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1 2 3 4 5 Alpha-Synuclein is a Target of Fic-mediated 6 Adenylylation/AMPylation: Implications for Parkinson's Disease 7 8 Anwesha Sanyala,g*, Sayan Duttab*, Aswathy Chandranb, Antonius Kollerc,h, Ali Camaraa, Ben G. 9 Watsona, Ranjan Senguptaa, Daniel Ysselsteinb,c, Paola Montenegrob,c, Jason Cannond, Jean- 10 Christophe Rochetb,e, and Seema Mattooa,f,1 11 12 aDepartment of Biological Sciences, bDepartment of Medicinal Chemistry and Molecular 13 c 14 Pharmacology, Purdue University, Herbert Irving Comprehensive Cancer Center, Columbia d 15 University Medical Center, New York, NY, School of Health Sciences, Purdue University, 16 ePurdue Institute for Integrative Neuroscience, and fPurdue Institute for Inflammation, 17 Immunology and Infectious Disease, Purdue University, 915 W State St., LILY G-227, West 18 Lafayette, IN 47907 19 20 *equal contribution 21 1 22 To whom correspondence should be addressed. E-mail: [email protected] 23 g 24 Present address: Ann Romney Center for Neurologic Diseases, Brigham and Women’s 25 Hospital, Harvard Medical School, Boston, MA 26 hPresent address: Northeastern University, Barnette Institute, Boston, MA 27 28 The authors declare no conflict of interest. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 *ResearchbioRxiv Highlights preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

HIGHLIGHTS:  Aggregated forms of the presynaptic αSyn cause neurotoxicity and induce ER stress in cellular and animal models of Parkinson’s disease.  We have identified αSyn as a novel target for the human Fic protein, HYPE, a key regulator of ER homeostasis.  HYPE adenylylates αSyn and reduces the aggregation of recombinant αSyn  Fic-mediated adenylylation/AMPylation is a possible mechanism by which cells cope with αSyn toxicity. bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Graphic abstract

Sanyal & Dua et al., 2019 – Graphic Abstract

AMP

AMPylaon αSynuclein αSynuclein

PPi monomers monomers ATP

oligomers oligomers

fibers fibers bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 ABSTRACT: 5 During disease, cells experience various stresses that manifest as an accumulation of 6 misfolded and eventually lead to cell death. To combat this stress, cells activate a 7 8 pathway called UPR (Unfolded Protein Response) that functions to maintain ER (endoplasmic 9 reticulum) homeostasis and determines cell fate. We recently reported a hitherto unknown 10 mechanism of regulating ER stress via a novel post-translational modification (PTM) called Fic- 11 mediated Adenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation 12 induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an AMP (adenosine 13 monophosphate) to the ER chaperone, BiP, to alter the cell’s UPR-mediated response to 14 misfolded proteins. Here, we report that we have now identified a second target for HYPE - 15 alpha-Synuclein (αSyn), a presynaptic protein involved in Parkinson’s disease (PD). Aggregated 16 αSyn has been shown to induce ER stress and elicit neurotoxicity in PD models. We show that 17 18 HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation in vitro, 19 suggesting a possible mechanism by which cells cope with αSyn toxicity. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 INTRODUCTION: 5 Post-translational modifications (PTMs) catalyzed by the Fic (filamentation induced by 6 cAMP) family of are fast being recognized as a central regulatory paradigm governing 7 8 diverse signal transduction pathways in both and [1]. Fic proteins 9 consist of a core HxFx(D/E)(G/A)N(G/K)RxxR motif, with the being essential for 10 catalytic activity [2]. Additionally, the activity of most Fic proteins is inhibited by inter- and intra- 11 molecular interactions with an inhibitory helix (α-inh), characterized by an (S/T)xxxE(G/N) motif 12 [3]. We previously discovered that some bacterially secreted Fic proteins could induce host cell 13 toxicity by covalently modifying mammalian Rho RhoA, Rac1, and Cdc42 with AMP 14 () [2], [4]. This adenylylation or "AMPylation" event renders the Rho 15 GTPases inactive, thereby inducing cytoskeletal collapse and allowing the bacteria to evade 16 phagocytosis [5], [6]. We further showed that the activity of Fic proteins was conserved in 17 18 eukaryotes. Specifically, we demonstrated that the sole Fic protein in humans, HYPE 19 (Huntingtin yeast interacting protein E) or FicD, functions as an adenylyltransferase [2, 4, 7]. 20 HYPE is a 52kDa protein and is classified as a Class II Fic protein, consisting of a canonical Fic 21 domain and an α-inh sequence that lies N-terminal to its Fic motif (Figure 1A). Accordingly, a 22 mutation of HYPE's His363 to Ala renders it enzymatically inactive, while a mutation of its 23 Glu234 to Gly renders it constitutively active [3], [7]. We showed that HYPE localizes to the 24 lumen of the ER () and adenylylates the Hsp70 chaperone, BiP (Binding 25 immunoglobulin protein) [7]. BiP monitors protein folding in cells and serves as a sentinel for the 26 27 activation of an ER stress response pathway called the UPR (unfolded protein response)[8]. 28 During disease, cells experience various stresses that manifest as an accumulation of 29 misfolded proteins and eventually lead to cell death [9]. To combat this stress, cells activate the 30 UPR pathway, which initially functions to restore normalcy to the cell. However, upon sustained 31 stress, the UPR triggers . Thus, maintenance of ER homeostasis is a critical aspect of 32 determining cell fate and requires a properly functioning UPR. It was recently reported that while 33 E234G-HYPE adenylylates BiP, WT-HYPE serves to reverse HYPE-mediated adenylylation of 34 BiP [10]. Thus, WT-HYPE is classified as a de-AMPylase and a H363A mutation in the wild type 35 background renders an inactive de-AMPylase. Together, the adenylyltransferase and de- 36 37 AMPylase activity of HYPE on BiP highlights the important role HYPE plays in tightly regulating 38 UPR and ER homeostasis in response to protein misfolding. 39 Proper protein folding entails a process where the nascent polypeptides are folded into a 40 three-dimensional functional identity. The organized native structure of the protein is at its 41 energy minimum state while the unfolded polypeptide has higher free energy [11]. Molecular 42 chaperones in the cytosol and ER, such as BiP, bind misfolded proteins with varying specificity 43 and mediate their folding [12]. Any changes in the system that destabilize the folding process 44 lead to the formation of misfolded species that lack a thermodynamically stable structure and 45 46 hence have a propensity to aggregate [13]. These aggregates are prone to further aggregation 47 by a process called nucleation, thus increasing in size at an accelerated rate [13]. Such 48 aggregates are a hallmark of various degenerative diseases like Parkinson’s disease (PD), 49 Alzheimer’s disease, Huntington's disease, type II diabetes, cystic fibrosis and others [13]. 50 Several lines of evidence suggest that accumulation of specific protein aggregates is toxic for 51 cells [14]. 52 PD is a progressive neurodegenerative disorder characterized by tremors, bradykinesia, 53 rigidity and instability of posture [15]. These motor symptoms are thought to arise due to the 54 toxic accumulation of aggregates of a presynaptic protein - alpha-Synuclein (αSyn) - in 55 56 dopaminergic neurons of the substantia nigra in the midbrain [16]. αSyn is encoded by the 57 SNCA gene and consists of a 140 amino acids (Figure 1B). It is an intrinsically disordered 58 protein that consists of an amphipathic N-terminal region and an acidic C-terminal region 59 flanking a highly hydrophobic central domain, also referred to as the NAC (Non-Amyloid-beta 60 Component of Alzheimer's disease) domain. Because of its natively unfolded structure, αSyn 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 has a high propensity to aggregate[13]. Further, point mutations in the SNCA gene that target 5 Ala53 in particular and those that result in Ala53Thr, Ala53Glu, Ala30Pro, Glu46Lys, His50Gln, 6 and Gly51Asp substitutions, as well as duplication or triplication of the gene, lead to familial 7 8 forms of PD due to enhanced aggregation of the protein [17]. , , O- 9 GlcNAcylation, and of αSyn are also reported, suggesting possible roles of 10 PTMs in PD [18] [53]. αSyn fibrils accumulate in structures called Lewy bodies in surviving 11 neurons [19]. Although Lewy bodies are a hallmark neuropathological feature of PD, evidence 12 suggests that the intermediate oligomeric species capable of forming fibrils may be more toxic 13 than Lewy bodies, potentially because of their ability to permeabilize lipid membranes [20-23]. 