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Weizmann Institute of Science
The ubiquitin-proteasome system is responsible for degradation of most of the cellular proteins, a process that is crucial for maintaining normal life course of the cell. Nevertheless, not all of the proteins degraded by the proteasome are ubiquitinated.
The most notable example is Ornithine Decarboxylase, ODC, the first and rate limiting enzyme in the polyamine biosynthesis pathway.
The degradation of ODC provides an alternative mechanism for targeting to the proteasome. ODC degradation by the proteasome is greatly stimulated by a polyamine-induced protein, termed Antizyme (Az), in a ubiquitin-independent manner.
ODC is active only as a homodimer. The heterodimerization of Az and ODC inactivates ODC as an intermediate step in the process of targeting it to degradation by the proteasome.
While ubiquitin recognition elements within the proteasome were extensively studied, the recognition element for the ODC and Az complex is still unknown.
The main goal of ourresearch is to shed light on two main aspects of this recognition process:
1) Identifying ODC recognition elements intrinsic or extrinsic to the proteasome.
2) Further characterization of the degradation signals within ODC and Az, which lead to ODC degradation.
In order to address these questions we use methods such as co-immunoprecipitatation, followed by 2D LCMS/MS, and different manipulations of the proteins.
We have also obtained yeast mutant strains, with deletions in proteasomal and non-proteasomal ubiquitin recognition elements, in order to investigate yeast ODC recognition and degradation by the yeast proteasome.
The Eukaryotic Initiation Factor 5A (eIF5A) is the only known protein containing the unusual amino acid hypusine. There are two known isoforms; both carry this post-translational modification which occurs by two consecutive enzymatic steps: first the deoxyhypusine synthase (DHPS) transfers the amino-butyl moiety from the polyamine spermidine to the lysin 50 of the protein, and then the deoxyhypusine hydroxylase (DOHH) irreversibly hydroxylates the deoxyhypusine residue.
eIF5A is indispensable for yeast growth: its depletion leads to growth arrest. eIF5A modification steps are evolutionarily conserved in all eukaryotes, and an eIF5A mutant which cannot be hypusinated, cannot complement yeast eIF5A knockout cells. In the light of these facts it is no wonder that hypusine formation is considered to be a primary task for the polyamine metabolism.
However, the exact roles of eIF5A and hypusine are still not clear.
The processes, in which the protein is involved in, have been largely debated in the past. Initially it was regarded as a translation initiation factor, but recent works pointed out its major role as a translation elongation factor.
What makes our research even more interesting is that both eIF5A isoforms can be associated with cancer. A weak expression of eIF5A-2 transcript can normally be found only in parts of the brain and testis, while the protein is expressed in several tumor cell lines. The expression of eIF5A-1 is, on the other hand, ubiquitous, but it is upregulated in colorectal carcinoma and its hypusination seems to be involved in a precursor form of vulva cancer.
Our aim is to enlighten the function of eIF5A and its modification, trying to carry out our experiments as physiologically as possible, the protein being normally fully hypusinated.
Some of the techniques used for achieving our goals are iso-electric focusing, co-immunoprecipitaion as well as tissue culture methods
