Feedback regulation of polyamine biosynthesis: a characterization at the molecular level

Abstract: The polyamines putrescine, spermidine and spermine are essential for cell growth and differentiation. The biosynthesis of polyamines are tightly regulated by feedback mechanisms involving two enzymes, namely ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). This thesis deals with the mechanisms behind the polyamine-mediated feedback control of these enzymes. The polyamines were found to regulate ODC at a translational level. Depletion of cellular polyamines resulted in an increase in the ODC synthesis, which was not explained by a change in the amount of ODC mRNA. AdoMetDC, on the other hand, was demonstrated to be regulated by polyamines at two different levels, namely by changes in the transcription/stability of the AdoMetDC mRNA as well as in the translational efficiency of the mRNA. Polyamine depletion induced an increase in AdoMetDC synthesis rate which was much larger than that of the AdoMetDC mRNA content. The translational control of AdoMetDC expression was found to be dependent on the presence of the unusually long GC rich 5´ untranslated region in the AdoMetDC mRNA. AdoMetDC has earlier been shown to be synthesized as a proenzyme, which then is converted into the two subunits of the active enzyme. Evidence was obtained in the present thesis that putrescine stimulates this process in vivo. It was furthermore demonstrated that the very rapid turnover of AdoMetDC may be affected by inhibitors of polyamine metabolism. Aminoguanidine, which is frequently used in cellular systems to inhibit any degradation of polyamines by a serum amine oxidase, was shown to apparently inhibit AdoMetDC by an irreversible mechanism which markedly stabilized the enzyme against proteolytic degradation. To obtain information on structures essential for the feedback control of ODC as well as for the rapid degradation of the enzyme, the ODC gene from the insect trypanosome C. fasciculata was cloned and characterized. C. fasciculata ODC is so far the only known parasite ODC with a rapid turnover. The protein, which had a 40% homology to mammalian ODC, was found to lack a carboxyterminal degradation domain generally believed to be necessary for the rapid turnover of the enzyme. Nevertheless, it was shown to retain its rapid turnover even when expressed in a mammalian system, indicating the presence of another degradation domain.