14 Such αSyn oligomers can also diffuse into the ER lumen and interact directly with BiP. 15 Accumulation of αSyn oligomers results in ER stress and activation of UPR in affected neurons 16 [24, 25]. 17 18 Our discovery that HYPE adenylylates BiP established HYPE as a new player in UPR 19 regulation in response to ER stress. We and others have now demonstrated that adenylylation 20 alters BiP's ATPase activity and activation of the downstream UPR cascades to reinstate ER 21 homeostasis [7], [26-28]. Given this critical role for HYPE in how cells cope with stress from 22 misfolded proteins and the direct correlation between αSyn accumulation, ER stress and PD 23 progression, we reasoned that HYPE may play a role in PD, possibly via UPR regulation or 24 direct interaction with αSyn. Indeed, a role for HYPE in neurodegeneration was recently 25 reported in a C. elegans model, where activation of HYPE’s adenylyltransferase activity was 26 27 shown to induce neuroprotective aggregative effects of proteins like amyloid beta (Aβ), mutant 28 Huntingtin (m-Htt), and αSyn through its manipulation of cytosolic Hsp70 chaperones [29]. We, 29 too, have shown previously that HYPE can directly bind to misfolded proteins [7], and global 30 proteomic analyses of adenylylated peptides by mass spectrometry indicate targets for HYPE 31 other than BiP and related heat shock proteins [30, 31]. However, αSyn has never been 32 identified as a target for HYPE or known to be adenylylated. 33 Here, we report αSyn as a bona fide novel target for HYPE. We show that HYPE directly 34 binds αSyn in vitro and adenylylates it at residues predominantly in its N-terminus. 35 Accordingly, analysis of rat midbrain sections and primary cultures reveals that HYPE is 36 37 enriched in neurons of the substantia nigra and colocalizes with markers for dopaminergic 38 neurons, sites where αSyn aggregates during PD. Importantly, adenylylation alters the structure 39 of αSyn fibrils and leads to inhibitory effects on αSyn fibrillation and αSyn-mediated membrane 40 permeabilization. Collectively, these results suggest that HYPE-mediated αSyn adenylylation 41 may be a mechanism by which affected cells cope with αSyn toxicity. This is the first report 42 identifying αSyn as a target for HYPE, and reinforces a role for Fic-mediated 43 adenylylation/AMPylation in neurodegeneration. Importantly, our data shed light on a possible 44 direct means of modifying and reducing toxicity from misfolded aggregates, which may function 45 46 in parallel with HYPE’s effect on chaperones. 47 48 49 RESULTS 50 51 HYPE is enriched in the substantia nigra in rat midbrain sections 52 Global transcriptomic and proteomic studies indicate that HYPE is expressed at 53 extremely low levels in all tissues, including neurons [2]. Indeed, endogenous HYPE is rarely 54 detected in most epithelial cells like HEK293 or HeLa by standard Western blotting or 55 56 immunofluorescence techniques [7]. To assess HYPE expression in neurons, we performed 57 immunohistochemistry on sections of rat midbrain using antibody generated against bacterially 58 purified full length human HYPE, where human and rat HYPE (FicD) share 90% 59 sequence identity. We observed strong staining for HYPE in sections corresponding to the 60 substantia nigra, a region where neuronal toxicity occurs due to αSyn aggregation during PD 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 (Figure 2A). HYPE’s staining pattern within the substantia nigra appeared neuronal under higher 5 magnification analysis (Figure 2A, red and blue boxes, and supplemental Figure 1). We also 6 saw evidence of HYPE staining in the pyramidal neurons of the hippocampus (as indicated in 7 8 Figure 2A). 9 10 HYPE is expressed in nigral dopaminergic neurons and localizes to the cell body 11 Toxicity induced by αSyn aggregation predominantly affects nigral dopaminergic 12 neurons [15]. We, therefore, assessed the localization of HYPE in primary dissociated neurons 13 derived from embryonic rat midbrain. Immunofluorescence analyses of HYPE revealed that it 14 colocalizes with the neuronal marker MAP2 (microtubule-associated protein 2) as detected by 15 fluorescence microscopy, confirming HYPE expression in neurons (Figure 2B), whereas HYPE 16 expression in surrounding non-neuronal cells (consisting primarily of astrocytes) was much 17 18 weaker. Interestingly, compared to MAP2, which is expressed across both the cell body and in 19 neurites, HYPE is detected predominantly in the neuronal cell body, but not to a large extent in 20 the dendrites (Figure 2B, upper panels). In contrast to HYPE, the chaperone protein BiP (also 21 an ER lumenal protein) displays ER-specific expression in all parts of the neuron and is present 22 at higher levels in surrounding astrocytes (Figure 2B, lower panels and Supplemental Figure 2). 23 Finally, we observed HYPE expression in dopaminergic neurons that stained positive for 24 hydroxylase (TH) (Figure 2C), indicating that HYPE is expressed in neurons that are 25 most vulnerable in PD. Together, these data support our hypothesized link between HYPE 26 27 activity and its relevance to PD. 28 29 HYPE interacts with αSyn and adenylylates it in vitro 30 αSyn is known to localize to the ER lumen and interact with BiP [24, 32, 33]. We 31 previously reported that HYPE interacts with and adenylylates BiP in the ER lumen, and alters 32 UPR activation presumably by altering BiP's ability to refold misfolded proteins [7]. Additionally, 33 we found that HYPE itself has the ability to bind to misfolded proteins [7]. Thus, αSyn is 34 correctly situated in the ER to allow interactions with HYPE. Given HYPE's role in UPR 35 regulation and its expression in regions of the brain that are associated with aggregation of 36 37 misfolded αSyn, we asked whether HYPE directly interacts with αSyn. Complementary co- 38 immunoprecipitation experiments using SH-SY5Y neuroblastoma-derived cells with antibody to 39 αSyn or to HYPE revealed a direct interaction between the two proteins (data not shown). Our 40 experience with HYPE informs us that in its enzymatically active state, it interacts transiently 41 with its substrates (like BiP); however, transient interactions can be captured in vitro by 42 assessing interactions using the catalytically dead E234G/H363A-HYPE mutant, which 43 functions as a substrate trap [7]. We, therefore, conducted biolayer interferometry with 44 bacterially expressed and purified His-tagged αSyn and untagged E234G/H363A-HYPE, with 45 46 αSyn immobilized on the anti-His antibody sensor, to determine HYPE's binding efficiency for 47 αSyn. E234G/H363A-HYPE displayed sustained binding to αSyn with a Kd of 15.3 + 0.04µM 48 (Figure 3A). 49 We next asked if HYPE could adenylylate αSyn. For this, untagged αSyn was purified as 50 oligomeric and as low molecular weight, mostly monomeric species (by passing the αSyn 51 solution prepared by dissolving the lyophilized protein over a 100kD-cut off filter). Monomeric 52 and oligomeric αSyn were incubated with E234G-HYPE or its enzymatically inactive 53 E234G/H363A mutant in an in vitro adenylylation reaction using α-32P-ATP as a 54 source. Both monomeric and oligomeric forms of αSyn were efficiently adenylylated by 55 56 enzymatically active E234G-HYPE but not by E234G/H363A HYPE (Figure 3B). Even though 57 equal concentrations of monomeric and oligomeric αSyn were used in this reaction, monomeric 58 αSyn appeared to be more efficiently adenylylated, as determined by stronger intensity of 59 radiolabelled bands corresponding to αSyn on the autoradiogram (Figure 3B). These data are 60 the first indication of αSyn as a novel target for HYPE-mediated adenylylation. 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 5 HYPE adenylylates αSyn at threonine residues predominantly in its N-terminus 6 Monomeric αSyn incubated with E234G-HYPE in an in vitro adenylylation reaction as 7 8 described above was digested with trypsin or chymotrypsin and subjected to LC-MS/MS mass 9 spectrometric analysis (see Methods). Peptides with a mass shift corresponding to the addition 10 of an AMP were found to be modified at amino acids T33, T54, and T75. Representative spectra 11 for peptides with an AMP modification at T33, T54, and T75 showing peaks at m/z 348.08 12 representing the protonated form of AMP and neutral losses from the peptides of adenine, 13 adenosine or AMP are shown in Figures 4A, B, and C. Interestingly, nearly all the αSyn 14 adenylylation sites identified lie in the N-terminus domain, a region critical for interacting with the 15 membrane and needed for stabilizing αSyn [34]. 16

