3 Results
Line 1 Line 2 Line 30.0
3.3 Investigation of the UPS function in vivo
3.3.3 Investigation of the UPS function in the KI and KO mice crossed with Ub G76V -GFP mice
and the heart and liver were excised. Extracted proteins were examined by Western blot using antibodies directed against ubiquitin and GFP (Fig. 3.43).
First, this Western blot analysis showed again that the steady-state levels of ubiquitinated proteins are much higher in the liver than in the heart under normal conditions. Second, systemic proteasomal inhibition resulted in increased levels of ubiquitinated proteins and GFP, both in the heart and the liver. Unexpectedly, the GFP-antibody detected several bands around 29 kDa, two bands in the heart samples and even three bands in the liver samples. The reason for this is not known at the moment.
3.3.3 Investigation of the UPS function in the KI and KO mice
function of the UPS in an ubiquitination-dependent manner and in the whole animal (in vivo), was to cross the KI and KO mice with the UbG76V-GFP mice, which express a reporter protein with a tag for efficient degradation by the UPS. To investigate whether an alteration or even an impairment of the UPS by the cMyBP-C mutants was present, the steady-state levels of ubiquitinated proteins and GFP were determined by Western blot and the 20S proteasome activities were measured.
3.3.3.1 Investigation of the UPS function in the KI mice crossed with UbG76V-GFP mice
Proteins were extracted from ventricles of very old WT x UbG76V-GFP (age ~ 79 wk, two males and females) and KI x UbG76V-GFP (age ~ 62 wk, four males and females) mice and analyzed by Western blot using an antibody directed against GFP (Fig. 3.44).
A 3.6-fold increase of the GFP level was found in the KI x UbG76V-GFP mice compared to WT indicating an impairment of the UPS. It was then investigated
Figure 3.44: Determination of the GFP level in ventricles of WT and KI mice crossed with UbG76V-GFP mice. Proteins were extracted from ventricles of WT and KI mice crossed with UbG76V -GFP mice. On the left, a blot stained with an antibody directed against GFP is shown. The first lane represents the molecular weight marker (MW). Below is the corresponding Ponceau. On the right, the quantitative analysis normalized to Ponceau and related to WT is shown. Bars represent the mean±SEM with *P<0.05 vs. WT, Student`s t-test. The number of animals is indicated in the bars.
WT KI
GFP
Ponceau 25
WT KI
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
4 8
*
GFP proteinlevel(AU)
MW WT KI
GFP
Ponceau 25
WT KI
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
4 8
*
GFP proteinlevel(AU)
MW
The steady-state levels of ubiquitinated proteins were elevated in the KI x UbG76V -GFP mice as compared to WT mice. Quantitative analysis showed a 2.6-fold increase in the amount of ubiquitinated proteins. Thus, the UPS was altered at the ubiquitination level in old KI x UbG76V-GFP mice.
To examine whether there is also an alteration of the UPS at the degradation level in these old mice, all three 20S proteasome activities were measured in the same animals as above (Fig. 3.46).
Figure 3.46: The 20S proteasome activities in ventricles of WT and KI mice crossed with UbG76V -GFP mice. Proteins were extracted from ventricles of WT and KI mice crossed with UbG76V-GFP mice. The 20S proteasome activities were measured using specific, fluorogenic substrates. Bars represent the mean±SEM. The number of animals is indicated in the bars.
Figure 3.45: Ubiquitination in ventricles of WT and KI mice crossed with UbG76V-GFP mice.
Proteins were extracted from ventricles of WT and KI mice crossed with UbG76V-GFP mice. On the left, a blot stained with an antibody directed against ubiquitin is shown. The first lane represents the molecular weight marker (MW). Below is the corresponding Ponceau. On the right, the quantitative analysis normalized to Ponceau and related to WT is shown. Bars represent the mean±SEM with
***P<0.001 vs. WT, Student`s t-test. The number of animals is indicated in the bars.
WT KI
Ponceau
WT KI
0.0 0.5 1.0 1.5 2.0 2.5 3.0
4 8
***
Ubiquitinatedproteins(AU)
MW
Ubiquitinated proteins 150
WT KI
Ponceau
WT KI
0.0 0.5 1.0 1.5 2.0 2.5 3.0
4 8
***
Ubiquitinatedproteins(AU)
MW
Ubiquitinated proteins 150
WT KI
0.0 0.5 1.0 1.5 2.0
4 8
Chymotrypsin-like activity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
4 8
Trypsin-likeactivity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
3 7
Caspase-likeactivity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
4 8
Chymotrypsin-like activity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
4 8
Chymotrypsin-like activity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
4 8
Trypsin-likeactivity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
4 8
Trypsin-likeactivity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
3 7
Caspase-likeactivity(AU)
WT KI
0.0 0.5 1.0 1.5 2.0
3 7
Caspase-likeactivity(AU)
All three 20S proteasome activities had a tendency to be elevated in old KI x UbG76V -GFP vs. WT mice, but the elevations were not significant. Thus, no significant alteration of the UPS was detected at the degradation level in old KI x UbG76V-GFP mice.
