Gali Prag
Affiliations
Tel Aviv University
Biography
Gali Prag, who is focusing on the interpretation mechanism of the ubiquitin signal, graduated and earned his PhD from the Hebrew University. With his supervisor Prof. Oppenheim Gali studied bacterial genetics and obtained two EMBO fellowships to determine the structures of several chitin degrading enzymes in complex with their native substrates. Gali completed his postdoctoral studies at the NIH with Dr. James Hurley, where he determined the first structures of ubiquitin-receptor complexes with ubiquitin and some of the ESCRT complexes. In 2008 he returned to Israel and established a structural-biology and bacterial genetic laboratory in TAU.
Abstract
Structural insight into mechanisms for deciphering ubiquitin signals
Gali Prag, Department of Biochemistry and Molecular Biology, Tel Aviv University, Israel
About 100 deubiquitylating enzymes (DUBs) rapidly reverse ubiquitylation signals, posing challenges to genetic biochemical and biophysical monitoring and characterization of post ubiquitylation events. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic ubiquitylation apparatus in E. coli strains that lack DUBs. We demonstrated that tethering split antibiotic resistance gene fragments onto ubiquitin and ubiquitylation substrates gives rise to bacterial growth under selective conditions.
Using this selection system we identified and characterized novel E3s, and ubiquitin-receptors, proteins that decode the ubiquitin signal into cellular responses. Tethering two different affinity-tags, one onto ubiquitin and the other onto the ubiquitylation substrates, provided a simple purification scheme that yielded milligram quantities of ubiquitylated proteins. Using this system we demonstrated that contrary to in-vitro assays that lead to spurious modification of several lysine residues of the proteasomal ubiquitin-receptor Rpn10, the reconstituted system faithfully recapitulates its monoubiquitylation on Lys-84 that is observed in-vivo. We determined the crystal structure of ubiquitylated-Rpn10. The structure elucidated a novel ubiquitin-binding domain (UBD) within Rpn10. Biophysical measurements and studies with the developed bacterial genetic system corroborated the structural data.
We found that one function of this novel UBD is to direct the ubiquitylation to Lys-84. Intriguingly, we showed that ubiquitylation at Lys84 ejects the Rpn10 receptor from the proteasome. We propose that Rpn10 functions as a soluble ubiquitin-receptor that brings ubiquitylated-cargo to degradation in the proteasome. Following cargo release and degradation, Rpn10 undergoes ubiquitylation on the proteasome; this process ejects the receptor from the proteasome, thus promoting the next cycle of Rpn10 dependent proteasomal protein degradation.
