5 Discussion
5.4 Discussion results 4.4
5.4.2 The effect of L11 on Hdm2 and HdmX
Ribosomal proteins as L11, L23, and L5 control Hdm2 by regulating its levels and activity as Ubiquitin ligase. Overexpression of these proteins induces the same effect as nucleolar stress, characterized by stabilization of p53 and Hdm2 [175, 177-178, 221, 223, 225, 227, 229]. One of the current hypotheses of how L11 mediates the inhibition of Hdm2 is the withdrawal of Hdm2 from the place where it has its major role as Ubiquitin ligase of p53 (nucleoplasm). In an alternative model Mu-Shui Dai et al proposed that ribosomal proteins block ubiquitination of substrates by binding between the RING domain of Hdm2 and the substrate (in this case p53)[175, 178]. To test the effect of L11 on the activity of Hdm2, we used an in vitro approach and tested the effect of the addition of L11 on the reactions. Hdm2-mediated ubiquitination of substrates as p53, HdmX and even Hdm2 itself was inhibited by addition of L11. With this
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approach, we demonstrated that in addition to relocalization, L11 binding to Hdm2 has a direct effect on the activity to ubiquitinated substrates. The Hdm2 RING domain alone which does not carry the L11 binding site is not able to be inhibited by L11. Interestingly, the formation of free Ubiquitin chains catalyzed by Hdm2 in presence of its Ubiquitin E2 was not affected. How does L11 block Hdm2-mediated substrated ubiquitination? A possible mechanism is an inhibitory effect of L11 on Hdm2 oligomerization which is required for its activity [264]. The RING domain of Hdm2 has also been reported to interact with the central acidic domain of Hdm2 [265].This interaction could be required for the proper accommodation of its RING allowing the ubiquitination of substrates. It has also been reported that Hdm2 is able to form Ubiquitin free chains as a monomer [218].
Further experiments were established in vitro to determine the effects of HdmX on the L11 inhibition of Hdm2. HdmX seems to help Hdm2 to escape from the L11-dependent inhibition. This may result in a partial rescue of the activity of Hdm2 and consequent substrate ubiquitination. The Hdm2/HdmX ligase complex can be more resistant to the effect of L11 due to HdmX does not interact with L11. Based on these observations, we propose a model where Hdm2 and HdmX oligomers mediate the ubiquitination of substrates (Figure 61).
However, dimers are probably sufficient to recognize the Ubiquitin E2s and to induce Ubiquitin chain formation. The contact of the Hdm2 oligomers (for simplification we consider tetramers in this case), requires a double interaction of dimers: on the one hand the RING dimerization and on the other hand RING interaction with the acidic domain of a second molecule (Figure 61). Six interaction sites stabilize the tetramer. L11 could interfere with the interaction of the RING with the acidic domain impairing four of the six contact sites resulting in the dissociation of tetramer, but not the RING dimer complexes. The dimers can still lead to the allosteric activation of the Ubiquitin E2 (i.e for chain formation of free Ubiquitin), but probably not longer initiate the ubiquitination of substrates (Figure 62A). In this scenario, due to HdmX do not interact with L11, the complex of HdmX/Hdm2 can not dissociate completely (Figure 62B), even when two of the interaction sites are disturbed. This may partly affect the nature of the Hdm2/HdmX complex, but ubiquitination of substrates and Ubiquitin chain
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formation may still be possible (Figure 62B).If the model is correct, how ubiquitination of in vitro translated HdmX is inhibited by L11 in vitro? In the model, we propose that an L11 insensitive complex consist of a tetramer of two heterodimers. The in vitro translated amounts of HdmX are very low compared to the concentration of the Hdm2 ligase in the reaction (~1/200). In these conditions, due to the high amounts of Hdm2 homodimers, the L11 non-sensible complex (tetramer of heterodimers) can not be formed, resulting in inhibition of HdmX ubiquitination. In cells, L11 inhibits Hdm2-mediated ubiquitination of p53, increasing p53 levels. Co-transfection of HdmX partially rescued Hdm2-mediated ubiquitination of p53, but did not affect significantly the L11-induced levels of p53. Other proteins are possibly involved in the outcome of Hdm2, HdmX and L11 on p53 ubiquitination and levels, which can explain the differences observed in vitro and in cellulo.
Our model is also supported by microscopie data where Hdm2 can relocalize HdmX and L11 at the same time into the nucleoplasm, suggesting the possibility of ternary complexes. HdmX did not affect the localization of L11 and vice versa. Further biochemical analysis is required to prove these initial observations. Co-immunoprecipitations of L11 and HdmX, in precense and absence of Hdm2 could give more evidences about the endogenous complex and its regulation.
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Figure 61. A model for binding of L11 to Hdm2 and HdmX complexes. A fully active oligomer of Hmd2 is stabilized by six surfaces of contact, including the RING-RING dimerization and acidic domain-RING interaction. This conformation is essencial for susbtrate recognition and ubiquitination. Upon nucleolar stress, ribosomal proteins as L11 interact with Hdm2 on its Zing finger domain dissociating acidic domain-RING interactions. In the case of the hetero tetrameric complex Hdm2/HdmX. L11 can not bind HdmX, therefore only two contact (acidic domain-RING) could be dissociated. Resulting in complex which still conserves 4 surfaces of interaction, is not dissociated, and consequently it kept actived. In the model, surfaces of interaction are indicated in yellow.
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(A)
(B)
Figure 62. A model for a selective inhibition of Hdm2 by L11. (Uper panel) The L11-mediated dissociation of the tetrameric form of Hdm2 specifically inhibits susbtrates ubiquitination, but the dimerization of the RING is not affected in consequence Hdm2 is able to activated allosterically the Ubiquitin E2s and induce Ubiquitin chains formation. (Lower panel).
The HdmX tetrameric complex is not dissociated by L11. Therefore, HdmX/Hdm2 complex is still able for susbtrates ubiquitination.
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