Polyamine analogues inhibit the ubiquitination of spermidine/spermine N1-acetyltransferase and prevent its targeting to the proteasome for degradation

Biochem J. 2001 Aug 15;358(Pt 1):137-45. doi: 10.1042/0264-6021:3580137.

Abstract

Spermidine/spermine N(1)-acetyltransferase (SSAT), a key enzyme in mammalian polyamine catabolism, undergoes rapid turnover (half-life approx. 30 min) and is highly inducible in response to polyamine analogues such as bis(ethyl)spermine (BE-3-4-3), which greatly stabilize the enzyme. Rapid degradation of SSAT in reticulocyte lysates was preceded by formation of a ladder of ubiquitinated forms, and required the production of high-molecular-mass complexes with ubiquitin (HMM-SSAT-Ubs). Mutation of all 11 lysines in SSAT separately to arginine demonstrated that no single lysine residue is critical for its degradation in vitro, but mutant K87R had a significantly longer half-life, suggesting that lysine-87 may be the preferred site for ubiquitination. Mutations at the C-terminus of SSAT, such as E171Q, resulted in marked stabilization of the protein, due to the lack of formation of the HMM-SSAT-Ubs. Addition of BE-3-4-3 prevented the accumulation of ubiquitin conjugates and the proteasomal degradation of wild-type SSAT. These results indicate that conformational changes brought about by the binding of polyamine analogues prevent the efficient polyubiquitination of SSAT, leading to a major increase in the amount of SSAT protein, and that alteration of the C-terminal end of the protein has a similar effect in preventing the productive interaction with an E2 or E3 component of the ubiquitin pathway.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetyltransferases / chemistry*
  • Animals
  • Arginine / chemistry
  • Binding Sites
  • Cysteine Endopeptidases / chemistry*
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Lysine / chemistry
  • Multienzyme Complexes / chemistry*
  • Mutation
  • Plasmids / metabolism
  • Polyamines*
  • Proteasome Endopeptidase Complex
  • Protein Binding
  • Protein Conformation
  • Rabbits
  • Rats
  • Reticulocytes / chemistry
  • Reticulocytes / metabolism
  • Spermidine / chemistry*
  • Spermine / chemistry*
  • Time Factors
  • Ubiquitins / chemistry
  • Ubiquitins / metabolism

Substances

  • DNA, Complementary
  • Multienzyme Complexes
  • Polyamines
  • Ubiquitins
  • Spermine
  • Arginine
  • Acetyltransferases
  • diamine N-acetyltransferase
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex
  • Lysine
  • Spermidine