Final remarks and outlook

Taken together, our results point to a mechanism that is rather based on the degradation of secreted proteins right after translation than on a feedback mechanism that inhibits translocation of maturated proteins when misfolded proteins are accumulating in the cytosol. We proved the cytosolic accumulation of FLAG-PSCA-HA to be independent of side effects from chemical proteasome inhibitors and its relevance for endogenously expressed proteins. Now, we begin to understand the mechanism behind this observation, but there are still many open questions.

First, it is necessary to reproduce Bag6 knockdown effects on FLAG-PSCA-HA precursor protein degradation and to identify other chaperones and E3 ligases leading to recruitment of PSCA to the 26S proteasome. It was shown for cytoplasmic PrP that N- or C-terminally located hydrophobic sequences are needed for its ubiquitination and proteasomal degradation (Hessa et al. 2011) and we should prove such a mechanism for PSCA. It was shown that proteins with extremely efficient signal sequences (e.g. Osteopontin) did not accumulate during MG132 treatment (Rane et al. 2004; Kang et al. 2006). Domain swapping experiments using

110 Osteopontin signal peptide and for FLAG-PSCA-HA protein during proteasome inhibition would help to understand the role of translocation efficiencies with respect to precursor compartmentalization and resulting MHC class I peptide assembly. Moreover, analyzing the accumulation of FLAG-PSCA-HA precursor protein during kifunensine treatment, which inhibits access of glycoproteins to ERAD, could shed more light on the participation of ERAD to the mislocalization process. Integration of proteins into Sec61 is inhibited by cotransin and investigation of FLAG-PSCA-HA glycosylation pattern during cotransin treatment, as compared to MG132 treatment, could help to identify possible overlapping pathways. Mislocalized PrP aggregates are believed to contribute to translocation inhibition of cytoplasmic PrP (Ma et al.

2002; Chakrabarti et al. 2011). Hence, it would be worthwhile to investigate whether cytosolic FLAG-PSCA-HA proteins aggregate during MG132 treatment, too. Additionally, the reversibility of FLAG-PSCA-HA precursor protein accumulation after removal of MG132 should be analyzed.

Huge proteasomal burden because of misfolded immunoglobulin chains, permanently activates the unfolded protein response and ERAD in multiple myeloma cells (Bianchi et al. 2009; Cenci et al. 2006), resulting in high sensitivity to proteasome inhibitors like bortezomib. An intracellular mechanism that degrades secretory proteins right after translation, to enhance the antigenic repertoire of MHC class I molecules, would further explain the extensive cytotoxic effect of proteasome inhibition. Identifying the mechanism of protein mislocalization would help to better understand the major developing processes of many human diseases. Several neurodegenerative disorders, like Alzheimer’s disease, Parkinson’s disease or Huntington’s disease, and various cancer types are caused by cellular accumulation of misfolded proteins (Selkoe 2003; Rubinsztein 2006; Soto et al. 2006; Dai et al. 2007; Morimoto 2008). Additionally, selective transport inhibition of ER-targeted proteins determines polycystic liver disease, which is characterized by progressive development of biliary epithelial cysts throughout the liver (Davila et al. 2004). Pathologies, like lysosomal storage disease (Sawkar et al. 2006) and cystic fibrosis (Koulov et al. 2010), result from misfolded proteins, too. Elucidating the reason why cells occasionally fail to compartmentalize misfolded cytotoxic species, endorsing them to interfere with normal cellular protein homoeostasis, will assist to shed light on the etiology of amyloid diseases.

111 7.2. Immunoproteasome precursor organization in murine astrocytes

The assembly of immunoproteasomes is an event that includes multiple steps. N-terminal pro-sequences and C-terminal extensions of the subunits render the proteasomes catalytically inactive until they are cleaved off in the final maturation step. This autocatalytic cleavage generates the functional active conformation of the 20S proteasome complex (Schmidtke et al.

1996). The assembly of immunoproteasome subunits seems to be partially impeded in the brain, which is indicated by the accumulation of immunoproteasome precursors (Kremer et al. 2010).

In this study, we generated polyclonal antibodies against the immunoproteasome subunits LMP2, LMP7 and MECL-1 and were able to confirm the findings of Kremer et al. This points to the existence of a post-translational mechanism, which regulates immunoproteasome formation in areas where uncontrolled inflammatory responses could cause considerable harm destroying fundamental, non-renewable cells. An enrichment of unprocessed immunoproteasome precursor proteins in lower fractions of density gradients of cytokine-induced astrocytes supports the hypothesis of incomplete proteasome formation and precursor proteins that accumulate in half-proteasomes (Chen & Hochstrasser 1995), stated by Kremer et al. Next, we used FLAG-tagged MECL-1 and detected the same effect, which proves the construct as an excellent tool for further research and immunoprecipitation experiments. Establishing a cell line that stably expresses the FLAG-tagged MECL-1 construct would be the next step towards the elucidation of this mechanism. Factors that regulate the suppression of immunoproteasome formation in brains are unknown, but such a mechanism may exist in other cells. PI31 is a protein that was identified as a suppressor of 20S and 26S proteasomes function (M Chu-Ping et al. 1992) and overexpression of PI31 in astrocytes could explain the precursor accumulation. Our results are contrary to this hypothesis. The amount of P31 in astrocytes was slightly reduced as compared to control cells. Clarifying the role of immunoproteasome expression and the mechanism of its reduced formation in brains could provide new insights into immune response regulation in immunoprivileged organs and help to understand how active immune tolerance can be achieved.

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8. Appendix