17 18 Adenylylation of αSyn shows inhibitory effects on aggregation and fibril formation 19 As mentioned earlier, αSyn aggregation is thought to play a key role in 20 neurodegeneration associated with PD. Therefore, to determine the functional consequence of 21 αSyn adenylylation, we asked whether adenylylation affects the kinetics of αSyn fibril formation. 22 To monitor fibrillation kinetics, we conducted an in vitro αSyn fibrillation assay using thioflavin T 23 (ThT), a fluorescent dye that forms a complex with the aggregating αSyn protein. ThT 24 fluorescence intensity directly correlates with the extent of fibrillation. Mouse WT αSyn and a 25 human A53T αSyn clinical mutant, which are known to display an increased rate of fibrillation, 26 27 were used as positive controls [35]. Data from representative fibrillation assays are presented in 28 Figure 5A and in supplemental Figure 3. αSyn alone or first adenylylated by E234G-HYPE was 29 allowed to incubate with ThT under shaking conditions to induce the formation of fibrils and 30 monitored for 5 days. As expected, untreated WT-αSyn formed aggregates reaching saturation 31 levels, as evidenced by a sigmoidal fluorescence trace with a distinct lag phase and plateau 32 (Figure 5A, blue line). In contrast, data from five of the seven biological replicates showed that 33 fibril formation by αSyn adenylylated by E234G-HYPE was significantly compromised, 34 displaying markedly reduced ThT fluorescence levels (Figure 5A; compare blue line vs green 35 line). αSyn left unmodified by incubating with catalytically inactive E234G/H363A-HYPE did not 36 37 show impaired fibrillation, indicating that the mere presence of HYPE does not affect the 38 fibrillation process (Figure 5A; purple line). Interestingly, incubation of αSyn with WT-HYPE also 39 displayed a slight reduction in fibrillation levels in some biological replicates (Figure 5A, red line 40 and supplementary Figure 3). Quantification of the end-point fibrillation levels, however, 41 indicated that αSyn fibrillation levels were significantly reduced only upon incubation with the 42 adenylylation-competent E234G-HYPE (Figure 5B). 43 The fibrillation results were verified by separating and quantifying the insoluble pellet 44 (aggregated αSyn fibrils) and supernatant (soluble, monomeric αSyn) fractions from each of the 45 46 fibrillation samples. Specifically, equal amounts of end-point fibrillation samples were subjected 47 to ultracentrifugation. The supernatant (S) and pellet (P) fractions were separated by SDS- 48 PAGE and analyzed by western blotting with antibody to αSyn, to distinguish the monomeric 49 and oligomeric αSyn proteins (Figure 5C). Quantification of monomeric αSyn in each of these 50 fractions revealed that adenylylation significantly reduced the amount of insoluble αSyn (Figure 51 5D). Depicted in Fig 5D is the quantification of monomeric αSyn in “Pellet” fractions normalized 52 against the corresponding αSyn only (100%) sample. The histogram corresponding to 53 adenylylated αSyn shows decreased insolubility of 74%. Interestingly, assessment of the 54 fibrillation data shown in Figure 5A normalized against the maximum fluorescence for each 55 56 individual sample revealed a similar lag time for all samples incubated with HYPE when 57 compared to αSyn alone (Figure 5E), whereas the lag times observed for the A53T and mouse 58 αSyn positive controls were markedly shorter. 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 Taken together, these data suggest that adenylylation specifically limits the rate of 5 elongation and the amount of αSyn fibrils formed, whereas it does not alter the lag time, a 6 measure of the rate of nucleation. 7 8 9 Adenylylation alters αSyn fibril morphology 10 Next, we examined the end-point fibrillation samples shown in Figure 5A by transmission 11 electron microscopy (TEM) to confirm that amyloid-like fibrils were formed by WT and HYPE- 12 treated αSyn, and to assess whether adenylylation had any effect on αSyn fibril structure. All the 13 samples displayed an unbranched fibril morphology (Figure 6). In agreement with the literature 14 [36], WT-αSyn fibrils were most frequently arranged as two fibrils (each with a diameter of ~10- 15 15 nm) wound around each other via a helical twist characterized by a well-defined pitch length 16 (Figures 6A). Fibrils with a similar twisted morphology were also observed in samples of αSyn 17 18 incubated with adenylylation-incompetent WT-HYPE or E234G/H363-HYPE (Figures 6B and 6C, 19 red arrows). In contrast, αSyn adenylylated by E234G-HYPE displayed a mixed population of 20 fibrils: whereas some paired fibrils had a twisted morphology (red arrows), others were arranged 21 in a parallel fashion without any twist (yellow arrows) (Figure 6D). These data suggest that αSyn 22 adenylylation can interfere with the protein’s ability to form typical twisted fibrils under the 23 conditions used here, possibly due to steric hindrance from the adenylylation PTM. 24 25 Adenylylation reduces membrane permeabilization 26 27 The pathological mechanism of action and physiological role of αSyn are poorly 28 understood. Interaction with membrane surfaces has been shown to promote the process of 29 self-aggregation and lead to disruption of bilayer integrity by lipid extraction, phenomena which 30 could play a central role in αSyn neurotoxicity [34, 37]. We have also observed that familial 31 mutants of αSyn that show greater neurotoxicity in primary neuronal cell cultures (e.g. A30P and 32 G51D) also display greater vesicle disruption [34, 37]. Thus, we asked whether adenylylation 33 alters αSyn’s ability to permeabilize the membrane. To this end, we compared unmodified WT 34 αSyn to that incubated with WT-, E234G-, or E234G/H363A-HYPE for their ability to disrupt 35 small unilamellar vesicles (SUV) using a calcein dye release assay. SUVs prepared from a 36 37 50:50 mixture (mol/mol) of egg phosphatidylglycerol (PG) and egg phosphatidylcholine (PC) 38 were loaded with calcein at a self-quenching concentration and incubated with the adenylylated 39 or unmodified αSyn variants. Permeabilization was assessed by real time measurement of the 40 increase in fluorescence intensity caused by dequenching of the released dye into the 41 surrounding buffer. Interestingly, αSyn adenylylated by E234G-HYPE showed a significant 42 decrease in vesicle leakage compared to the non-adenylylated samples (Figure 7), suggesting 43 that adenylylation may be a protective mechanism to combat the toxic effects of vesicle 44 disruption associated with membrane-induced αSyn aggregation. 45 46 47 DISCUSSION 48 The discovery of Fic proteins as evolutionarily conserved adenylyltransferases has 49 brought about a renewed interest in adenylylation/AMPylation, and underscores the importance 50 of this previously underappreciated post-translational modification (PTM). Documentation of 51 adenylylation in signal transduction is growing. Fic proteins represent one of three known types 52 of adenylyltransferases, the other two being bacterial synthase adenylyltransferase 53 (GS-AT) and the newly discovered Sel-O pseudokinase [38, 39]. PTMs catalyzed by Fic 54 proteins are now being recognized as a universal signaling paradigm that regulates critical 55 56 processes like bacterial pathogenesis, cellular trafficking, drug tolerance, and prokaryotic and 57 eukaryotic stress response [1]. Further, our discovery of BiP as the elusive target for the human 58 Fic protein, HYPE, has unveiled Fic-mediated adenylylation as a critical step in UPR regulation 59 and the cell’s ability to cope with misfolded proteins [7]. Further, through its ability to manipulate 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 chaperone activity, Fic-mediated adenylylation has recently been linked to neurodegeneration 5 [29]. 6 As mentioned earlier, HYPE localizes to the ER lumen. To date, BiP is the only known 7 8 ER-localized physiological target for HYPE. However, some cellular and proteomic analyses 9 have identified additional targets – such as and cytosolic Hsp70 and Hsp40 family 10 chaperones - that localize to cellular compartments other than the ER [30], [31], [40]. How 11 HYPE interacts physiologically with these non-ER localized proteins is unclear [54], though a 12 recent report reveals that a mass exodus of ER resident proteins occurs in response to ER Ca2+ 13 depletion [41]. Additionally in C. elegans, HYPE is reported to be partially present in the cytosol 14 in addition to the ER lumen [29]. 15 Here, we identify αSyn, a presynaptic protein involved in Parkinson’s disease 16 pathogenesis, as a novel target for the human Fic protein, HYPE, and propose a direct role for 17 18 adenylylation in modulating neurotoxicity. During PD, accumulation and aggregation of 19 misfolded αSyn manifests as the formation of Lewy bodies and is thought to lead to the death of 20 dopaminergic neurons in the substantia nigra region of the brain, potentially via a mechanism 21 involving vesicle disruption. Accordingly, we see that HYPE is present in dopaminergic neurons 22 and enriched in the substantia nigra of normal rat brains (Figure 2). While αSyn is predominantly 23 a cytosolic protein, it is known to transgress membranes upon oligomerization [24, 37]. αSyn 24 has been reported to enter the ER lumen, where it interacts with BiP and is suggested to trigger 25 a UPR response [24]. Thus, we predict that HYPE and αSyn likely interact directly in the ER 26 27 lumen. Alternatively, released cytosolic pools of HYPE may interact with αSyn. Indeed, we 28 confirmed a direct interaction between HYPE and αSyn using immunoprecipitation and biolayer 29 interferometry. While this binding affinity was relatively weak (in the μM range; Figure 3), it is in 30 keeping with the binding affinity of HYPE for BiP [52]. It remains to be determined whether in a 31 physiological setting, the interaction between HYPE and αSyn could be enhanced by the 32 presence of BiP. 33 Our LC-MS/MS analysis identified three sites of adenylylation on αSyn – T33, T54 and 34 T75. We attempted to validate these sites in vitro using site directed mutants of each of these 35 sites. However, these adenylylation site-mutants of αSyn could still be modified in an in vitro 36 37 adenylylation reaction (data not shown), likely because of secondary-site modifications, as is 38 often seen with Fic and other enzymes [6] [7]. Interestingly, all the adenylylation sites on αSyn 39 that we identified localized to domains of αSyn that are critical for membrane association and 40 have also been reported as sites of O-GlcAcylation [53]. Primarily an intrinsically disordered 41 protein, binding to the phospholipid membrane induces a structural rearrangement causing 42 αSyn to form a helical structure [42-45]. A partial disruption of αSyn-membrane interactions 43 contributes to αSyn aggregation at the membrane surface, a process that is apparently coupled 44 to vesicle disruption [34, 37, 42, 46, 47]. We, therefore, assessed the physiological implications 45 46 of αSyn adenylylation in PD using an in vitro liposome-based assay for measuring phospholipid 47 membrane permeability, and found that adenylylation of αSyn reduces αSyn-mediated 48 membrane permeabilization (Figure 7). In a previous study, substitutions at threonine residues 49 in the amphipathic helical domain, including T33, were found to disrupt αSyn membrane binding 50 [48]. By analogy with these earlier findings, adenylylation of αSyn threonine residues could 51 perturb αSyn-lipid interactions. This effect, as well as a potential adenylylation-mediated 52 disruption of interactions between neighboring αSyn molecules at the membrane surface, could 53 lead to a decrease in the protein’s ability to elicit vesicle permeabilization [34]. 54 Of note is that adenylylation led to a decrease in the thioflavin T fluorescence in 55 56 solutions of αSyn incubated under fibrillation conditions (Figure 5). We also observed a change 57 in the fibril morphology of adenylylated αSyn (Figure 6). Typical αSyn fibrils display a bundling 58 pattern that consists of twisted fibrils [49, 50]. We found that adenylyated αSyn fibrils existed 59 largely as bundles of parallel fibrils (Figure 6D), suggesting (as one possibility) that addition of 60 an AMP causes steric hindrance that prevents the fibrils from becoming closely intertwined. Our 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 observation that αSyn adenylylation influences fibril morphology also implies that the difference 5 in thioflavin T fluorescence shown in Figure 5 may reflect a combined effect of, in part, a 6 difference in the quantum yield of the dye bound to the different fibril polymorphs, as well as a 7 8 decrease in the extent of fibrillation of the adenylylated protein. 9 Collectively, our findings indicate that αSyn adenylylation interferes with αSyn fibril 10 formation in solution and with αSyn-mediated vesicle disruption, and changes the preferred 11 morphology of the fibrils. Evidence suggests that each of these effects could impact αSyn 12 pathology and interfere with αSyn neurotoxicity [49, 50]. Thus, physiologically, we propose that 13 adenylylation may be triggered as a protective response to cope with the formation of toxic αSyn 14 aggregates. 15 As mentioned earlier, HYPE plays a key role in regulating the cell’s response to ER 16 stress, and αSyn aggregation is also known to trigger ER stress and the UPR pathway via its 17 18 interactions with BiP. An important question remains whether HYPE-mediated adenylylation of 19 αSyn occurs in response to ER stress or whether adenylylation of αSyn is independent of UPR 20 activation. Preliminary quantitative RT-PCR analysis of cDNA isolated from human control and 21 PD patient samples revealed that HYPE’s transcriptional levels did not correlate with other UPR 22 associated transcripts (data not shown). Experiments are underway to determine whether HYPE, 23 which we have shown to bind misfolded proteins directly in vitro, may bind and adenylylate αSyn 24 independently of the UPR response. 25 In summary, we have identified αSyn as a novel target for the human Fic protein, HYPE. 26 27 In doing so, we have uncovered a potential neuroprotective role for Fic-mediated adenylylation 28 in PD that may function concurrently with HYPE’s effects on chaperone activity. It remains to be 29 determined whether the interaction of HYPE with αSyn is connected to ER stress. However, our 30 discovery of a new, physiologically relevant target for HYPE provides significant insights for 31 catalysis and substrate recognition, and opens a new line of research for combatting PD. Given 32 the central role HYPE plays in cell fate, targeting HYPE as opposed to αSyn directly allows a 33 chance to preempt the effects of αSyn aggregation and protect the cell from potential stress- 34 related damage. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 LEGENDS 5 6 Figure 1: A) Schematic representation of human HYPE. HYPE’s Fic motif with the catalytic 7 8 H363 and its α-inh domain (yellow box) with E234 are indicated. SS/TM, signal 9 sequence/transmembrane domain. TPR, tetratricopeptide domain. Fic, filamentation induced by 10 cAMP domain. B) Schematic representation of human alpha-Synuclein (αSyn). Familial mutants 11 associated with Parkinson’s disease (A30P, E46K, H50Q, G51D, A53E, and A53T) are 12 indicated. Sites of adenylylation (T33, T54, and T75) are indicated in red. αSyn’s N-terminal (N- 13 term), central hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid), and C- 14 terminal (C-term) domains are shown. Schematic representations for HYPE and αSyn are not to 15 scale. 16

