It was reported that in response to IFN-γ treatment, polyubiquitination of newly translated proteins is enhanced, accompanied by remodeling of the UPS (Seifert et al., 2010). Thereby, the authors observed an accumulation of polyubiquitin conjugates which they claim are only efficiently cleared in IP proficient cells. As a consequence they suggested that this enhanced proteolytic capacity of the IP preserves cell viability during IFN-γ induced oxidative stress.
This proposed new function of the immunoproteasome raised a number of fundamental questions that are difficult to resolve with the present understanding of proteasome function.
Therefore some key findings of Seifert et al. were reinvestigated.
3.4.1 Ubiquitin conjugate degradation during immunoproteasome neosynthesis
Seifert et al. reported that IFN-γ induced polyubiquitin conjugates accumulate due to a transient decrease in proteasome activity during the shift from 26S SP to IP (Seifert et al., 2010). Within this time period of up to 48 hours of IFN-γ treatment the amount of ubiquitin conjugates of total lysate from wild type and LMP7 deficient MEFs was monitored. Cell lysates were separated by SDS-PAGE and analyzed by immunoblot (Figure 37 A).
Densitometric analysis of total polyubiquitin specific signal normalized to the loading control is shown in Figure 37 B for four independent experiments (three experiments were performed by Michael Basler, Biotechnology Institute Thurgau, Switzerland). With this experiment no significant differences between wild type and LMP7 deficient MEFs, with respect to ubiquitin conjugates during 48 hours of cytokine treatment, visualized by ubiquitin “smear” in immunoblot were detectable in contrast to Seifert et al.. The efficiency of IFN-γ induction as well as LMP7 deficiency by the induction or lack of expression of LMP7 in wild type and LMP7-deficient MEFs respectively was controlled by immunoblot. As seen in Figure 37 C, immunoblots of total cell lysates of both wild type and LMP7 deficient MEFs treated with IFN-γ for up to 48 hours showed LMP7 induction, as expected.
Figure 37: The amount of high molecular weight polyubiquitin conjugates does not change in response to IFN-γ. (A) Immunoblot analysis of polyubiquitin conjugates in MEFs from LMP7 knockout and C57BL/6 wild type mice at different times after exposure to IFN-γ; α-tubulin served as loading control. One representative experiment out of four independent experiments with similar outcomes is shown. (B) Bar graph showing the ubiquitin levels determined by densitometric analyses of four different immunoblot reproductions comparable to the one shown in (A); shown are the mean values +/- SD obtained after normalization to α-tubulin and relative to the value for C57BL/6 wild type mice before IFN-γ stimulation (BL6 WT 0 hour). Unpaired student´s t-test. * p<0.05. (C) The induction of LMP7 with 200 U/ml IFN-γ was confirmed by immunoblot analysis; α-tubulin served as loading control.
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-/-3.4.2 ALIS induction and degradation during immunoproteasome neosynthesis
Seifert et al. reported that, similar to the induction of polyubiquitin conjugates, the formation of ALIS is induced by IFN-γ. Therefore it was analyzed by immunofluorescence whether inclusions are formed differently in MEF cells generated from wild type and LMP7-deficient mice. MEF cells from C57BL/6 wild type and LMP7-deficient mice were grown on coverslips and treated with IFN-γ (200 U/ml) for up to 48 hours. Cells were fixed, permeabilized and stained for ubiquitin with an anti-ubiquitin antibody (FK2) which detects mono- and polyubiquitinated conjugates (Figure 38 A). With confocal microscopy ubiquitin positive structures were quantified by measuring the total area of extranuclear, FK2 positive structures larger than 0.5 µm2. The number of ALIS per cell was calculated by dividing the total area of FK2 fluorescence of all ALIS per cell by the minimum ALIS-size of 0.5 µm2. Statistical analysis was performed for three independent experiments resulting in a total of 210 analyzed cells (Figure 38 B). As reported by Seifert et al., we could also detect inclusion formation after IFN-γ treatment in both wild type and LMP7 deficient MEFs. However, in contrast to their data, we found that the number of aggregates per cell increased to the same extent in LMP7-deficient and wild type MEFs.
Figure 38: The number of ALIS per cell increased to the same extent in LMP7-deficient and wild type MEFs. (A) LMP7 knockout and wild type C57BL/6 MEFs were treated with IFN-γ for the indicated time period. Formation of ALIS was visualized after fixation by immune staining for ubiquitin (FK2) (green). Accumulation of ubiquitin positive aggregates (arrow heads) was detectable after IFN-γ exposure. Scale bar 10 µm. Pictures were analyzed with ImageJ software. (B) Statistical evaluation of ALIS per cell was performed for three independent experiments; N>210 cells. Unpaired student´s t-test. * p<0.05.
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4 Discussion
No well-defined immunological function for FAT10 has been reported to date. However, FAT10 was shown to exert important pathophysiological roles as it was reported to promote obesity, aging and tumorogenesis (Canaan et al., 2014; Gao et al., 2014). Because induced FAT10 expression was found to mediate these adverse effects, it is tempting to speculate that this might be the reason for its strict regulation. The transient nature of FAT10 expression, its assumed irreversible conjugation and subsequent degradation undermines the apparent necessity of a timely and locally restricted expression. On the other hand, however, it remains the question for what reason FAT10 expression is induced in the first place that would warrant the potential risk? The expression profile of FAT10 indicates that this benefit might be of immunological relevance and might be considered as the primary function of FAT10.