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Intracellular protein degradation and mechanisms of cancer

One main focus of research in our laboratory is the involvement of the ubiquitin proteasome system (UPS) in differentiation and the pathogenesis of malignant transformation. To de-convolute the process which is enormously complex and involves numerous distinct mechanisms, we decided to study two different, yet related processes: (i) activation of the NF-κB transcriptional regulator, and (ii) evasion of apoptosis.
(i) The transcriptional activator NF-κB has a strong anti-apoptotic activity. It promotes cell division, migration, motility angiogenesis, and adhesion, and it mediates the inflammatory response. Not suprisingly, it is constitutively up-regulated in many malignancies. It is activated via a two step ubiquitin-mediated mechanism: (a) processing of the precursor proteins p105 and p100 to the active subunits, p50 and p52, respectively, and (b) signal-induced degradation of the inhibitor IκBα that sequesters NF-κB inactive in the cytosol. Signal-induced degradation of the inhibitor results in translocation of the active factor to the nucleus where it initiates specific transcription. We are particularly interested in the mechanism(s) that lead to processing of the precursor proteins. These events are exceptional, as in most cases the ubiquitin system destroys its target substrates completely, while here it processes them in a limited manner to release short, active subunits from longer inactive precursors. We aim to identify the characteristics of the p105 and p100 molecules that render them resistant to degradation but sensitive to processing, and to purify and characterize the ubiquitin ligases, E3s, involved. We hypothesize that the ligases are up-regulated in different malignancies, contributing to the high level of NF-κB in these tumors. For our publications on NF-κB and other transcription regulators, see Refs. A(i) 1-15, A(iii) 1-2, and A(iv) 1-6.
(ii) Evasion of apoptosis or gradual development of resistance to genotoxic stimuli such as irradiation or chemotherapeutic agents, is a hallmark of malignant cells as their behavior becomes more aggressive. We are studying the regulation of Inhibitors of Apoptosis Proteins, many of them are RING finger ubiquitin ligases, E3s, that inhibit apoptosis by targeting caspases for ubiquitination and subsequent degradation. A feature characteristic to many RING finger E3s is their ability to catalyze auto-ubiquitination and target themselves for degradation. Thus, their activity is balanced between self destruction and destruction of their substrates. The activity of IAPs must be tightly regulated, as their untoward activation will inhibit apoptosis, while decreased activity will induce it. A group of recently discovered pro-apoptotic proteins, cytosolic Reaper, Grim and Hid in Drosophila, and mitochondrial Smac/Diablo and Omi/Htr2A in mammalian cells, act to down regulate them, probably by stimulating their auto-ubiquitination and targeting them for degradation. In contrast, NF-κB for example, acts to increase IAPs expression. We have recently shown that Reaper regulates both the fly dIAP1 and the mammalian XIAP via stimulating their auto-ubiquitinating activity, thus shifting the balance towards self destruction. Furthermore, a short peptide that contains the N-terminal AVAF box of Reaper fused to an internal Tryptophan box of this protein, can, with high efficiency, mimic the activity of the full length protein. Our aim is to better understand the regulation of IAPs, so it will be possible to down-regulate them and re-sensitize malignant cells to apoptosis-inducing agents. For our publications on regulation of apoptosis, see Refs. A(ii) 1-2.

Out studies on the involvement of the UPS in pathogenesis of malignancies led us to directly analyze the mechanisms involved in the degradation of oncoproteins such as c-Myc, and tumor suppressors, such as p27 [Refs. A(iii) 1-2].