17 18 Figure 2: HYPE is expressed in adult and embryonic rat midbrain neurons. A) Top panel: 19 Coronal section of a rat brain probed with antibody to HYPE and visualized with peroxide-based 20 secondary staining shows strong HYPE expression in the substantia nigra and hippocampus. 21 Scale bar = 2 mm. Bottom panels: Higher magnification views of HYPE staining in the nigral 22 region. Scale bars in the red and blue boxes represent 100 μm and 50 μm, respectively. B) 23 Immunofluorescence imaging of primary midbrain cultures from E17 rat embryos shows co- 24 localization of HYPE (green, top panel) or another ER lumenal protein, BiP (green, bottom 25 panel), with the neuronal marker MAP2 (red). HYPE shows a preferential neuronal expression, 26 27 whereas BiP is expressed highly in both neurons and surrounding glial cells. Both HYPE and 28 BiP selectively localize to the cell body in neurons. Scale bar = 50 µm. C) Immunofluorescence 29 showing co-localization of HYPE (red) with the dopaminergic neuronal marker TH (tyrosine 30 hydroxylase; green). 31 32 Figure 3: HYPE binds to and adenylylates αSyn. A) Steady state binding affinity 33 determination for E234G/H363A-HYPE incubated with His6-tagged αSyn immobilized on HIS1K 34 sensors were obtained by Biolayer Interferometry. B) In vitro adenylylation assay with purified 35 HYPE and αSyn using α32P-ATP as a nucleotide source showing adenylylation of αSyn by 36 37 enzymatically active E234G-HYPE but not by its catalytically dead mutant E234G/H363A-HYPE. 38 39 Figure 4: Fragmentation of AMP-modified peptides. Figures A, B, and C represent the 40 fragmentation of AMP-modified peptides in MS2 (top) and MS3 (bottom) of the neutral loss 41 fragment of AMP. A) MS2 and MS3 fragmentation of peptide T33AMPKEGVLYVGSK. B) MS2 42 and MS3 fragmentation of peptide TKEGVVHGVAT54AMPVAEK. C) MS2 fragmentation of m/z 43 755.36 of peptide TKEQVTNVGGAVVTGVT75AMPAVAQK. Annotated are fragments that lose 44 AMP, adenosine or adenine, either from 1+ ions or 2+ ions (2+). The most abundant ion (-AMP) 45 46 was subsequently isolated and fragmented again to produce fragments to identify the peptide. B 47 and y ions are annotated as well as their respective sequences/amino acids. 48 49 Figure 5: HYPE-mediated adenylylation of αSyn apparently inhibits fibril elongation 50 without altering the lag time. A) Representative kinetic traces of fibrillation assays conducted 51 with purified αSyn adenylylated in the presence of E234G HYPE (green line) or left unmodified 52 as αSyn alone (blue line) show a significant reduction in the yield of ThT fluorescence – 53 potentially reflecting inhibition of fibril elongation – as a result of αSyn adenylylation. Control 54 samples consist of αSyn incubated in the presence of WT HYPE (red line) or catalytically dead 55 56 E234G/H363A HYPE (purple line) prior to fibrillation. B) Quantification of endpoint ThT signal 57 intensities obtained from N=3 fibrillation assays. Asterisk * indicates a p value = 0.02. C) 58 Endpoint fibril samples from (A) were ultracentrifuged, and soluble αSyn in the supernatant (S) 59 fraction and insoluble/aggregated αSyn in the pellet (P) fraction were analyzed by SDS-PAGE. 60 D) Quantification of the bands corresponding to monomeric αSyn in the pellet fractions of the 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 gel shown in (C). Adenylylation of αSyn following incubation with E234G-HYPE results in a 5 decrease (74% vs. 100%) in the amount of αSyn seen in the pellet fraction, suggesting that 6 more αSyn is soluble/not aggregated. E) Normalized ThT values indicate that the lag time of 7 8 fibrillation is unaltered by HYPE-mediated adenylylation, as the curves for WT αSyn and αSyn 9 incubated with various HYPE samples overlap. In contrast, control samples of mouse WT αSyn 10 (dark blue line) or human A53T αSyn mutant (orange line) exhibit a shorter lag time, indicating a 11 much faster rate of nucleation. 12 13 Figure 6: Adenylylation alters the morphology of αSyn fibrils. Transmission electron 14 microscopy images of fibrils formed by human WT αSyn. The protein was untreated (A) or 15 incubated with WT HYPE (B), its catalytically inactive E234G/H363A mutant, (C) or the 16 constitutively adenylylation competent E234G mutant (D). Fibrils formed by αSyn largely consist 17 18 of bundles of fibril pairs that are intertwined, with each twist occurring at regular pitch lengths 19 (red arrows). However, significant levels of fibrils formed by adenylylated αSyn (incubated with 20 E234G) do not display this twist and are instead structured as parallel fibril pairs (yellow arrows). 21 Magnified images of fibrils showing one representative twist (A-C) or one parallel fibril 22 polymorph (D) are shown beside each image. Scale 200nm. 23 24 25 Figure 7: Adenylylation reduces αSyn’s ability to disrupt membranes. Calcein-loaded PG: 26 27 PC (1:1) SUVs were incubated with adenylylated or non-adenylylated (control) αSyn and 28 examined for an increase in calcein fluorescence (excitation and emission wavelengths, 485 nm 29 and 515 nm). The data are presented as relative membrane permeabilization after 12 h, 30 normalized with respect to non-adenylylated WT αSyn control. Mean ± SEM, n = 4 (each with 3 31 technical replicates). 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 Supplemental Legends: 5 6 Figure S1: Additional coronal sections of a rat brain (n=2 and n=3) probed with antibody to 7 8 HYPE and visualized with peroxide-based secondary staining. HYPE-specific staining of the 9 substantia nigra is shown at 10X and 20X magnification. Scale bars represent 100 µm and 50 10 µm, respectively 11 12 Figure S2: Immunofluorescence images of a rat primary midbrain culture stained for HYPE 13 (green, left panel) or BiP (red, middle panel). The right panel consists of a merged image 14 showing HYPE and BiP immunoreactivity as well as DAPI-stained nuclei. HYPE is preferentially 15 expressed in neurons, whereas BiP staining reflects glial and neuronal expression. Both 16 proteins appear to be preferentially expressed in neuronal cell bodies. Scale bar represents 100 17 18 µm. 19 20 Figure S3: Kinetic traces of a fibrillation assay conducted with purified human WT αSyn 21 adenylylated in the presence of E234G HYPE (green line) or left unmodified as αSyn alone 22 (blue line) show a significant reduction in the yield of ThT fluorescence – potentially reflecting 23 inhibition of fibril elongation – as a result of αSyn adenylylation. Control samples consist of αSyn 24 incubated in the presence of WT-HYPE (red line) or catalytically dead E234G/H363A HYPE 25 (purple line) prior to fibrillation. The background fluorescence for the ThT only control against 26 27 which all samples were blanked is also shown (light blue line). 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 KEYWORDS: Fic / Adenylylation / AMPylation / Parkinson's Disease / αSynuclein 5 6 7 8 9 ABBREVIATIONS: 10 11 AMP: Adenosine monophosphate 12 BiP: Binding immunoglobulin protein 13 BLI: Biolayer Inferometry 14 ER: Endoplasmic reticulum 15 FIC: Filamentation induced by cAMP 16 GFP: Green fluorescence protein 17 18 GS-AT: Glutamine synthase adenylyltransferase 19 HYPE: Huntingtin yeast interacting protein E 20 LC-MS: Liquid chromatography – mass spectrometry 21 MAP2: Microtubule associated protein 2 22 NAC: Non-amyloid-beta component of Alzheimer’s disease 23 PD: Parkinson’s Disease 24 PTM: Post-translational modification 25 SUV: Small unilamellar vesicles 26 27 TEM: Transmission electron microscopy 28 TH: Tyrosine hydroxylase 29 UPR: Unfolded protein response 30 WT: wild type 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 MATERIALS AND METHODS 5 6 Cloning and plasmids 7 8 For bacterial expression, WT αSyn/pT7-7 plasmid was procured from Addgene 9 (www.addgene.com; plasmid #36046). This vector was also used as a backbone for cloning 10 His6-tagged WT-αSyn by PCR amplification. 11 12 Protein expression and purification 13 Untagged human WT αSyn, its A53T variant, and mouse WT αSyn purified as previously 14 described [34]. Oligomeric species of untagged αSyn were separated from low molecular weight, 15 mostly monomeric, species by passing the protein over a 100kDa cut-off filter. His -tagged αSyn 16 6 was expressed in E. coli BL21-DE3 (Stratagene) in LB medium containing 100 µg/ml of 17 o 18 ampicillin to a density of A600 = 0.6. Protein expression was induced for 3 hours at 37 C with 0.4 19 mM IPTG. Cells were lysed in lysis buffer (50 mM HEPES pH 8.0, 250 mM NaCl, 5 mM 20 imidazole, 10% (v/v) glycerol) containing 1mM PMSF and purified using a cobalt resin. Resin 21 was washed with wash buffer (50 mM HEPES pH 8.0, 250 mM NaCl, 40 mM imidazole,10% 22 (v/v) glycerol) and αSyn-His protein was eluted from the resin using elution buffer (50 mM 23 HEPES pH 8.0, 250 mM NaCl, 350 mM imidazole, 10% (v/v) glycerol) and further purified by ion 24 exchange chromatography using 100 mM Tris-HCl pH 7.5 with a salt gradient from 10 mM-1M 25 NaCl. Fractions containing αSyn were purified by size exclusion chromatography in buffer 26 27 containing 100 mM Tris-HCl pH 7.5, 100 mM NaCl to separate monomer and the void-volume oligomer. Protein concentrations were measured using the Bradford method. Purity was 28 o 29 determined by SDS-PAGE and proteins were stored at -80 C. 30 Recombinant HYPE and its mutants were purified as previously described [7]. 