The mechanism of toxicity of Norspermidine in Arabidopsis
Sayas E1*, Alabadí D1, del Pozo JC2, Ferrando A1, Serrano R1
1 Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-C.S.I.C
2 Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid
*ensamon@upvnet.upv.es
Polyamines are small polycationic molecules found in all organisms where they play essential functions not well understood. On the other hand, high concentrations of polyamines are toxic by mechanisms
also unknown. The uncommon polyamine norspermidine (NE) has been used as toxic cation to select resistant mutants affected in cation homeostasis (Alejandro et al, 2007, EMBO J 26: 3203) and therefore
it is important to know the mechanisms of NE toxicity. This is also important to ascertain the use of NE as anti-tumoral drug (Prakash et al, 1988, Anticancer Res 8: 563). NE treatment triggers a transcriptional
response similar to that observed when cytosolic misfolded proteins accumulate. Accordingly, seedlings treated with NE have increased levels of insoluble proteins. This toxic effect can be explained if NE
were denaturing proteins or disturbing the Ubiquitin Proteasome System (UPS). In vivo NE treatment reduced the levels of polyubiquitinated proteins and in vitro NE inhibited the ubiquitination reaction,
probably interfering with E3 enzymes. Further evidence was obtained with a DELLA protein fused with Green Fluorescence Protein (GFP-RGA), that is degraded in the presence of gibberellins (GA) via the
UPS (Silverstone et al, 2001, Plant Cell 13: 1555). By using confocal microscopy and western blot we showed that after a previous NE treatment, RGA is stabilized in the presence of GAs. These results
suggest that one mechanism of NE toxicity is blocking the degradation of misfolded proteins by inhibition of their ubiquitination. The structural similarity between NE and lysine residues of substrate proteins of
the UPS may explain this effect.
A
B
kDa
kDa
kDa
NE inhibits poliubiquitination. Western blot of total poliubiquitinated protein A) seedlings subjected to NE
(24h, 3mM) or mock treated with or without the proteasome inhibitor MG132 (50uM, 6h) B) time course of
seedlings treated with NE (3mM).
NE inhibits ubiquitination of E3 enzyme in vitro.
Immunoblot analysis of the in vitro ubiquitination of a
recombinant E3 enzyme.
E
NE stabilizes DELLA protein GFP-RGA in the
presence of Giberellins.
Confocal images of root tips of seedlings treated
with A) mock B) 3mM, 24h NE C) 1uM, 3h GA3 D)
1uM, 3h GA3, 3mM, 24h NE.
E. Western blot showing GFP-RGA levels.
1) mock treated seedlings
2) NE (3mM, 24h)
3) GA3 (1uM, 3h) and NE (3mM, 24h)
4) GA3 (1uM, 3h)
kDa
1
2
3
4
5
6
130
95
72
55
43
34
26
NE treatment increases the insoluble protein
fraction. Total protein of seedlings treated with
3mM, 24h NE (1) or mock (2). Insoluble protein
fraction of seedlings treated with NE (3) or mock
(4). Soluble protein fraction of seedlings treated
with NE (5) or mock (6)
Suggested model of NE toxicity in
Arabidopsis.
Under normal conditions, proteins
targeted for degradation are
polyubiquitinated by the E2-E3 enzyme
complex. NE, due to its similarity to a
free lysine residue, could interact with
the E2-E3 enzyme complex and thus
preventing the correct degradation of
misfolded or denaturated proteins by
these enzymes. These misfolded
proteins could then form insoluble toxic
aggregates, which in turn would trigger
a Heat Shock response.