Angelman Syndrome: Proteomics Analysis of an UBE3A Knockout Mouse and Its Implications (Proteomic Analysis in Neurological Disorders) Part 2

Results

In this project, the effects of the loss of UBE3A proteins were investigated using Ube3a knockout mice. Two-D DIGE method [Figure 1] was used to identify candidate substrates of Ube3a in the cerebellum and hippocampus; a total of 94 proteins and 74 proteins were initially found differentially expressed in the cerebellum or hippocampus of the Ube3a knockout mice, respectively. Next, the protein candidates were tested with the following filtering criteria: 1) proteins appeared in all 2-D DIGE runs examined and 2) the one-way Analysis of Variance (ANOVA) compared protein expression level between the wild type and AS mice indicating the difference is statistically significant. The differentially expressed proteins were then identified by MALDI-TOF with a threshold score of 63. A total of 10 proteins that were successfully identified and fulfilled the filtering criteria are listed in Table 4. An additional 4 proteins, including Mash1, NeuroD, Pax6 and VDR, were not detected in 2-D DIGE runs but were investigated in this project due to the fact that these proteins were shown to be highly associated with one of the proteins identified in this project, CaBP (14).

Fig. 1. Work flow of project

Differentially expressed proteins identified by the 2-D DIGE could be due to the direct, indirect or both direct and indirect effects from the loss of Ube3a expression. Ube3a is known to be involved in proteasome-dependent as well as proteasome-independent functions. Therefore, Western blot analysis was performed to verify the differentially expressed proteins identified by 2-D DIGE analysis and further studied at the mRNA level by Real-Time RT-PCR. It helps to understand that these protein candidates may be affected at the protein level due to impaired protein degradation mechanism (4). Alternatively, these proteins may be affected at the transcriptional level, as Ube3a is also involved in transcriptional regulation (15).

By comparing protein samples from wild type mice and AS mice in the 2-D DIGE study, protein candidates that showed differential expression were found and identified by the MALDI-TOF method. Western blot was then employed to confirm the differential expression observed in 2-D DIGE. Real time RT-PCR was used to detect any differences at the transcriptional level in the protein candidates identified.

Table 4. Differentially expressed proteins detected by 2-D DIGE

2-D DIGE and silver staining

After the Decyder software analysis was performed by using analytical gel (CyDye labelled), another set of protein electrophoresis (preparative gel) were performed using 600 μg of unlabelled protein sample. The protein samples used in the preparative gel were the same as those used for 2-D DIGE analysis. After electrophoresis, silver staining was conducted to visualize protein spots on the gel. Typically, 800-1000 protein spots were visualized on each gel [Figure 2].

Fig. 2. Silver staining of acrylamide gel after SDS-electrophoresis

Protein extract (600 μg) from cerebellum was loaded in a first dimension IPG strip (pH3-11, NL, 24 cm; Running time: 15.5 hr, approx 47 kVh) and resolved in 12.5% acrylamide gel (Running time: 5.15 hr, 10 W per gel). Proteins were visualized by silver staining. These gels are called preparative gels, and they contain more protein content to allow for subsequent analyses including silver staining and MALDI-TOF. CyDye labelled proteins were run on analytical gels which are scanned by lasers and thus do not require a high quantity of proteins.

Detection and identification of differentially expressed protein in AS versus wild type brain tissue

A total of ten differentially expressed proteins were detected by using 2-D DIGE from protein samples extracted from the cerebellum or hippocampus of wild type mice and AS mice (Ube3a knockout). The protein spots were recovered from silver-stained acrylamide gels run in parallel, and identification was made based on the protein profile generated by MALDI-TOF [Figure 3] with a threshold score of 63. All candidates were confirmed at least twice in separate 2-D DIGE runs and MALDI-TOF identification. Another four bHLH proteins, including Mash1, NeuroD, Pax6 and VDR, were also studied in this project, as the proteins are highly related to CaBP (14).

A total of eight proteins studied are involved in REDOX reactions, including HSP70, SOD2, MDH, LDH, VDR, GSTs-Mu1, ATP5a1 and CaBP. Four of the bHLH proteins, VDR, Pax6, Mash1 and NeuroD are involved in neuronal cell differentiation, while NSF is crucial in synaptic vesicle transmission and learning processes that are controlled by the hippocampus [Figure 4]. TPI1 is involved in energy production, while Cofilin 1 is involved in actin disassembly and may also be involved in neuronal signal transduction.

Fig. 3. Protein identification by MALDI-TOF

MS spectrum for one of the proteins identified- CaBP. After peptide detection, the peptide profile was used to match the NCBI database for protein identification.

Fig. 4. 2-D DIGE results and inter-connected functions of the identified proteins. This figure shows differentially expressed proteins categorized by their functions. Some proteins are known to be involved in multiple pathways and functions.