טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHayat Ortal
SubjectDefining the cellular context of Lys11-linked polyubiquitin
signals
DepartmentDepartment of Biology
Supervisor Professor Michael Glickman
Full Thesis textFull thesis text - English Version


Abstract

Lys11-linked polyubiquitin is a fascinating ubiquitin linkage because its levels are elevated during a very narrow window of the cell cycle and it is necessary for the cell cycle progression toward the anaphase. Interference with Lys11 signaling is correlated with certain cancers.

The ubiquitin molecule is one of the most conserved proteins in cells, unique because of its action as a posttranslational modification of other proteins. Ubiquitin chain elongation can occur through seven Lys and the N-terminus residues. The different linkages have different conformations and therefore signal for different cellular outcomes.

Lys11-linkages are the third most abundant linkages in cells, after the more abundant Lys48- and the Lys63-linkages. Although there are specific receptors for the Lys48- and the Lys63-linkages, no such receptor specific for the Lys11-linkage type has been described, making it almost impossible to isolate Lys11-linked polyubiquitin-conjugates.

Our first aim was to characterize substrates carrying the Lys11 signal on them and to define the Lys11 signal. In order to do so we aimed to generate a tool that would enable us to isolate Lys11-linked conjugates from cells, so they can be further analyzed for their target substrates and unique receptors.

We thought of using a protein known to associate with the Lys11-linkages, the deubiquitinating enzyme (DUB) OTUD7B, also called Cezanne, which was described to hydrolyze the Lys11-linkage type with high specificity. Amplification of the gene locus occurs in some breast cancer tumors. Overexpression of the OTUD7B in these tumors correlates with the tumor malignancy and metastasis formation tendency.

We have used the inactive OTU domain of the OTUD7B enzyme in an approach termed Inactive Catalytic Domain Capture (ICDC). Validating high specificity of this domain towards the Lys11-linkage type from a series of enzymatically synthesized ubiquitin chains confirmed its application as a “Trap”.

This tool not only binds Lys11-linkages but also can sequester linkages from being processed by active DUBs, indicating the high affinity characteristic of a receptor of the K11-Trap. The K11-Trap can also protect the Lys11-linked ubiquitin chains on a substrate from being processed by proteasome-associated DUBs, suggesting it might protect substrates from the proteasome.

K11-Trap was able to fish-out the proteins carrying Lys11-linkages from HeLa cells extract. We used the proteasome inhibitor, MG132, in order to see if interfering with the proteasome activity will affect the cellular levels of Lys11-linkages. We found that the proteasome inhibition did cause accumulation of Lys11-linkages, together with the expected accumulation of Lys48-linkages, in HeLa cells. Furthermore, together with the Lys11-linkages also Lys48-linked ubiquitin species eluted, suggesting a mixture of these two ubiquitin linkages on the same substrate.

All our findings highlight the potency of the K11-Trap that we have engineered to specifically bind Lys11-linkages and serve as a tool for isolation of Lys11-linked ubiquitin-conjugates. The involvement of this specific linkage type and of proteins processing it during cell cycle and in tumor malignancy and metastasis formation, makes it an interesting research reagent, a possible diagnostic tool, and therapeutic target.