Eine Plattform für die Wissenschaft: Bauingenieurwesen, Architektur und Urbanistik
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A systematic proteomic approach to the VCP/p97-cofactor interaction landscape reveals p97 functions in stress response signaling
The ubiquitin-directed AAA+ ATPase VCP/p97 facilitates degradation of proteins in diverse cellular pathways, including ER-associated degradation, DNA damage response, ribosomal quality control, transcription factor activation, signaling, autophagy and apoptosis. To fulfil its functions in these different pathways, p97 forms alternative multiprotein complexes with distinct sets of adapter proteins and substrate processing cofactors. However, how p97 cooperates with this host of cofactor proteins, the exact composition of cofactor complexes and whether this determines functional specificity remains poorly understood from a biological as well as mechanistic perspective. In this study, we used a systematic proteomic approach to shed light on the p97-cofactor system and its targets. We generated isogenic HEK293 cell lines inducibly expressing individual p97 cofactors. From these cell lines, affinity purifications (APs) of 23 cofactor proteins were performed in parallel, followed by data-independent quantitative SWATH mass spectrometry (SWATH-MS) to identify associated proteins. With our approach, we were able to confirm basic principles of cofactor complex assembly represented by core complexes of mutually exclusive major substrate adapters, which are complemented by accessory cofactors. Accessory modulators, like deubiquitinating enzymes, were identified to bind several core complexes whereas targeting factors such as membrane proteins recruit cofactor complexes to a specific cellular site or organelle. From our findings, we propose that cofactor complex assembly might be based on mechanistic requirements for specialized regulations rather than on specific biological pathways. However, the unexpected complexity of p97-cofactor interactions could be the result of an exceptionally high dynamic within the system. Nevertheless, our MS analysis identified numerous known and novel interacting proteins and allowed us to arrange distinct cofactors into functional modules together with specific sets of additional ...
A systematic proteomic approach to the VCP/p97-cofactor interaction landscape reveals p97 functions in stress response signaling
The ubiquitin-directed AAA+ ATPase VCP/p97 facilitates degradation of proteins in diverse cellular pathways, including ER-associated degradation, DNA damage response, ribosomal quality control, transcription factor activation, signaling, autophagy and apoptosis. To fulfil its functions in these different pathways, p97 forms alternative multiprotein complexes with distinct sets of adapter proteins and substrate processing cofactors. However, how p97 cooperates with this host of cofactor proteins, the exact composition of cofactor complexes and whether this determines functional specificity remains poorly understood from a biological as well as mechanistic perspective. In this study, we used a systematic proteomic approach to shed light on the p97-cofactor system and its targets. We generated isogenic HEK293 cell lines inducibly expressing individual p97 cofactors. From these cell lines, affinity purifications (APs) of 23 cofactor proteins were performed in parallel, followed by data-independent quantitative SWATH mass spectrometry (SWATH-MS) to identify associated proteins. With our approach, we were able to confirm basic principles of cofactor complex assembly represented by core complexes of mutually exclusive major substrate adapters, which are complemented by accessory cofactors. Accessory modulators, like deubiquitinating enzymes, were identified to bind several core complexes whereas targeting factors such as membrane proteins recruit cofactor complexes to a specific cellular site or organelle. From our findings, we propose that cofactor complex assembly might be based on mechanistic requirements for specialized regulations rather than on specific biological pathways. However, the unexpected complexity of p97-cofactor interactions could be the result of an exceptionally high dynamic within the system. Nevertheless, our MS analysis identified numerous known and novel interacting proteins and allowed us to arrange distinct cofactors into functional modules together with specific sets of additional ...
A systematic proteomic approach to the VCP/p97-cofactor interaction landscape reveals p97 functions in stress response signaling
Hülsmann, Julia (Autor:in) / Meyer, Hemmo
17.03.2020
Hochschulschrift
Elektronische Ressource
Englisch
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