Mechanisms of polyamine-dependent control of ornithine decarboxylase and its antizyme
Zusammenfassung der Projektergebnisse
Polyamines are organic cations with essential functions in all organisms e.g. by balancing negative charges of nucleic acids. They are especially important in dividing cells, which is why rapidly replicating cancer cells have a particularly high demand of polyamines. The homodimeric ornithine decarboxylase (ODC), which converts ornithine into the diamine putrescine, is a rate-limiting enzyme in the biosynthesis of polyamines. ODC levels are controlled by a conserved homeostatic feedback control system involving an ODC binding protein called antizyme. After our identification of the ODC antizyme (OAZ1) gene in bakers' yeast, we have analyzed the mechanisms, by which polyamines concentrations influence the levels of ODC. We have discovered three processes in which polyamines act to down-regulate ODC. First, polyamines promote synthesis of Oaz1 protein at the level of OAZ1 mRNA translation. We identified a novel mechanism, in which polyamines bind to nascent Oaz1 polypeptide thereby preventing stalling of ribosomes on the OAZ1 mRNA and thus promote completion of Oaz1 synthesis. Secondly, we found that polyamines inhibit ubiquitin-dependent degradation of Oaz1. We have identified and characterized the ubiquitylation enzymes that target Oaz1 for degradation and found this process to be controlled in synchrony with the cell division cycle. Thirdly, we discovered that polyamines promote targeting of ODC by Oaz1 beyond their effects on Oaz1 synthesis or stability. We could show that polyamines directly promote Oaz1-mediated degradation of ODC at the level of the proteasome both in vivo and in vitro. We found that recognition of the ODC by the proteasome involves its N-terminal unstructured domain, which is essential for degradation and sufficient for proteolytic targeting of reporter proteins. Other data suggest that Oaz1, beyond enhancing exposure of the ODC N-terminal domain, moreover promotes ODC degradation by contributing an additional proteasome binding site. In summary, the findings of this project established the ODC antizyme polypeptide as an intracellular sensor of polyamine concentrations with a critical function in the homeostatic feedback regulation of polyamine biosynthesis.
Projektbezogene Publikationen (Auswahl)
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(2009) Catalytic Mechanism and Assembly of the Proteasome. Chem Rev. 109, 1509-1536
. Marques, A.J., Palanimurugan, R., Matias, A.C., Ramos, P.C., and Dohmen, R.J.
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(2011). Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Nature 477, 490-494
Kurian, L., Palanimurugan, R., Gödderz, D., and Dohmen R.J.
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(2011). The N-terminal unstructured domain of yeast ODC functions as a transplantable and replaceable ubiquitin-independent degron. J. Mol. Biol. 407, 354-367
Gödderz, D., Schäfer, E., Palanimurugan, R., and Dohmen, R.J.
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(2012) Ubiquitin Family Modifiers and the proteasome: Reviews and Protocols. Springer Protocols: Methods in Molecular Biology Volume 832
Dohmen, R.J., and Scheffner, M. (Eds.)
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(2012) Ultrafiltration based in vitro assay for determining polyamine binding to purified proteins. Protocol Exchange
Palanimurugan R and Dohmen R.J.
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(2013). A conserved protein with AN1 zinc-finger and ubiquitin-like domains modulates Cdc48 (p97) function in the ubiquitinproteasome pathway. J. Biol. Chem. 47, 33682–33696
Sá-Moura, B, Funakoshi, M., Tomko Jr., R.J., Dohmen, R.J., Wue, Z. Peng, J., and Hochstrasser, M.
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(2013). Hsp70 nucleotide exchange factor Fes1 is essential for ubiquitindependent degradation of misfolded cytosolic proteins. Proc. Nat. Acad. Sci. U.S.A. 110, 5975-5980
Gowda, N.K.C, Kandasamy, G., Froehlich, M.S., Dohmen, R.J., and Andréasson, C.
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(2014). Cotranslational polyamine sensing by nascent ODC antizyme. In Ito, K. (ed.) Regulatory Nascent Polypeptides. Springer Press
Palanimurugan, R., Kurian, L., Hegde, V., and Dohmen, R.J.
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Ubiquitin, Ubiquitin-Like Proteins, and Proteasome-Mediated Degradation. Molecular Cell Biology / Bradshaw, Ralph A.; Stahl, Philip D. (Hrsg.). - Waltham : Elsevier, 2016. - (Encyclopedia of Cell Biology ; 1). - S. 582-595. - ISBN 978-0-12-394796-3
Dohmen, R.J., Huibregtse, J.M., and Scheffner, M.