Non-canonical ubiquitination, and regulation of transcription and of elements of the ubiquitin system

An additional major focus of interest in our laboratory involves “non-canonical” modes of ubiquitination. We identified a novel mode of ubiquitination in which modification occurs at the N-terminal residue of the substrate rather than on internal lysine residues. This modification is essential for the degradation of naturally occurring lysine-less proteins, such as the cell cycle regulator p16INK4a, that cannot be modified otherwise. We have also shown that the auto-ubiqutinating activity of Ring1B, a ubiquitin ligase that is a part of the Polycomb transcriptional repressive complex PRC1, does not serve to target the protein for degradation, but rather to activate its ligase activity towards histone H2A. Accordingly, the chains generated are not the ”canonical” lysine48-based chains recognized by the 26S proteasome for degradation, but rather lysine6, 27-based mixed and most probably doubly branched chains that activate the ligase by recruiting to the complex additional, yet to be identified proteins. The finding that the auto-ubiquitinating activity of the Ring1B ligase does not lead to its degradation, implies the ligase itself must be degraded following targeting by an exogenous ligase. Not only that we are searching for this ligase, but this finding opened a whole new line of research in the laboratory - how the components of the ubiquitin system are regulated – or how the controllers are being controlled. For our publications on “non-canonical” mechanisms of ubiquitination and regulation of the components of the ubiquitin system, see Refs. A(iv) 1-6 and A(v) 1-2.

For our reviews on the UPS, see Refs. B, 1-6.

Selected articles:
A.   Original, peer-reviewed articles

    i.  Activation of the NF-κB transcriptional regulator by the ubiquitin system:

  1. Orian, A., Whiteside, S., Israël, A., Stancovski, I., Schwartz, A. L. and Ciechanover, A. (1995). Ubiquitin-Mediated Processing of NF-κB Transcriptional Activator Precursor: Reconstitution of a Cell Free System and Identification of the Ubiquitin-Carrier Protein, E2, and a Novel Ubiquitin-Protein Ligase, E3, Involved in Conjugation. J. Biol. Chem. 270, 21707-21714.
  2. Alkalay, I., Yaron, A., Hatzubai, A., Orian, A., Ciechanover, A., and Ben Neriah, Y. (1995). Stimulation-Dependent IκBα Phosphorylation Marks the NF-κB Inhibitor for Degradation via the Ubiquitin-Proteasome Pathway. Proc. Natl. Acad. Sci. USA 92, 10599-10603.
  3. Gonen, H., Stancovski, I., Shkedy, D., Hadari, T., Bercovich, B., Bengal, E., Mesilati, S., Abu-Chatoum, O., Schwartz, A. L., and Ciechanover, A. (1996). Isolation, Characterization, and Partial Purification of a Novel Ubiquitin-Protein Ligase, E3: Targeting of Protein Substrates via Multiple and Distinct Recognition Signals and Conjugating Enzymes. J. Biol. Chem. 271, 302-310.
  4. Yaron, A., Gonen, H., Alkalay, I., Hatzubai, A., Jung, S., Beyth, S., Mercurio, F., Manning A.M., Ciechanover, A.*, Ben-Neriah, Y.* (1997). Inhibition of NF-κB Cellular Function via Specific Targeting of the IκBα-Ubiquitin Ligase. EMBO J. 16, 6486-6494.
    *senior corresponding authors
  5. Orian, A., Schwartz. A.L., Israël, A., Whiteside, S., Kahana, C., and Ciechanover, A. (1998). Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain. Mol. Cell. Biol. 19, 3664-3673.
  6. Gonen, H., Bercovich, B., Orian, A., Carrano, A., Takizawa, C., Yamanaka, K., Pagano, M., Iwai, K., and Ciechanover, A. (1999). Identification of the Ubiquitin Carrier Proteins, E2s, Involved in Signal-Induced Conjugation and Subsequent Degradation of IκBα. J. Biol. Chem. 274, 14823-14830.
  7. Orian, A., Gonen, H., Bercovich, B., Fajerman, I., Eytan, E., Israël, A., Mercurio, F., Iwai, K, Schwartz, A.L., and Ciechanover, A. (2000). SCFβ-TrCP Ubiquitin Ligase-Mediated Processing of NF-κB p105 Requires Phosphorylation of its C-Terminus by IκB Kinase. EMBO J. 19, 2580-2591.
  8. Cohen, S., Orian, A., and Ciechanover, A. (2001). Processing of p105 is Inhibited by Docking of p50 Active Subunits to the Ankyrin Repeat Domain, and Inhibition is Alleviated by Signaling via the C-Terminal Phosphorylation/Ubiquitin-Ligase Binding Domain. J. Biol. Chem. 276, 26769-26776.
  9. Amir, R.E., Iwai, K., and Ciechanover, A. (2002). The NEDD8 pathway is required for for
    SCFβ-TrCP- mediated processing of the NF-κB precursor p105. J. Biol. Chem. 277, 23253-23259.
  10. Amir, R.E., Haecker, H., Karin, M., and Ciechanover, A. (2004) Mechanism of Processing of the NF-κB2 p100 Precursor: Identification of the Specific Polyubiquitin Chain-Anchoring Lysine Residue and Analysis of the Role of NEDD8-Modification on the SCFβ-TrCP Ubiquitin Ligase. Oncogene 23, 2540-2547.
  11. Cohen, S., Achbert-Weiner, H., and Ciechanover, A. (2004). Dual Effect of IKKβ-Mediated Phosphorylation on p105 Fate: SCFβ-TrCP- Dependent Degradation and SCFβ-TrCP -Independent Processing.
    Mol. Cell. Biol. 24, 475-486.
  12. Fajerman, I., Schawartz, A.L., and Ciechanover, A. Degradation of the Id2 Developmental regulator: Targeting via N-Terminal Ubiquitination.Biochem. Biophys. Res. Commun. 314, 505-512.
  13. Ben-Saadon, R., Fajerman, I., Ziv, T., Hellman, U., Schwartz, A.L., and Ciechanover, A. (2004). The Tumor Suppressor Protein p16INK4a and the Human Papillomavirus oncoprotein E7-58 are Naturally Occurring Lysine-Less Proteins that are Degraded by the Ubiquitin System: Direct Evidence for Ubiquitination at the N-Terminal Residue. J. Biol. Chem. 279, 41414-41421.
  14. Cohen, S., Lahav-Baratz, S., and Ciechanover, A. (2006). Two Distinct Ubiquitin-Dependent Mechanisms are involved in NF-κB p105 Proteolysis. Biochem. Biophys. Res. Commn. 345, 7-13.
  15. Kravtsova-Ivantsiv, Y., Cohen, S., and Ciechanover, A. (2009). Modification by Single Ubiquitin Moieties Rather Than Polyubiquitination is Kravtsova-Ivantsiv, Y., Cohen, S., and Ciechanover, A. Modification by Single Ubiquitin Moieties Rather Than Polyubiquitination is Sufficient for Proteasomal Processing of the p105 NF-κB Precursor. Mol. Cell. 33, 496-504.

  ii.  Regulation of apoptosis by the ubiquitin system

  1.  Ryoo, H.-D., Bergmann, A., Gonen, H., Ciechanover, A., and Steller, H. (2002). Regulation of Drosophila IAP1 Degradation and Apoptosis by Reaper and UbcD1. Nature Cell Biology 4, 432-438.
  2. Herman- Bachinsky, Y., Ryoo, H.D., Ciechanover, A.*, and Gonen, H. (2007). Regulation of the Drosophila Ubiquitin Ligase DIAP1 is Mediated via Several Distinct Ubiquitin System Pathways. Cell Death and Differentiation. 14, 861-871.  
    *Senior correspoinding author

  iii.  Degradation of oncoproteins by the ubiquitin system

  1. Gross-Mesilaty, S., Reinstein, E., Bercovich, B., Tobias, K.E., Kahana, C., and Ciechanover, A. (1998). Basal and Human Papillomavirus E6 Oncoprotein-Dependent Accelerated Degradation of Myc Proteins by the Ubiquitin Proteolytic Pathway. Proc. Natl. Acad. Sci. USA 95, 8058-8063.
  2. Ben-Izhak, O., Lahav-Baratz, S., Meretyk, S., Ben-Eliezer, S., Sabo, E., Dirnfeld, M., Cohen, S., and Ciechanover, A. (2003). Inverse Relationship Between Skp2 Ubiquitin Ligase and the Cyclin Dependent Kinase Inhibitor p27Kip1 in Prostate Cancer. J. Urol. 170, 241-245.