31 Specifically, wild type and HYPE mutant proteins were expressed in E. coli BL21-DE3-RILP 32 (Stratagene) in LB medium containing 50 μg/ml of kanamycin (pSMT3) to a density of A600 = 0.6. 33 Protein expression was induced for 12-16 hours at 18oC with 0.4 mM IPTG. Cells were lysed in 34 lysis buffer (50 mM HEPES pH 8.0, 250 mM NaCl, 5 mM imidazole) containing 1 mM PMSF 35 protease inhibitor and purified using a cobalt resin. Resin was washed with wash buffer (50 mM 36 37 HEPES pH 8.0, 250 mM NaCl, 20 mM imidazole). His6-SUMO tag was cleaved by incubating 38 proteins with ULP1 at 4°C. The protein mixture was diluted in wash buffer without imidazole and 39 re-applied to a cobalt column, and flow through containing HYPE was further purified by ion 40 exchange chromatography using 100 mM Tris-HCl pH 7.5 with a salt gradient from 10 mM – 1 41 M NaCl. Fractions containing HYPE were purified by size exclusion chromatography in buffer 42 containing 100 mM Tris-HCl pH 7.5, 100 mM NaCl. Protein concentrations were measured 43 using the Bradford method. Purity was determined by SDS-PAGE and proteins were stored at - 44 80oC. 45 46 47 Biolayer interferometry 48 The binding kinetic assays were performed on an Octet Red 384 instrument (ForteBio, 49 Menlo Park, CA) with the Biolayer Interferometry (BLI) technique. The assays were carried out 50 using Anti-pentaHis (HIS1K) biosensors (ForteBio, Menlo Park, CA) at 30 °C with the plate 51 shaking speed at 1000 rpm. HIS1K sensors were dipped into the wells of 20 mM His6-αSyn in 52 HEPES buffer (50 mM HEPES, 150 mM NaCl, pH7.5) for 2 minutes to load αSyn on the sensors, 53 followed by incubation in buffer for a minute to build a baseline. Next, the sensors were dipped 54 into wells of N-terminally His tagged HYPE in HEPES Buffer at a concentration ranging from 1 55 56 µM to 150 µM for 2 minutes to obtain association curves. The sensors were then dipped back to 57 the baseline HEPES buffer wells for 3 minutes to obtain dissociation curves. The data were 58 analyzed by the ForteBio data analysis software v.9.0.0.12. The binding constants (Kd) and 59 kinetic parameters were determined by globally fitting data to the 1:1 fitting model. 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 5 6 In vitro adenylylation assays 7 8 0.5 μg of E234G or E234G/H363A HYPE (aa 102-458) protein was incubated either 9 alone or with 5 μg of untagged αSyn monomer or oligomer in an adenylylation reaction 10 containing 5 mM HEPES pH 7.5, 1 mM manganese chloride tetrahydrate, 0.5 mM EDTA and 11 0.01 mCi α-32P-ATP for 15 minutes. Reactions were stopped with SDS PAGE loading buffer, 12 separated on 4-20% polyacrylamide gels and visualized by autoradiography. 13 14 Mammalian cell culture assays 15 HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) 16 supplemented with 5% (v/v) fetal bovine serum (Life Technology) and 100 g/ml 17 o 18 penicillin/streptomycin (Sigma-Aldrich) at 37 C with 5% CO2. Fugene HD (Promega) was used 19 for transfections according to the manufacturer’s directions. Cells were transfected for 30 hrs. 20 Primary midbrain cultures were prepared and treated as previously described [34]. 21 22 Mass spectrometry 23 For in-gel protein digestion, gel bands were cut out, destained, reduced with 5mM DTT 24 and alkylated with 10mM iodoacetamide and digested with either trypsin at 37C or chymotrypsin 25 at room temperature essentially as described by Shevchenko et al. [51] with minor 26 27 modifications. For LC-MS/MS analysis, the dried peptide mix was reconstituted in a solution of 28 2% formic acid (FA) for MS analysis. Peptides were loaded with the autosampler directly onto a 29 2cm C18 PepMap pre-column (Thermo Scientific, San Jose, CA) which was attached to a 50cm 30 EASY-Spray C18 column (Thermo Scientific). Peptides were eluted from the column using a 31 Dionex Ultimate 3000 Nano LC system with a 1.5 min gradient from 2% buffer B to 5% buffer B 32 (100% acetonitrile, 0.1% formic acid), followed by a 15 min gradient to 20% of B, followed by a 2 33 min gradient to 30% of B. The gradient was switched from 30% to 85% buffer B over 1 min and 34 held constant for 1 min. Finally, the gradient was changed from 85% buffer B to 98% buffer A 35 (100% water, 0.1% formic acid) over 1 min, and then held constant at 98%t buffer A for 15 more 36 37 minutes. The application of a 2.0 kV distal voltage electrosprayed the eluting peptides directly 38 into the Thermo Fusion Tribrid mass spectrometer equipped with an EASY-Spray source 39 (Thermo Scientific). Mass spectrometer-scanning functions and HPLC gradients were controlled 40 by the Xcalibur data system (Thermo Scientific). Initial experiments were performed to identify 41 AMP modified peptides by running each digest separately with a data dependent method to 42 acquire as many MS/MS in a 3 second span. This data was analyzed for the presence of AMP 43 modification with either inspect (proteomics.ucsd.edu) or Thermo Proteome Discoverer 2.1 and 44 looking for a modification of 329.05252 on either tyrosine, threonine or or by manually 45 46 looking at MS/MS scans and looking for the specific signal for AMP (m/z 348.08). Three MS/MS 47 scans were identified that contained AMP, however, the most abundant peak was identified as 48 the neutral loss of AMP from the parent mass. Therefore an analysis was conducted that 49 targeted these neutral loss ions for an additional MS3 scan to identify the peptide sequence and 50 position of the AMP modification. 51 52 Immunocytochemistry and immunohistochemistry (IHC) 53 Primary midbrain cultures were fixed with 4% (w/v) paraformaldehyde and blocked with 54 1X PBS containing 10% (v/v) Fetal Bovine Serum (FBS), 1% (w/v) BSA and 0.3% (v/v) Triton X- 55 56 100 for 1 hour. This was followed by treatment with primary antibodies - mouse anti-HYPE 57 (Abcam cat# ab67163, 1:100), chicken anti-MAP2 (Encor cat# CPCA-MAP2, 1:2000) or rabbit 58 anti-TH (Phosphosolutions cat# 2025-THRAB, 1:1000) diluted in 1X PBS containing 1% (w/v) 59 BSA and incubated overnight at 4°C. The cells were washed and incubated with secondary 60 antibodies Alexa anti-chicken 594, Alexa anti-rabbit 488 or Alexa anti-mouse 594 for 1 hour at 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 room temperature, washed, and imaged using the CYTATION 3 Cell Imaging Reader or Nikon 5 Ti confocal microscope 6 Rat brain sections (40 μm) were obtained using a microtome and stored in cryo- 7 8 protectant solution at -20°C. On the day of the experiment, sections were transferred to a 9 separate plate and washed six times with PBS. All incubations were conducted while shaking at 10 100 rpm. The sections were blocked with 10% Normal Donkey Serum (NDS) in 0.3% PBS- 11 Triton for 90 minutes, treated with mouse primary antibody for HYPE (Abcam cat# ab67163, 12 1:100) diluted in PBS containing 1% NDS and 0.3% Triton for 24 hours at 4°C. The following 13 day, the sections were washed three times with PBS, incubated with biotinylated secondary 14 antibody anti-mouse (Jackson ImmunoResearch, cat# 715-065-151, 1:200) for 90 minutes at 15 room temperature, washed with PBS and incubated in the premade ABC solution from 16 17 VECTASTAIN Elite ABC HRP Kit (Vector Laboratories, cat# PK-7200) for 60 minutes. A solution 18 of DAB (diaminobenzidine) and H2O2 was prepared using the DAB Peroxidase Substrate Kit 19 (Vector Laboratories, cat# SK-4100). The sections were were then treated with the DAB solution 20 until a brown color developed, washed to remove non-specific staining and transferred to a slide. 21 The tissues were allowed to dry overnight and were dehydrated using a series of solutions of 22 increasing ethanol concentrations (70-100%), followed by treatment with Histoclear, mounted 23 using Histomount and dried overnight before microscopy using the Olympus BX53 light 24 microscope. 25 26 27 αSyn fibrillation 28 Lyophilized untagged αSyn was dissolved in PBS (pH 7.4) with 0.02% [w/v] NaN3, and 29 dialyzed against the same buffer (24 h at 4°C) to remove excess salt. The solution was filtered 30 by successive centrifugation steps through a 0.22 µm spin filter and a 100 kDa centrifugal filter 31 to isolate low molecular weight (mostly monomeric) protein. This low molecular weight αSyn 32 was incubated in PBS for an hour with or without HYPE as outlined above. Fibrillation solutions 33 of αSyn were prepared by diluting the adenylation reaction mixture (or blank) to a final 34 concentration of 35 µM in the wells of a 96-well plate. To determine the extent of αSyn 35 36 fibrillation, thioflavin T (ThioT, final concentration, 20 µM) was added to the fibrillation solutions, 37 which were incubated at 37 °C with shaking at the ‘normal’ setting in a Spectra Fluor Plus or 38 Genios plate reader (Tecan, Upsala, Sweden). HYPE was not removed from the fibrillation 39 mixture as it did not interfere with fibrillation or show increased ThioT fluorescence on its own. A 40 nylon ball was added to avoid inhomogeneous mixing of samples. ThioT fluorescence was 41 measured with excitation at 440 nm and emission at 490 nm. Mean ThioT fluorescence data 42 determined from 2-4 technical replicates were normalized using the following equation: 43 44 45 normalized fluorescence= 46 47 48 where Ft is the ThioT fluorescence emission intensity at time t, and Fmin and Fmax are the 49 minimum and maximum fluorescence intensities during the incubation, respectively. 