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

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.

SUBMITTER: Coleman CS

PROVIDER: S-EPMC1222041 | biostudies-other | 2001 Aug

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Tau stabilizes microtubules; however, in Alzheimer's disease (AD) and tauopathies, tau becomes hyperphosphorylated, aggregates, and results in neuronal death. Our group recently uncovered a unique interaction between polyamine metabolism and tau fate. Polyamines exert an array of physiological effects that support neuronal function and cognitive processing. Specific stimuli can elicit a polyamine stress response (PSR), resulting in altered central polyamine homeostasis. Evidence suggests that elevations in polyamines following a short-term stressor are beneficial; however, persistent stress and subsequent PSR activation may lead to maladaptive polyamine dysregulation, which is observed in AD, and may contribute to neuropathology and disease progression. METHODS:Male and female mice harboring tau P301L mutation (rTg4510) were examined for a tau-induced central polyamine stress response (tau-PSR). The direct effect of tau-PSR byproducts on tau fibrillization and oligomerization were measured using a thioflavin T assay and a N2a split superfolder GFP-Tau (N2a-ssGT) cell line, respectively. To therapeutically target the tau-PSR, we bilaterally injected caspase 3-cleaved tau truncated at aspartate 421 (AAV9 Tau ΔD421) into the hippocampus and cortex of spermidine/spermine-N1-acetyltransferase (SSAT), a key regulator of the tau-PSR, knock out (SSAT-/-), and wild type littermates, and the effects on tau neuropathology, polyamine dysregulation, and behavior were measured. Lastly, cellular models were employed to further examine how SSAT repression impacted tau biology. RESULTS:Tau induced a unique tau-PSR signature in rTg4510 mice, notably in the accumulation of acetylated spermidine. In vitro, higher-order polyamines prevented tau fibrillization but acetylated spermidine failed to mimic this effect and even promoted fibrillization and oligomerization. AAV9 Tau ΔD421 also elicited a unique tau-PSR in vivo, and targeted disruption of SSAT prevented the accumulation of acetylated polyamines and impacted several tau phospho-epitopes. Interestingly, SSAT knockout mice presented with altered behavior in the rotarod task, the elevated plus maze, and marble burying task, thus highlighting the impact of polyamine homeostasis within the brain. CONCLUSION:These data represent a novel paradigm linking tau pathology and polyamine dysfunction and that targeting specific arms within the polyamine pathway may serve as new targets to mitigate certain components of the tau phenotype.

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

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