Watson, B. G. | bioRxiv Science

bioRxiv2019

Alpha-Synuclein is a Target of Fic-mediated Adenylylation/AMPylation: Implications for Parkinson’s Disease

During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called UPR (Unfolded Protein Response) that functions to maintain ER (endoplasmic reticulum) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification (PTM) called Fic-mediated Adenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an AMP (adenosine monophosphate) to the ER chaperone, BiP, to alter the cells UPR-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE - alpha-Synuclein (Syn), a presynaptic protein involved in Parkinsons disease (PD). Aggregated Syn has been shown to induce ER stress and elicit neurotoxicity in PD models. We show that HYPE adenylylates Syn and reduces phenotypes associated with Syn aggregation in vitro, suggesting a possible mechanism by which cells cope with Syn toxicity.\n\nHIGHLIGHTSO_LIAggregated forms of the presynaptic protein Syn cause neurotoxicity and induce ER stress in cellular and animal models of Parkinsons disease.\nC_LIO_LIWe have identified Syn as a novel target for the human Fic protein, HYPE, a key regulator of ER homeostasis.\nC_LIO_LIHYPE adenylylates Syn and reduces the aggregation of recombinant Syn\nC_LIO_LIFic-mediated adenylylation/AMPylation is a possible mechanism by which cells cope with Syn toxicity.\nC_LI\n\nGraphic Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2018

Kinetic And Structural Parameters Governing Fic-Mediated Adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78

Fic (filamentation induced by cAMP) proteins regulate diverse cell signaling events by post-translationally modifying their protein targets, predominantly by the addition of an AMP (adenosine monophosphate). This modification is called Fic-mediated Adenylylation or AMPylation. We previously reported that the human Fic protein, HYPE/FicD, is a novel regulator of the unfolded protein response (UPR) that maintains homeostasis in the endoplasmic reticulum (ER) in response to stress from misfolded proteins. Specifically, HYPE regulates UPR by adenylylating the ER chaperone, BiP/GRP78, which serves as a sentinel for UPR activation. Maintaining ER homeostasis is critical for determining cell fate, thus highlighting the importance of the HYPE-BiP interaction. Here, we study the kinetic and structural parameters that determine the HYPE-BiP interaction. By measuring the binding and kinetic efficiencies of HYPE in its activated (Adenylylation-competent) and wild type (de-AMPylation-competent) forms for BiP in its wild type and ATP-bound conformations, we determine that HYPE displays a nearly identical preference for the wild type and ATP-bound forms of BiP in vitro and preferentially de-AMPylates the wild type form of adenylylated BiP. We also show that AMPylation at BiPs Thr366 versus Thr518 sites differentially affect its ATPase activity, and that HYPE does not adenylylate UPR accessory proteins like J-protein ERdJ6. Using molecular docking models, we explain how HYPE is able to adenylylate Thr366 and Thr518 sites in vitro. While a physiological role for AMPylation at both the Thr366 and Thr518 sites has been reported, our molecular docking model supports Thr518 as the structurally preferred modification site. This is the first such analysis of the HYPE-BiP interaction and offers critical insights into substrate specificity and target recognition.

biochemistry↗