Current Research

Ubiquitin and Proteasome mediated degradation

Our group is part of the Rappaport – Technion Integrated Cancer Center – R-TICC (LINK) and we work in tight collaboration with many of its researchers. Our studies focus on the role of the ubiquitin system (that was discovered in our laboratory and was culminated with the awarding of the 2004 Nobel Prize in Chemistry) in basic mechanisms of diseases, cancer and neurodegeneration in particular. Our research is a nice and elegant example of how the answer to one question leads to the next, which is rather unexpected. Further, it is an example of how curiosity-driven basic research leads – by different groups – to its translation to efficient drugs to combat severe human diseases.

One area of studies in our laboratory is the mechanism by which the ubiquitin system activates NF-κB, a major transcription factor that is upregulated in many tumors and is thought to sustain, though not to initiate the malignant process.  We identified KPC1 as the ubiquitin ligase, E3, that catalyzes ubiquitin-mediated cleavage of the inactive p105 precursor of NF-κB to the active p50 subunit. Surprisingly, generation of excess p50 subunits by KPC1 shows strong tumor suppressive properties (1). We are now studying the tumor suppressive mechanism, and it appears that it is the recruitment of the immune system which plays an important role this process. We are developing small molecules that may mimic the process with the hope that their application can also lead to tumor suppression.

We went on to discover that a rather ‘non-canonical’ modification by monoubiquitination drives the KPC1-mediated processing of p105 rather than the well accepted, known, and well-studied polyubiquitination (2). This prompted a study of the mechanisms that underlie the cellular “choice” of the preferred tagging mechanism. We found that short proteins can settle with monoubiquitination where a single ubiquitin moiety can stabilize them on the proteasome, whereas longer substrates require a longer chain, or multiple single ubiquitin moieties (multiple mono-ubiquitinations) (3). A systematic screen confirmed our hypothesis, and unraveled a much broader mode of modification than previously thought – a large proportion of the proteome is targeted by monoubiquitination or multiple monoubiquitinations (4).

Another subject which has attracted our attention and in which we invest significant efforts is the fate of the ubiquitin system components themselves, whether and how the predator becomes a prey? Their level clearly affects their activity, which in turn affects the processes they control.  We have shown that the ubiquitin moieties in the polyubiquitin chains are partially degraded along with the substrate as part of the conjugate (5,6). A deeper analysis demonstrated that the proximal moieties of the polyubiquitin chain are degraded, whereas the more distal ones are holding the substrate to the proteasome securing its processive degradation and are then recycled for re-use (7). In an additional study we have shown that under stress, the proteasome is subjected to autophagy that digests its two sub-complexes – the 20S and the 19S (8-12). Interestingly, the proteasome has to be ubiquitinated prior to being engulfed by the autophagic vacuoles (8,11). We are now looking for the ubiquitin ligase that carries out this activity, as its regulation/modulation may have an effect on the growth of tumors by targeting the proteasome for destruction, thus cutting short the supply of amino acids to the cell, resulting in its death. Preliminary results This may affect selectively malignant cells that are more dependent on supply of nutrients for their survival.    The entry into the field of autophagy brought us to a deeper study of the inter-relationships and cross talk between the ubiquitin and the autophagic systems (10). Preliminary results show that a cooperation between the two systems is necessary in order to maintain viability under stress. Since cancer cells are constantly under nutrient and oxygen deficiency, this cross talk is crucial for their survival on one hand, but provides a promising plethora of potential drug targets on the other.

In an independent line of research we are studying the involvement of the ubiquitin system in neurodegenerative disorders – the hallmark of which is accumulation in the brain of abnormal/mutated and typically aggregated proteins. Out of several diseases, we decided to study Huntington’s disease where several good experimental tools – probes, cells, and animal models – are available. Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine–adenine–guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin–proteasome system, suggesting that mHtt is marked for proteasomal  degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses.We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination—attenuation of the pathogenic effect of mHtt (13).

 References:

