Analyzing the influence of elevated intracellular cAMP concentrations on SNAPIN

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The current literature suggests important role of SNAPIN in the proper lysosome functionality (see section 2.5). Therefore, the present study aimed at further elucidating the importance of SNAPIN and the identified downregulated phospho-site at S133 in different processes such as the internalization and secretion of acidic hydrolases as well as lysosome positioning.

5.1.3. Analyzing the influence of elevated intracellular cAMP concentrations on

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This was consistent among all replicates. Figure 5.4B shows the quantitative analysis of the Western Blot signals. For each replicate and condition, the SNAPIN intensity was normalized on the α-Tubulin signal. Afterwards, the means of the relative SNAPIN intensities were calculated and plotted as a bar chart for both conditions. The variability among the replicates is shown by the standard deviation. Additionally, the relative SNAPIN intensities of the single replicates are indicated as dots. By comparing the FSK/db-cAMP treated sample with the control, no difference in the mean relative SNAPIN intensity was observed. A two sample t-test revealed no significant differences in the relative SNAPIN intensities obtained for the PKA-stimulated and the non-stimulated group (homoscedastic, p-value 0.7898, significance threshold 0.05). Therefore, the results indicate that the SNAPIN protein level was not affected by the treatment.

Figure 5.4: Investigating the influence of FSK/db-cAMP treatment on SNAPIN protein level.

Cells were either incubated for 30 min with FSK/db-cAMP or DMSO (control). The experiment was performed in triplicates (n=3). Whole cell proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane.

SNAPIN detection was performed by using an anti-SNAPIN antibody (10 s exposure) (A). α-Tubulin signals were used as a loading control (1 s exposure) (A). For each condition and replicate, SNAPIN intensities were normalized on the corresponding α-Tubulin signals. The relative SNAPIN intensities of the FSK/db-cAMP treated sample group and the control are depicted as bar charts (B). The variability among the replicates is shown by the standard deviation (B).

Additionally, the relative SNAPIN intensities of the single replicates are indicated as dots (B). Statistical analysis was performed by a t-test (p-value 0.7898). (A.U.: arbitrary units; n.s.: not significant (p-value > 0.05))

5.1.3.2. Investigating SNAPIN’s association to the lysosomes upon FSK/db-cAMP treatment In phosphoproteomic studies, protein levels within the starting material are usually quantified by investigating the non-phospho-enriched fraction of the sample. Especially after organelle enrichment, stimulation dependent changes in the subcellular protein localization might be misinterpreted as phospho-site regulation although the phosphorylation status of the whole protein population remains unaltered. Within the present study, SNAPIN was not detected in the non-phospho enriched proteomic data set of the membrane lysosomal fractions (see table 5.4). In order to investigate cAMP-mediated changes in lysosome association of SNAPIN,

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HEK 293 cells were either treated with FSK/db-cAMP or DMSO. Afterwards, the lysosomes were enriched before proteins present in the lysosomal fraction were separated via SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. The experiment was performed in three biological replicates. SNAPIN levels were investigated by immune detection. The signals obtained after incubating the membrane with an antibody directed against the lysosomal protease Cathepsin D (CTSD) were used for normalizing the SNAPIN level to the amounts of lysosomes present within the different samples after the enrichment procedure. For the heavy chain of mature CTSD, a mass of approx. 31-35 kDa was expected (Huang et al. 1979; Hasilik and Neufeld 1980).

SNAPIN was detected at 15 kDa in all samples (Figure 5.5A). After incubating the upper part of the membrane with the anti-CTSD antibody, signals at approx. 35 kDa were observed. These signals were attributed to the heavy chain of CTSD. Figure 5.5B depicts the results of the signal quantification. For each replicate, SNAPIN intensities were normalized to the CTSD signal (indicated as dots). The average SNAPIN intensities identified for the two sample groups are shown as bar charts (+/- standard deviation). By comparing the FSK/db-cAMP treated sample group with the control, the SNAPIN level tends to be reduced in the lysosomal fraction upon elevation of the intracellular cAMP level. However, when applying statistical analysis (two sample t-test), the difference is not significant (homoscedastic, p-value 0.1095, significance threshold 0.05). It is reasonable to assume that the insignificance is predominantly caused by the relative SNAPIN intensity observed in the first replicated of the non-stimulated group (1.1).

Compared to the other two replicates of the control, the relative SNAPIN intensity detected in this sample is strongly reduced, thus lowering the mean value and increasing the standard deviation in the non-stimulated group.

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Figure 5.5: Investigating the influence of elevated intracellular cAMP levels on the lysosome-association of SNAPIN.