3.3.3.2 Investigation of the UPS function in the KO mice crossed with UbG76V -GFP mice
Proteins were extracted from very old WT x UbG76V-GFP (age ~ 83 wk, all females) and KO x UbG76V-GFP (age ~ 77 wk, all females) mice and analyzed by Western blot using an antibody directed against GFP (Fig. 3.47).
The GFP protein level was increased (1.5-fold) in old KO x UbG76V-GFP mice vs. WT mice, but not significantly. As in Fig. 3.43, the GFP-antibody detected unexpectedly two bands around 29 kDa. The reason for this is not known at the moment.
Figure 3.47: Determination of the GFP level in ventricles of WT and KO mice crossed with UbG76V-GFP mice. Proteins were extracted from ventricles of WT and KO mice crossed with UbG76V -GFP mice. On the left, a blot stained with an antibody directed against -GFP is shown. The first lane represents the molecular weight marker (MW). Below is the corresponding Ponceau. On the right, the quantitative analysis normalized to Ponceau and related to WT is shown. Bars represent the mean±SEM. The number of animals is indicated in the bars.
25MW
WT KO
GFP
Ponceau
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
GFP proteinlevel(AU)
MW
25MW 25
WT KO
GFP
Ponceau
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
GFP proteinlevel(AU)
MW
The level of ubiquitinated proteins was 2-fold higher in old KO x UbG76V-GFP mice vs. WT mice. Thus, like in the old KI x UbG76V-GFP mice, the UPS was altered at the ubiquitination level in old KO x UbG76V- GFP mice.
Finally, all three 20S proteasome activities were measured in the same animals as above to analyze whether there is also an alteration of the UPS at the degradation level (Fig. 3.49).
All three 20S proteasome activities tended to be higher in old KO x UbG76V-GFP mice
Figure 3.48: Ubiquitination in ventricles of WT and KO mice crossed with UbG76V-GFP mice.
Proteins were extracted from ventricles of WT and KO mice crossed with UbG76V-GFP mice. On the left, a blot stained with an antibody directed against ubiquitin is shown. The first lane represents the molecular weight marker (MW). Below is the corresponding Ponceau. On the right, the quantitative analysis normalized to Ponceau and related to WT is shown. Bars represent the mean±SEM with
***P<0.001 vs. WT, Student`s t-test. The number of animals is indicated in the bars.
WT KO
0.0 0.5 1.0 1.5 2.0
4 8
*
Chymotrypsin-like activity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
Trypsin-likeactivity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
Caspase-likeactivity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
4 8
*
Chymotrypsin-like activity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
4 8
*
Chymotrypsin-like activity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
Trypsin-likeactivity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
Trypsin-likeactivity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
Caspase-likeactivity(AU)
WT KO
0.0 0.5 1.0 1.5 2.0
5 8
Caspase-likeactivity(AU)
Figure 3.49: The 20S proteasome activities in ventricles of WT and KO mice crossed with UbG76V-GFP mice. Proteins were extracted from ventricles of WT and KO mice crossed with UbG76V -GFP mice. The 20S proteasome activities were measured using specific, fluorogenic substrates. Bars represent the mean±SEM with *P<0.05 vs. WT, Student`s t-test. The number of animals is indicated in the bars.
WT KO
Ponceau
WT KO
0.0 0.5 1.0 1.5 2.0 2.5
5 8
***
Ubiquitinatedproteins(AU)
MW
Ubiquitinated proteins 150
WT KO
Ponceau
WT KO
0.0 0.5 1.0 1.5 2.0 2.5
5 8
***
Ubiquitinatedproteins(AU)
MW
Ubiquitinated proteins
WT KO
Ponceau
WT KO
0.0 0.5 1.0 1.5 2.0 2.5
5 8
***
Ubiquitinatedproteins(AU)
MW
Ubiquitinated proteins 150