  1. Shabek, N., Herman-Bachinsky, Y., Buchsbaum, S., Lewinson, O., Haj-Yahya, M., Hejjaoui, M., Lashuel, H.A., Sommer, T., Brik, A., and Ciechanover, A. (2012). The Size of the Proteasomal Substrate Determines whether its Degradation will be Mediated by Mono- or Polyubiquitylation.  Mol. Cell 48, 87-97 (PDF).
  2. Braten, O., Livneh, I., Ziv, T., Admon, A., Kehat, I., Caspi, L., Gonen, H., Bercovich, B., Godzik, A., Jahandideh, S., Jaroszewski, L. Sommer, T., Kwon, Y.T. Guharoy, M., Tompa, P., and Ciechanover, A. (2016). Numerous Proteins with Unique Characteristics are Degraded by the 26S Proteasome Following Monoubiquitination. Proc. Natl. Acad. Sci. USA 113, E4639-E4647. (PDF).
  3. Shabek, N., Bachinsky-Herman, Y., and Ciechanover, A. (2009). Ubiquitin Degradation with its Substrate and as a Monomer in a Ubiquitination-Independent Mode Provide Clues to Proteasome Regulation.  Natl. Acad. Sci. USA. 106, 11907-11912 (PDF).
  4. Braten, O., Livneh, I., Ziv, T., Admon, A., Kehat, I., Caspi, L., Gonen, H., Bercovich, B., Godzik, A., Jahandideh, S., Jaroszewski, L. Sommer, T., Kwon, Y.T. Guharoy, M., Tompa, P., and Ciechanover, A. (2016). Numerous Proteins with Unique Characteristics are Degraded by the 26S Proteasome Following Monoubiquitination. Proc. Natl. Acad. Sci. USA 113, E4639-E4647 (PDF).
  5. Shabek, N., Bachinsky-Herman, Y., and Ciechanover, A. (2009). Ubiquitin Degradation with its Substrate and as a Monomer in a Ubiquitination-Independent Mode Provide Clues to Proteasome Regulation.  Natl. Acad. Sci. USA. 106, 11907-11912 (PDF).
  6. Shabek, N., and Ciechanover, A. (2010). Degradation of Ubiquitin: The Fate of the Cellular Reaper.  Cell Cycle 9, 523-530 (PDF).
  7. Sun, H., Mali, S.M., Singh, S.K., Meledin, R., Brik, A., Kwon, Y.T., Kravtsova-Ivantsiv, Y., Bercovich, B., and Ciechanover, A. (2019). Diverse Fate of Ubiquitin Chain Moieties: The Proximal is Degraded with the Target, and the Distal Protects the Proximal From Removal, and Recycles.
    Proc. Natl. Acad. Sci. USA 116, 7805-7812. doi: 10.1073/pnas.1822148116. PMID: 30867293. http://www.pnas.org/cgi/doi/10.1073/pnas.1822148116 (PDF).  Was highlighted by an editor-invited commentary: van der Heden, van Noort G.J., Gan, J., and, Ovaa, H. (2019). Synthetic ubiquitinated proteins meet the proteasome: Distinct roles of ubiquitin in a chain. Proc Natl Acad Sci USA. 116, 7614-7616. doi: 10.1073/pnas.1903405116. PMID: 30926663
  8. Cohen-Kaplan, V., Livneh, I., Avni, N., Fabre, B., Ziv, T., Kwon, Y. T., Ciechanover, A. (2016). p62- and Ubiquitin-Dependent Stress-Induced Autophagy of the Mammalian 26S Proteasome. Natl. Acad. Sci. USA 113, E7490-E7499 (PDF).
  9. Cohen-Kaplan, V., Ciechanover, A.*, and Livneh, I. (2016). p62 at the Crossroad of the Ubiquitin-Proteasome System and Autophagy. Oncotarget 7, 83833-83834 doi: 0.18632/oncotarget.13805. PMID: 27974671 (PDF).
    *Corresponding author
  10. Cohen-Kaplan, V., Livneh, I., Avni, N., Cohen-Rosenzweig, C., and Ciechanover, A. (2016). The Ubiquitin-Proteasome System and Autophagy: Coordinated and Independent Activities.  Int. J. Biochem. Cell Biol. 79, 403-418 (PDF). http://dx.doi.org/10.1016/j.biocel.2016.07.019
  11. Cohen-Kaplan, V., Ciechanover, A.*, and Livneh, I. (2017). Stress-induced Polyubiquitination of Proteasomal Ubiquitin Receptors Targets the Proteolytic Complex for Autophagic Degradation. Autophagy 13, 1-2 (PDF).
    *Corresponding author
  12. Livneh, I., Cohen-Kaplan, V., Cohen-Rosenzweig, C., Avni, N., and Ciechanover, A. (2016). The Life Cycle of the 26S Proteasome – From Birth, through Regulation and Function, and on to its Death.  Cell Res. 26, 869-885. doi: 10.1038/cr.2016.86 (PDF ).
    1. Hakim-Eshed, V., Boulos, A., Cohen-Rosenzweig, C., Libo Yu-Taegerd, L.,
  13. Ziv, T.,  Kwon, Y.-T., Riess, O., Nguyen, H.H.P., Ziv, N.E., and Ciechanover, A. (2020). Site-specific ubiquitination of pathogenic huntingtin attenuates its deleterious effects. Proc. Natl. Acad. Sci,  USA. 117, 18661-18669 www.pnas.org/cgi/doi/10.1073/pnas.2007667117 (PDF).
  14. Kravtsova-Ivantsiv, Y., Goldhirsh, G., Ivantsiv A., Ben Itzhak, O., Kwon, Y.-T., Pikarsky, E., and Ciechanover, A., (2020). Excess of the NF-ĸB p50 subunit generated by the ubiquitin ligase KPC1 suppresses tumors via PD-L1– and chemokines-mediated mechanisms. Proc. Natl. Acad. Sci,  USA.  www.pnas.org/cgi/doi/10.1073/pnas.2019604117 (PDF).