50 51 Synthetic vesicle disruption assay and on-bead adenylylation 52 Calcein loaded Small unilamellar vesicles (SUVs) (diameter ~ 90 nm) were prepared as 53 described previously [37]. We opted to examine SUVs composed of egg PG and egg PC (1:1 54 mol/mol) because they contain anionic lipids necessary for αSyn membrane interactions and 55 56 this lipid composition is compatible with producing stable 90 nm vesicles. Egg PG and egg PC 57 suspended in chloroform were mixed in a round bottom flask. The chloroform was evaporated 58 under a nitrogen stream and further dried under vacuum for 1 h. Dried lipids were suspended in 59 a solution of calcein (170 mM) prepared in water using NaOH to adjust the pH to 7.4. The final 60 osmolality was 280 mOsm/kg. The liposomes were sized by extruding the suspension through a 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 50 nm pore size Whatman membrane. Calcein-containing vesicles were isolated from free 5 calcein via gel filtration through a Sephadex G-50 column pre-equilibrated with PBS, pH 7.4, 6 0.02 % (w/v) NaN3 (280 mOsm/kg). Fractions containing isolated vesicles were pooled and 7 8 stored at 4 °C until use. Calcein-loaded vesicles were found to be very stable, with no 9 spontaneous dye leakage observed over several weeks. The size of the vesicles was confirmed 10 by dynamic light scattering. 11 To avoid any interference of HYPE on vesicle permeabilization, the adenylation reaction 12 was done in PBS for one hour as mentioned previously (the adenylation method need to added 13 at the beginning) but with Ni-agarose bead bound HYPE. Untagged αSyn was removed from 14 the beads by removing the supernatant after centrifugation of the samples. To remove any non- 15 specifically bound αSyn from the beads, the sample was washed with small volumes of PBS 16 and pooled with the supernatant as mentioned above. αSyn concentration was calculated with 17 18 BCA assay and absorbance measurement at 280nm. 19 For membrane disruption experiments, monomeric αSyn variants (40 µM, unmodified 20 and modified) were incubated with calcein-loaded PG:PC SUVs (protein-to-lipid ratio, 1:20 21 mol/mol) at 37°C in a total volume of 40 µL. Samples were analyzed with a TECAN 22 fluorescence plate reader (excitation wavelength, 485 nm; emission wavelengths, 530 nm). To 23 determine the maximum dye release, vesicles were lysed by adding 3 µL of 1 % (v/v) Triton X- 24 100. The percentage leakage at time t was determined using the following equation: 25 26 27 percentage leakage= 28 29 30 where It is the fluorescence emission intensity at time t, I0 is the intensity at time 0, and Imax is 31 the maximal intensity determined after detergent lysis of the vesicles. αSyn-mediated leakage 32 reached a plateau after 12-16h, and the plateau value was used to compare the leakage among 33 the HYPE-modified and unmodified samples. 34 35 36 Western blot analysis of fibril samples 37 A 150 L aliquot of the fibrillation sample was centrifuged for 60min at 100,000 x g 38 (Beckman Coulter Optima MAX-XP) to precipitate the fibrils. The supernatant was collected to 39 analyze the residual monomeric content. The fibril pellet was dissolved in 150 L PBS and 40 vortexed gently and used for analysis. Appropriate amounts of the samples were mixed with 41 Laemmli sample buffer and denatured proteins were separated via SDS-PAGE on a 12 %(w/v) 42 polyacrylamide gel and transferred to an Immobilon-FL PVDF membrane (pore size, 0.45 m). 43 44 The membrane was probed with mouse anti-αSyn (BD Biosciences; 1:1,500), and an anti- 45 mouse AP-linked secondary antibody (Cell Signaling Technology, 1: 3,000). To visualize the 46 bands, the membrane was exposed to ECF substrate and imaged using a Typhoon imaging 47 system (GE Life Sciences, Pittsburgh, PA). Band intensities were quantified using ImageJ 48 software (NIH, Bethesda, MD), and averages calculated by quantifying each band in triplicate. 49 50 Transmission Electron Microscopy (TEM) analysis of αSyn amyloid-like fibrils 51 The morphology of the αSyn amyloid-like fibrils was analyzed by negative stain 52 biological TEM. 3 L of the αSyn sample solution of interest (35 M, directly from the fibrillation 53 54 assay) was pipetted on a discharged carbon-coated copper TEM grid (Electron Microscopy 55 Sciences, CF400CU; 400 mesh) and incubated for 1 min. Subsequently, the sample was 56 washed with deionized water carefully without letting it dry and stained with a 1% (w/v) 57 phosphotungstic acid (PTA) solution (3 L), which was left in contact with the protein on the grid 58 for 1 min. The excess solution was then removed by blotting with filter paper, and the sample 59 was imaged using an FEI Tecnai G2 20 Transmission Electron Microscope operating at 200 KV. 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 5 6 7 8 9 Acknowledgments 10 We thank the Biophysical Analysis Laboratory at Purdue University’s Bindley 11 Biosciences Center for use its Octet Red 384 for BLI analysis, and Dr. Chris Gilpin at the 12 Purdue Electron Microscopy Core for use of the FEI Tecnai G2 20 TEM. Finally, we are grateful 13 to members of the Mattoo and Rochet labs for their helpful discussions. 14 15 16 Declaration of interest The authors declare no conflict of interest with the contents of this 17 18 article. 19 20 21 Funding information This work was funded in part by the National Institute of General 22 Medical Sciences of the National Institute of Health (R01GM10092), an Indiana Clinical and 23 Translational Research Grant (CTSI-106564), and a Purdue Institute for Inflammation, 24 Immunology, and Infectious Disease Core Start Grant (PI4D-209263) to SM; a grant from the 25 Branfman Family Foundation to JCR; a grant from the National Institutes of Health 26 27 (R01ES025750) to JRC; grants from the Purdue Research Foundation (PRF-209104) and the 28 Cancer Prevention Internship Program to AS, and Eli Lilly-Stark Neuroscience Research 29 Institute-CTSI pre-doctoral fellowship to SD. 30 31 32 Author contributions 33 S.M. conceived the study. S.M., J-C.R., A.S., and S.D. designed the study. A.S., S.D., 34 A.C., A.C., and B.G.W. conducted the experiments. D.Y. and P.M. provided initial samples of 35 purified αSyn and followed protocols developed by J.R.C. for handling rat brain samples. R.S. 36 37 helped with TEM experiments. A.K. conducted mass spectrometric analyses. S.M. and J-C.R. 38 supervised the study. S.M. and A.S. wrote the manuscript. All authors revised and agreed to the 39 final version of the manuscript. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 REFERENCES: 5 6 1. Casey AK, Orth K. Enzymes Involved in AMPylation and deAMPylation. Chem Rev. 7 8 2018;118:1199-215. 9 2. Worby CA, Mattoo S, Kruger RP, Corbeil LB, Koller A, Mendez JC, et al. The fic domain: 10 regulation of cell signaling by adenylylation. Mol Cell. 2009;34:93-103. 11 3. Engel P, Goepfert A, Stanger FV, Harms A, Schmidt A, Schirmer T, et al. Adenylylation 12 control by intra- or intermolecular active-site obstruction in Fic proteins. Nature. 13 2012;482:107-10. 14 4. Mattoo S, Durrant E, Chen MJ, Xiao J, Lazar CS, Manning G, et al. Comparative 15 analysis of Histophilus somni immunoglobulin-binding protein A (IbpA) with other fic 16 domain-containing enzymes reveals differences in substrate and nucleotide specificities. 17 18 J Biol Chem. 2011;286:32834-42. 19 5. Zekarias B, Mattoo S, Worby C, Lehmann J, Rosenbusch RF, Corbeil LB. Histophilus 20 somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar 21 epithelial barrier. Infect Immun. 2010;78:1850-8. 22 6. Xiao J, Worby CA, Mattoo S, Sankaran B, Dixon JE. Structural basis of Fic-mediated 23 adenylylation. Nat Struct Mol Biol. 2010;17:1004-10. 24 7. Sanyal A, Chen AJ, Nakayasu ES, Lazar CS, Zbornik EA, Worby CA, et al. A novel link 25 between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and 26 27 the unfolded protein response. J Biol Chem. 2015;290:8482-99. 28 8. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein 29 response. Nat Rev Mol Cell Biol. 2007;8:519-29. 30 9. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin 31 Invest. 2002;110:1389-98. 32 10. Preissler S, Rato C, Perera L, Saudek V, Ron D. FICD acts bifunctionally to AMPylate 33 and de-AMPylate the endoplasmic reticulum chaperone BiP. Nat Struct Mol Biol. 34 2017;24:23-9. 35 11. Mallamace F, Corsaro C, Mallamace D, Vasi S, Vasi C, Baglioni P, et al. Energy 36 37 landscape in protein folding and unfolding. Proc Natl Acad Sci U S A. 2016;113:3159-63. 