Investigating the essential roles of Ubiquitin-like molecules and the Ubiquitin Proteasome System
We are interested on the role of ubiquitin and ubiquitin like molecules such as SUMO and NEDD8 in the regulation of physiological and pathological processes, more specifically during the transition of one stade to the other.
Figure 1. Principle of the ubiquitin-traps (TUBEs). TUBEs efficiently purify ubiquitylated proteins from cell extracts. (a) Schematic illustrating traditional GST pulldown, with use of DUB inhibitors IAA and NEM. (b) Schematic illustrating method of lysis in presence of TUBEs and absence of IAA/NEM. TUBEs are allowed to form complexes with poly-ubiquitylated proteins during lysis, and subsequently intact complexes are pulled down using glutathione agarose beads. (c) HEK 293 cells were pretreated with proteasome inhibitor, and stimulated with TNF-α. Cells were lysed in presence IAA/NEM, lysates were clarified and incubated with glutathione agarose beads bound to 20 ug GST-fusion protein. Eluted proteins were immunoblotted for IκBα and poly-ubiquitin. (d) HEK 293 cells were treated as above, and subjected to pulldown using the method described in (b ), adding 20 ug of purifed GST-fusion protein to lysis reaction. Asterisk indicates band corresponding to IκBα on reblotted membrane.
Figure 2. Implementations for Mass Spectrometry identification of Ubiquitylated proteins and interacting cellular factors using TUBEs.
Da Silva-Ferrada E, Torres-Ramos M, Aillet F, Campagna M, Matute C, Rivas C, Rodríguez MS, Lang V. (2011) Role of Monoubiquitylation on the Control of IκBα Degradation and NF-κB Activity. PLoS One. 6(10): Oct 12.Da Silva-Ferrada E, Torres-Ramos M, Aillet F, Campagna M, Matute C, Rivas C, Rodríguez MS, Lang V. (2011) Role of Monoubiquitylation on the Control of IκBα Degradation and NF-κB Activity. PLoS One. 6(10): Oct 12.http://www.ncbi.nlm.nih.gov/pubmed/22022389
In vivo requirements of the UBL modifier SUMO on SALL proteins function during steroidogenesis: D. melanogaster as a model system.