library of nucleic acid fragments from nucleic acid encoding a desired polypeptide, and more especially a library of essentially, randomly sampled fragments of coding DNA sequence predominantly of defined size range and a method for selecting cloned gene fragments from the library that encode soluble protein domains.

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