38 12. Hellman R, Vanhove M, Lejeune A, Stevens FJ, Hendershot LM. The in vivo association 39 of BiP with newly synthesized proteins is dependent on the rate and stability of folding 40 and not simply on the presence of sequences that can bind to BiP. J Cell Biol. 41 1999;144:21-30. 42 13. Auer S, Dobson CM, Vendruscolo M. Characterization of the nucleation barriers for 43 protein aggregation and amyloid formation. HFSP J. 2007;1:137-46. 44 14. Herczenik E, Gebbink MF. Molecular and cellular aspects of protein misfolding and 45 46 disease. FASEB J. 2008;22:2115-33. 47 15. Cuenca L, Gil-Martinez AL, Cano-Fernandez L, Sanchez-Rodrigo C, Estrada C, 48 Fernandez-Villalba E, et al. Parkinson's disease: a short story of 200 years. Histol 49 Histopathol. 2018:18073. 50 16. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alpha- 51 synuclein in Lewy bodies. Nature. 1997;388:839-40. 52 17. Lashuel HA, Overk CR, Oueslati A, Masliah E. The many faces of alpha-synuclein: from 53 structure and toxicity to therapeutic target. Nat Rev Neurosci. 2013;14:38-48. 54 18. Ma MR, Hu ZW, Zhao YF, Chen YX, Li YM. Phosphorylation induces distinct alpha- 55 56 synuclein strain formation. Sci Rep. 2016;6:37130. 57 19. Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury PT, Jr. Acceleration 58 of oligomerization, not fibrillization, is a shared property of both alpha-synuclein 59 mutations linked to early-onset Parkinson's disease: implications for pathogenesis and 60 therapy. Proc Natl Acad Sci U S A. 2000;97:571-6. 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 20. Fusco G, Chen SW, Williamson PTF, Cascella R, Perni M, Jarvis JA, et al. Structural 5 basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers. Science. 6 2017;358:1440-3. 7 8 21. Karpinar DP, Balija MB, Kugler S, Opazo F, Rezaei-Ghaleh N, Wender N, et al. Pre- 9 fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in 10 Parkinson's disease models. EMBO J. 2009;28:3256-68. 11 22. Volles MJ, Lee SJ, Rochet JC, Shtilerman MD, Ding TT, Kessler JC, et al. Vesicle 12 permeabilization by protofibrillar alpha-synuclein: implications for the pathogenesis and 13 treatment of Parkinson's disease. Biochemistry. 2001;40:7812-9. 14 23. Winner B, Jappelli R, Maji SK, Desplats PA, Boyer L, Aigner S, et al. In vivo 15 demonstration that alpha-synuclein oligomers are toxic. Proc Natl Acad Sci U S A. 16 2011;108:4194-9. 17 18 24. Colla E, Jensen PH, Pletnikova O, Troncoso JC, Glabe C, Lee MK. Accumulation of 19 toxic alpha-synuclein oligomer within endoplasmic reticulum occurs in alpha- 20 synucleinopathy in vivo. J Neurosci. 2012;32:3301-5. 21 25. Mercado G, Valdes P, Hetz C. An ERcentric view of Parkinson's disease. Trends Mol 22 Med. 2013;19:165-75. 23 26. Ham H, Woolery AR, Tracy C, Stenesen D, Kramer H, Orth K. Unfolded protein 24 response-regulated Drosophila Fic (dFic) protein reversibly AMPylates BiP chaperone 25 during endoplasmic reticulum homeostasis. J Biol Chem. 2014;289:36059-69. 26 27 27. Preissler S, Rato C, Chen R, Antrobus R, Ding S, Fearnley IM, et al. AMPylation 28 matches BiP activity to client protein load in the endoplasmic reticulum. Elife. 29 2015;4:e12621. 30 28. Preissler S, Rohland L, Yan Y, Chen R, Read RJ, Ron D. AMPylation targets the rate- 31 limiting step of BiP's ATPase cycle for its functional inactivation. Elife. 2017;6. 32 29. Truttmann MC, Pincus D, Ploegh HL. Chaperone AMPylation modulates aggregation 33 and toxicity of neurodegenerative disease-associated polypeptides. Proc Natl Acad Sci 34 U S A. 2018;115:E5008-E17. 35 30. Broncel M, Serwa RA, Bunney TD, Katan M, Tate EW. Global Profiling of Huntingtin- 36 37 associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic 38 Approach. Mol Cell Proteomics. 2016;15:715-25. 39 31. Truttmann MC, Cruz VE, Guo X, Engert C, Schwartz TU, Ploegh HL. The 40 Protein FIC-1 Is an AMPylase That Covalently Modifies Heat- 41 Shock 70 Family Proteins, Translation Elongation Factors and Histones. PLoS Genet. 42 2016;12:e1006023. 43 32. Grassi D, Howard S, Zhou M, Diaz-Perez N, Urban NT, Guerrero-Given D, et al. 44 Identification of a highly neurotoxic alpha-synuclein species inducing mitochondrial 45 46 damage and mitophagy in Parkinson's disease. Proc Natl Acad Sci U S A. 47 2018;115:E2634-E43. 48 33. Gorbatyuk MS, Shabashvili A, Chen W, Meyers C, Sullivan LF, Salganik M, et al. 49 Glucose regulated protein 78 diminishes alpha-synuclein neurotoxicity in a rat model of 50 Parkinson disease. Mol Ther. 2012;20:1327-37. 51 34. Ysselstein D, Joshi M, Mishra V, Griggs AM, Asiago JM, McCabe GP, et al. Effects of 52 impaired membrane interactions on alpha-synuclein aggregation and neurotoxicity. 53 Neurobiol Dis. 2015;79:150-63. 54 35. Mohite GM, Kumar R, Panigrahi R, Navalkar A, Singh N, Datta D, et al. Comparison of 55 56 Kinetics, Toxicity, Oligomer Formation, and Membrane Binding Capacity of alpha- 57 Synuclein Familial Mutations at the A53 Site, Including the Newly Discovered A53V 58 Mutation. Biochemistry. 2018;57:5183-7. 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 36. Li B, Ge P, Murray KA, Sheth P, Zhang M, Nair G, et al. Cryo-EM of full-length alpha- 5 synuclein reveals fibril polymorphs with a common structural kernel. Nat Commun. 6 2018;9:3609. 7 8 37. Ysselstein D, Dehay B, Costantino IM, McCabe GP, Frosch MP, George JM, et al. 9 Endosulfine-alpha inhibits membrane-induced alpha-synuclein aggregation and protects 10 against alpha-synuclein neurotoxicity. Acta Neuropathol Commun. 2017;5:3. 11 38. Kingdon HS, Shapiro BM, Stadtman ER. Regulation of . 8. ATP: 12 glutamine synthetase adenylyltransferase, an that catalyzes alterations in the 13 regulatory properties of glutamine synthetase. Proc Natl Acad Sci U S A. 1967;58:1703- 14 10. 15 39. Sreelatha A, Yee SS, Lopez VA, Park BC, Kinch LN, Pilch S, et al. Protein AMPylation 16 by an Evolutionarily Conserved Pseudokinase. Cell. 2018;175:809-21 e19. 17 18 40. Lewallen DM, Steckler CJ, Knuckley B, Chalmers MJ, Thompson PR. Probing 19 adenylation: using a fluorescently labelled ATP probe to directly label and 20 immunoprecipitate VopS substrates. Mol Biosyst. 2012;8:1701-6. 21 41. Trychta KA, Back S, Henderson MJ, Harvey BK. KDEL Receptors Are Differentially 22 Regulated to Maintain the ER Proteome under Calcium Deficiency. Cell Rep. 23 2018;25:1829-40 e6. 24 42. Bodner CR, Maltsev AS, Dobson CM, Bax A. Differential phospholipid binding of alpha- 25 synuclein variants implicated in Parkinson's disease revealed by solution NMR 26 27 spectroscopy. Biochemistry. 2010;49:862-71. 28 43. Jao CC, Der-Sarkissian A, Chen J, Langen R. Structure of membrane-bound alpha- 29 synuclein studied by site-directed spin labeling. Proc Natl Acad Sci U S A. 30 2004;101:8331-6. 31 44. Bussell R, Jr., Eliezer D. A structural and functional role for 11-mer repeats in alpha- 32 synuclein and other exchangeable lipid binding proteins. J Mol Biol. 2003;329:763-78. 33 45. Davidson WS, Jonas A, Clayton DF, George JM. Stabilization of alpha-synuclein 34 secondary structure upon binding to synthetic membranes. J Biol Chem. 1998;273:9443- 35 9. 36 37 46. Lee JH, Hong CS, Lee S, Yang JE, Park YI, Lee D, et al. Radiating amyloid fibril 38 formation on the surface of lipid membranes through unit-assembly of oligomeric species 39 of alpha-synuclein. PLoS One. 2012;7:e47580. 40 47. Reynolds NP, Soragni A, Rabe M, Verdes D, Liverani E, Handschin S, et al. Mechanism 41 of membrane interaction and disruption by alpha-synuclein. J Am Chem Soc. 42 2011;133:19366-75. 43 48. Perrin RJ, Woods WS, Clayton DF, George JM. Interaction of human alpha-Synuclein 44 and Parkinson's disease variants with phospholipids. Structural analysis using site- 45 46 directed mutagenesis. J Biol Chem. 2000;275:34393-8. 47 49. Peelaerts W, Bousset L, Van der Perren A, Moskalyuk A, Pulizzi R, Giugliano M, et al. 48 alpha-Synuclein strains cause distinct synucleinopathies after local and systemic 49 administration. Nature. 2015;522:340-4. 50 50. Bousset L, Pieri L, Ruiz-Arlandis G, Gath J, Jensen PH, Habenstein B, et al. Structural 51 and functional characterization of two alpha-synuclein strains. Nat Commun. 2013; 52 4:2575. 53 51. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins 54 silver-stained polyacrylamide gels. Anal Chem. 1996; 68:850-8. 55 56 52. Sanyal A, Zbornik EA, Watson BG, Christoffer C, Ma J, Kihara D. and Mattoo S, Kinetic 57 And Structural Parameters Governing Fic-Mediated Adenylylation/AMPylation of the 58 Hsp70 chaperone, BiP/GRP78. Biorxiv 494930; doi: https://doi.org/10.1101/494930 59 53. Levine PM, Galesic A, Balana AT, Mahul-Mellier A-L, Navarro MX, De Leon CA, et al. 60 αSynculein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 as potential inhibitors of Parkinson’s disease. Proc Nat Acad Sci. 2019; 5 doi:10.1073/pnas.1808845116 6 54. Sengupta R, Poderycki MJ, and Mattoo S. Cryo-APEX: An electron tomography tool for 7 8 subcellular localization of membrane proteins. bioRxiv 522482; 9 doi: https://doi.org/10.1101/522482 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Figure 1

