BOULAN
BIO-INT
Marie
Tutorial 3 BIO331
Figure 7: Impact of Mangiferine (MA) on the degradation of the protein Nrf2.
Description:
In order to understand the impact of Mangiferine on the degradation of the protein Nrf2, the presence
of Nrf2 was detected by western blot on SDS/PAGE on lysates of cells treated or not with either 50 μM
Mangiferine (MA) or 10 μM MG132 or with a combination of both 50 μM MA with 10 μM MG132 for
4 hours. The Nrf2 proteins were revealed with anti-Nrf2, while -β-actin, used as a loading control was
also detected (A)
From the western blot, two types of Nrf2 can be identified: Nfr2 (molecular weight: 60 kDA), Nrf2-Ub
(molecular weight: 100 kDA). Based on the analysis of signal intensities, the level of Nfr2 increases
when cells are treated with MA while no Nrf2-Ub is detected. In contrast, when cells are treated with
MG132 an increased expression of both Nrf2 and Nrf2-Ub is observed. Lastly, when cells are treated
with both MA and MG132 the amount of Nrf and 2 Nrf2-Ub is similar to that observed in cells treated
with MG132 alone.
The relative level of Nfr2 and Nfr2-ub is then quantified, with untreated cells used as a reference,
(value 1). As shown in graph B, a 2 fold increase in Nrf2 is observed with MA alone, while a 4 fold
increase is observed in cells treated with MG132 alone or with both MG132 and MA.
Interpretation: While an increase in the level of Nrf2 is observed after treatment with either MA or
MG132, only accumulation of Nrf2-Ub is observed in cells treated with MG132.
From the course we know that the ubiquitin-proteasome pathway is the major pathway of protein
degradation in eukaryotic cells. Ubiquitin is a marker that targets proteins for rapid proteolysis. Polyubiquitinated proteins are recognized and degraded by a large protease complex called proteasome.
The results previously described indicate that MA increases Nrf2 protein level by interfering with
protein degradation and or/stability. However, since an increase of Nfr2-Ub proteins is only observed
in cells treated with MG132, only MG132 inhibits protein degradation though the inhibition of the
ubiquitin-proteasome protein degradation pathway allowing an accumulation of Nrf2-Ub.
The level of Nrf2-Ub is more significant when MG132 is used. When proteins are degraded by the
proteasome it is because they are labeled with poly-ubiquitin chains. The fact that the proteins are
marked with these chains can explain a larger presence observed. We can think that Nfr2-Ub are is
degarded through polyubiquitinylation and therefore that MG132 stops the activity of the
proteasome.
While MA blocks protein degradation, it is through a different way. One possibility is that it could
prevent polyubiquitinylation to occur. This would explain why and accumulation of Nrf2 is observed
after treatement with MA, but why no accumulation of polyubiquitinylated protein is observed.
However, it seems that the efficiency of MA to prevent protein degradation is lower that that of
MG132.
Conclusion: MA and MG132 both interrupt the ubiquitin-proteasome protein degradation
mechanisms but acting at two different levels of this degradation pathway.
-Mangiferine inhibits the action of ubiquitin
- MG132 reduces/stops proteasome activity.
Bien mais du coup, vous n’allez pas tout à fait jusqu’au bout de votre analyse..