Lysosomes were enriched from HEK 293 cells, treated with FSK/db-cAMP or DMSO (control). Proteins present within the lysosomal fractions were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. Afterwards, SNAPIN (15 s exposure time) and CTSD signals (2 s exposure time) were visualized by immune detection (A). For signal quantification, SNAPIN intensities of each replicate were normalized to the corresponding CTSD signals (B; indicated as dots). The average relative SNAPIN intensity for the stimulated and non-stimulated sample group are depicted as bar charts (B). Statistical analysis was performed by a t-test (p-value 0.1095). (A.U.: arbitrary units; n.s.: not significant (p-value > 0.05))

5.1.3.3. Investigating cAMP-mediated SNAPIN dephosphorylation with 2D-gel electrophoresis

cAMP-mediated changes in the SNAPIN phosphorylation status were investigated by 2D-gel electrophoresis. In the first dimension, proteins were separated according to their isoelectric point (PI). As protein phosphorylation lowers the PI, these proteoforms migrate further to the anode (acidic pH) during IEF compared to their less-phosphorylated counterpart. After IEF, proteins were further separated via SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. A SNAPIN specific antibody was used for the detection of the different proteoforms. In addition to the FSK/db-cAMP treated sample and the DMSO control, a non-stimulated protein lysate was incubated with calf intestine alkaline phosphatase in order to visualize the migration behavior of dephosphorylated SNAPIN isoforms during 2D-gel electrophoresis. The experiment was performed in two biological replicates.

Figure 5.6A depicts the SNAPIN isoforms detected at 15 kDa after incubating the samples of the first replicate with the SNAPIN antibody. The signals corresponding to the highest phosphorylated proteoforms are indicated by the open arrowheads. Five distinct SNAPIN isoforms were observed after FSK/db-cAMP treatment. Compared to that, six proteoforms were detected in the DMSO control. Changes in the relative signal intensities were observed for the first (open arrowheads) and the third (arrow) isoform. In case of the FSK/db-cAMP treated sample, the intensity of the third proteoform is higher compared to the first one. By comparing these two isoforms in the DMSO control, a slightly stronger signal intensity of the first

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proteoform was detected. Additionally, the signal pattern of the isoform with the highest PI is less distinct in the DMSO control compared to the FSK/db-cAMP sample. The proteoform pattern observed in the phosphatase treated sample was similar to that obtained upon elevation of the intracellular cAMP levels.

The signals obtained in the second replicate are shown in figure 5.6B. Compared to the first replicate, the background signal is higher thus making the precise detection of the different SNAPIN isoforms more difficult. However, when comparing the first and the third SNAPIN proteoforms in both conditions, the isoform with the highest phosphorylation status seemed to be more abundant in the DMSO control. In case of the phosphatase treated sample, the third isoform was not detected in the second replicate.

The different relative signal intensities of the first and the third SNAPIN proteoform identified in both samples and replicates indicate that SNAPIN is dephosphorylated after FSK/db-cAMP treatment. This finding is further supported by the pattern observed in the phosphatase treated sample of the first replicate in which the third isoform is also highly abundant. Nevertheless, the absence of the third isoform in the phosphatase treated sample of the second replicate indicates that the dephosphorylation in the first replicate was not complete. The detection of at least two proteoforms after phosphatase treatment indicates the existence of two non-phosphorylated SNAPIN isoforms (expected unmodified SNAPIN PI = 9.35; phosphosite.org, retrieved 09.05.2019). In addition to these findings, SNAPIN also seems to be phosphorylated upon FSK/db-cAMP treatment as the signal pattern of the proteoforms with the highest PI appears to be less blurry compared the DMSO control.

Figure 5.6: Investigating changes in the abundance of different SNAPIN proteoforms after FSK/db-cAMP treatment by 2D-gel electrophoresis.

Cells were either incubated in medium containing FSK/db-cAMP or DMSO. Cell lysate treated with calf intestine alkaline phosphatase was used in order to identify the migration behavior of dephosphorylated isoforms. Proteins were first separated according to their PI and subsequently based on their molecular mass. Afterwards, proteins were transferred onto a nitrocellulose membrane followed by immune detection of SNAPIN. The signals shown in this figure were obtained at different exposure times (biological replicate 1 (A): FSK/db-cAMP 3 min; DMSO 5min(for the same exposure time of the FSK/db-cAMP and DMSO sample of the first replicate see Appendix; Phosphatase 1 min) (biological replicate 2 (B): FSK/db-cAMP 3 min; DMSO 3min; Phosphatase 3 min). Furthermore, the contrast (black/white value) between background and signal was adjusted for the signals of the second replicate.

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