  iv.  Degradation of MyoD and N-terminal ubiquitination

  1. Abu Hatoum, O., Gross-Mesilaty, S., Breitschopf, K., Hoffman, A., Gonen, H., Ciechanover,   A.*, and Bengal, E. (1998). Degradation of the Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA-Binding. Mol. Cell. Biol. 18, 5670-5677.          

             *Senior corresponding author.
 

     2.     Breitschopf, K., Bengal, E., Ziv, T., Admon, A., and Ciechanover, A. (1998). A Novel Site for  
             Ubiquitination: The N-Terminal Residue and Not Internal Lysines of MyoD is Essential for 
             Conjugation and Degradation of the Protein. EMBO J. 17, 5964-5973.

     3.     Aviel, S., Winberg, G., Massucci, M., and Ciechanover, A. (2000). Degradation of Epstein-Barr
             Virus Latent Membrane Protein 1 (LMP1) by the Ubiquitin-Proteasome Pathway: Targeting via 
             Ubiquitination of the N-Terminal Residue J. Biol. Chem. 275, 23491-23499.

     4.     Reinstein, E., Scheffner, M., Oren, M., Schwartz, A.L., and Ciechanover, A. (2000) Degradation  
             of the E7 Human Papillomavirus Oncoprotein by the Ubiquitin-Proteasome System:    
             Targeting  via  Ubiquitination of the N-Terminal Residue. Oncogene 19, 5944-5950.

     5.     Ben-Saadon, R., Fajerman, I., Ziv, T., Hellman, U., Schwartz, A.L., and Ciechanover, A. (2004).   
             The Tumor Suppressor Protein p16INK4a and the Human Papillomavirus oncoprotein E7-58   
             are Naturally Occurring Lysine-Less Proteins that are Degraded by the Ubiquitin System: 
             Direct Evidence for Ubiquitination at the N-Terminal Residue. J. Biol. Chem. 279, 41414-41421.

     6.     Sadeh, R., Breitschopf, K., Bercovich, B., Zoabi, M., Kravtsova-Ivantsiv, Y., Kornitzer, D.,  
             Schwartz, A.L., and Ciechanover, A. (2008). The N-Terminal Domain of MyoD is Necessary and
             Sufficient for its Nuclear Localization-Dependent Degradation by the Ubiquitin System. Proc.  
             Natl. Acad. Sci. USA 105, 15690-15695.

 

   v.  Regulation of components of the ubiquitin system

  1. Ben Saadon, R., Zaarur, D., Ziv, T., and Ciechanover, A. (2006). The Polycomb Protein Ring1B Generates Self Atypical Mixed Ubiquitin Chains Required for its In Vitro Histone H2A Ligase Activity. Mol. Cell. 24, 701-711.
  2. Shabek, N., Iwai, K., and Ciechanover, A. (2007). Ubiquitin is Degraded by the Ubiquitin System as aMonomer and as Part of its Conjugated Target. Biochem. Biophys. Res. Commun. 363, 425-31.

