Functional Characterization of Interaction Partners of the Co-Chaperone BAG3

Abstract

Protein homeostasis (proteostasis) describes the cellular balance between protein synthesis, function and degradation. The stress inducible co-chaperone BAG3 is a key factor in proteostasis, in particular under mechanical stress. In cooperation with the actin binding protein Synaptopodin 2 (SYNPO2), BAG3 is a central element of filamin (an actin crosslinking protein) homeostasis in muscle cells and immune cells. Here BAG3 is involved in the degradation of defective filamin via chaperone assisted selective autophagy (CASA), as well as in the induction of filamin transcription via the Hippo pathway. SYNPO2 can directly bind to the WW-domain of BAG3 and through its N-terminal PDZ-domain SYNPO2 is enabled to recruit complexes mediating the formation of autophagosomes. SYNPO2 belongs to the SYNPO family of actin binding proteins. Four isoforms of SYNPO2 exist, of which three contain an N-terminal PDZ-domain, whereas all four isoforms have a centrally located proline-rich motif (PPXY-motif). SYNPO2 was identified to interact with BAG3 via its PPXY-motif in a peptde screen for novel binding partners of the BAG3 WWdomain. Another SYNPO family member, Synaptopodin (SYNPO), was also identified in this peptide screen. SYNPO has three isoforms of which all contain two subsequent PPXYmotifs. Furthermore, SYNPO has been described to interact with the PDZ-domain containing protein MAGI-1 (Membrane Associated Guanylate Kinase Inverted 1). In this work SYNPO could be confirmed as novel BAG3 binding partner. Because SYNPO2 is not expressed ubiquitously in all cell types, it should be clarified whether SYNPO, together with BAG3, is similarly involved in autophagic protein degradation as shown for SYNPO2. In HeLa cells, which do not express SYNPO2, SYNPO turnover was monitored in the context of inhibition of the proteasome or autophagy. In contrast to SYNPO2, which is rapidly degraded via autophagy upon inhibition of the proteasome or mechanical stress, SYNPO protein levels remained stable upon inhibiton of the proteasome or autophagy. Furthermore, no association of SYNPO with autophagic marker proteins such as LC3 or p62 could be observed in HeLa cells. These findings imply, that SYNPO functions differently from SYNPO2 under the chosen experimental conditions. Challenging the cells with increased cellular stress to enhance the induction of BAG3 mediated degradation could potentially have a different effect on SYNPO turnover. Thus, similar functions of SYNPO and SYNPO2 in protein degradation can not be excluded. Immunofluorescent staining of SYNPO in HeLa cells reveals a punctate pattern. These SYNPO punctae are largely increased in size upon overexpression of the largest of the three SYNPO isoforms: SYNPOc. These SYNPOc punctae often show small pointed protrusions and sometimes appear as ring like structures. Furthermore, it could be observed that BAG3 is recruited to these SYNPOc punctae. For elucidation of the molecular functions of SYNPO punctae and the role of SYNPO – BAG3 interaction, immunofluorescent costaining experiments with several marker proteins for e.g. aggresomes, the cytoskeleton, lysosomes or endosomes were conducted. A co-localization of SYNPOc punctae with the early endosome marker EEA1 (early endosome antigen 1) could be observed pointing to a potential involvement of SYNPO in endosomal transport or processing. In addition, new binding partners of SYNPO could be identified via immunoprecipitation followed by mass spectrometry, one of them being the actin associated protein Annexin A2 (ANXA2). These findings further underscore a potential involvement of SYNPO in endosomal transport, as ANXA2 has been previously described to play a role in endosomal trafficking. The exact mechanism of SYNPO involvement in endosomal transport, and how BAG3 might contribute to this process, remains to be a topic for further investigation. Besides confirming SYNPO as a novel binding partner of BAG3, this work provides fundamental information on SYNPO behavior in HeLa cells. The nature of BAG3 – SYNPO interaction could be further characterized and new interaction partners of SYNPO could be identified. In addition, this work provides first experimental data linking SYNPO, possibly together with BAG3, to endosomes, shedding light in potential molecular functions of SYNPO in HeLa cells.