B.  Review Articles

  1. Ciechanover, A., and Brundin, P. (2003). The Ubiquitin-Proteasome System in Neurodegenerative Diseases: Sometimes the Chicken, Sometimes the Egg. Neuron 40, 427-446.
  2. Ciechanover, A., and Ben-Saadon R. (2004). N-Terminal Ubiquitination: More Protein Substrates Join In.
    Trends Cell Biol. 14, 103-106.
  3. Ciechanover, A. (2005). From the Lysosome to Ubiquitin and the Proteasome: The Rise of Proteolysis.
    Nature Rev. Mol. Cell Biol. 6, 79-86.
  4. Ciechanover, A. (2005). Intracellular Protein Degradation: From a Vague Idea through The Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting (Nobel Lecture).
    Cell Death Differ. 12, 1178-90.
  5. Reinstein, E., and Ciechanover, A. (2006). Protein Degradation and Human Diseases - the Ubiquitin Connection. Annal. Int. Med. 145, 676-684.
  6. Schwartz, A.L. and Ciechanover, A. (2008). Targeting proteins for destruction by ubiquitin System: Implications for Human Pathobilogy. Annu. Rev. Toxicol. Pharmacol. 49, 73-96.

C.   Book chapters and methods

  1. Ciechanover, A., and Kornitzer, D. (2003). Proteasomes/Ubiquitination. In: Handbook of Cell Signalling (Ralph A. Bradshaw, and Edward A. Dennis, eds.). Elsevier Science, San Diego, California, USA, and Academic Press. Vol. 3, Chapter 290, pp. 129-133.
  2. Ciechanover, A. (2005). N-terminal ubiquitination. In: Methods in Molecular Biology Volume 301. Series: Ubiquitin-Proteasome Protocols (C. Patterson and D.M. Cyr, eds.). Human Press, pp. 255-270.
  3. Ciechanover, A. (2005). Methods in Protein Ubiquitination. In: Cell Biology: A Laboratory Handbook (J. Celis, ed.). 3rd Edition. Elsevier Science and Academic/Harcourt Press. Vol. 4, pp. 351-360. 
  4. Ciechanover, A. (2005). Intracellular Protein Degradation: From a Vague Idea thru the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting.
    In: Les Prix Nobel. The Nobel Foundation, Stockholm, Sweden. pp.151-175.
  5. Kornitzer, D., and Ciechanover, A. (2008). Proteasomes/Ubiquitination. In: Handbook of Cell Signalling, 2nd ed. (Ralph A. Bradshaw, and Edward A. Dennis, eds.). Elsevier Science, San Diego, California, USA, and Academic Press (in press).

D.  Biography

  1. Ciechanover, A. (2005). Biography. In: Les Prix Nobel.
    The Nobel Foundation, Stockholm, Sweden. pp. 125-150.

    See also the Nobel Prize website:
    http://nobelprize.org/nobel_prizes/chemistry/laureates/2004

E.  Books edited

  1. Mayer, R.J., Ciechanover, A., Rechsteiner, M., and (2005). Protein Degradation Handbook, Volume 1 (R.J. Mayer, A. Ciechanover, and M. Rechsteiner, Eds.).
    Volume 1. Wiley-VCH, Weinheim, Germany. pp. I-XVI, 1-377.
  2. Mayer, R.J., Ciechanover, A., and Rechsteiner, M. (2006). Protein Degradation Handbook, Volume 2 (R.J. Mayer, A. Ciechanover, and M. Rechsteiner, Eds.).
    Volume 2. Wiley-VCH, Weinheim, Germany. pp. I-XIV, 1-286.
  3. Mayer, R.J., Ciechanover, A., and Rechsteiner, M. (2006). Protein Degradation Handbook, Volume 3 (R.J. Mayer, A. Ciechanover, and M. Rechsteiner, Eds.).
    Volume 3. Wiley-VCH, Weinheim, Germany. pp. I-XIV, 1-238.
  4. Mayer, R.J., Ciechanover, A., and Rechsteiner, M. (2008). Protein Degradation Handbook, Volume 4 (R.J. Mayer, A. Ciechanover, and M. Rechsteiner, Eds.).
    Volume 4. Wiley-VCH, Weinheim, Germany. pp. I-XV, 1-242.