A: 363 HPFIDGNGRTSR

1 45 108 180 263 381 458 N TM TPR TPR FIC C

TVAIEG 234

B: 1 T33 T54 60 T75 95 140 N-term NAC C-term

A30P E46K A53T/A53E G51D H50Q bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Figure 2

A:

hippocampus

substana nigra

2mm

100µm 50µm bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal & Dua et al., 2019 – Figure 2

B: HYPE% MAP2% Merge%

50µm

BiP% MAP2% Merge%

50µm

C: HYPE TH Merge bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Figure 3

A: B:

Adenylylated HYPE

Adenylylated αSyn

HYPE

Kd= 15.30uM + 0.04uM αSyn bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Figure 4

Sanyal, Dua et al., 2019 - Figure 4A

AMP (K)T33 KEGVLYVGSK(T)

tk160708_Seema_3T_target 07/10/16 17:02:55

tk160708_Seema_3T_target #22466-22657 RT: 27.53-27.76 AV: 39 NL: 1.19E3 F: ITMS + c NSI r Full ms2 [email protected] [200.0000-1800.0000] -[AMP+18] 1162.688 100

90

80

70

60

OH 50 N OH N 2NH 40 O N N O Relative Abundance OH -[adenosine]2+ 30 348.036 P + OH H 2+ O -[AMP+18] 20 -[adenosine] -[adenine] 630.004 745.959 1046.674 10 581.992 691.071 804.988 1242.745 250.173 295.564 413.650 465.098 543.213 892.817 937.524 995.594 1084.582 1295.554 1374.756 1428.675 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z

tk160711_Seema_MS3_target #13956-14101 RT: 27.60-27.87 AV: 48 NL: 5.70E1 F: ITMS + c NSI r Full ms3 [email protected] [email protected] [200.0000-2000.0000]

y y 1144.6997 100 LSGVYLVG 8 E 9 90 b b b b 80 T(-18)KEGVLY 7 V 8 G 9 S 10 70

60

50

40 822.5719 1016.6357 872.6632 Relative Abundance 30 951.6371 1163.7351 20 929.5061 690.4864 1119.5898 634.6769 773.4918 1034.5321 10 417.1026 733.5463 465.2737 525.1282 579.4188 788.7186 846.4811 903.5431 970.4457 0 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal & Dua et al., 2019 – Figure 4B

AMP (K)TKEGVVHGVAT54 VAEK(T) tk160708_Seema_3T_target 07/10/16 17:02:55

tk160708_Seema_3T_target #20260-20297 RT: 24.90-24.94 AV: 7 NL: 6.87E3 2+ F: ITMS + c NSI r Full ms2 [email protected] [200.0000-1800.0000] -[AMP+18] 754.103 100

90

80

70

60

50

2+ 40 OH -[adenosine] N 851.191 Relative Abundance OH N 30 2NH 803.182 2+ O -[adenine] 20 N N O OH 601.270 348.080 P H+ 860.032 10 OH O 573.877 610.328 250.184 503.133 327.292 359.242 454.420 657.805 736.634 947.169 979.757 1061.322 1148.832 1187.673 1277.772 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z

tk160711_Seema_MS3_target #12644-12718 RT: 25.05-25.19 AV: 25 NL: 5.56E1 F: ITMS + c NSI r Full ms3 [email protected] [email protected] [200.0000-2000.0000]

745.3719 100 y y y y y 90 KEA 3 VT(-18)A 6 VGH 893.47299 V 10 VGE 13 80 b b b b b 70 TKEGV 5 V 6 HGV 9 A b10 T(-18)V 12 AE 14 2+ 60 y13 639.4684 50 614.4327 907.5611

40 689.8413 993.6427 Relative Abundance 30 600.5500 979.7031 1160.7780 1361.1188 20 515.3869 347.3144 1107.4791 1279.0474 567.2601 855.7319 10 240.1989 414.4299 1379.0012

0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal & Dua et al., 2019 – Figure 4C

AMP (K)TKEQVTNVGGAVVTGVT75 AVAQK(T) tk160708_Seema_3T_target 07/10/16 17:02:55

tk160708_Seema_3T_target #25595-25675 RT: 31.26-31.36 AV: 17 NL: 9.88E3 F: ITMS + c NSI r Full ms2 [email protected] [200.0000-1800.0000] -[AMP+18]2+ 1070.3457 100

90

80 -[adenosine]2+ 70

60 1119.3053

50 2+ 40 -[adenine] 1167.3821 Relative Abundance 30 OH N OH N 20 2NH O 811.3639 N 348.1052 10 N O OH 829.5255 951.1994 406.3535P 516.3514+ 599.3596 701.2516 1203.6402 250.1164 OH H 1384.8721 1540.9741 1624.0121 1794.0888 0 O 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

tk160711_Seema_MS3_target #15795-15846 RT: 31.15-31.25 AV: 18 NL: 6.74E1 F: ITMS + c NSI r Full ms3 [email protected] [email protected] [200.0000-2000.0000] y9 856.6287 100 y14 y y y y y 1239.7962 y 90 KQAVAT(-18)V 7 G 8 T V 10 V 11 AG 13 G VNTV 18 Q y19 80 70 TKEQVTNV b8 GGAV b12 V b13 T b14 GVT(-18)A b18 V b19 A b20 60 955.6237 1283.8092 50

40 1185.6901 755.6363 1048.6270 Relative Abundance 30 698.4931 1384.7400 1556.2702 20 653.5496 1695.6217 802.8520 1794.9623 440.2375 1157.6521 1454.0285 10 339.2646 609.4991 1865.1897

0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Figure 5

Fibrilation SetFibrilation 4_Non normalized Set 4_Non normalized Fibrilation Set 4_Endpoint analysis B: * 5000 A: 5000 5000 αonlySyn aSynonly only aSyn 4000 4000 ++ WT WT HYPE HYPE + WT HYPE 4000 ++ E234G EG HYPE HYPE + EG HYPE 3000 3000 3000 ++ E234G/H363A HA HPYE HYPE+ HA HPYE 2000 2000 fluorescence 2000

ThT 1000 1000 1000 Endpoint Fluorescence 0 0 0 0 50 100 150 0 50 100 150 Non normalized ThioT fluorescence

Non normalized ThioT fluorescence Time (h) Time (h) only aSyn α Syn only + WT HYPE + EG HYPE + HA HPYE HYPE + WT HYPE + WT + E234G HYPE +E234G/ + E234G/H363A C: αSyn +WT +E234G H363A S P S P S P S P Fibrilation Set 4_Normalized Fibrilation Set 4_Normalized Syn oligomer 75 kD α 1.5 E: 1.5 αaSynSyn onlyonly 37 kD aSyn only ++ WT WT HYPE HYPE 20 kD αSyn monomer + WT HYPE 1.0 ++ E234G EG HYPE HYPE 1.0 + EG HYPE ++ E234G/H363A HA HPYE HYPE Pellet + HA HPYE A53T-A53Tα aSynSyn only D: 14000 * 0.5 A53T aSyn 0.5 MouseMouse WT αSyn only 13000 Mouse 12000 ThT fluorescence (normalized) ThT 100% 11000 0.0 100% 187% 208% 175% 96% 0 0.050 100 150 10000 92% 0 50 100 150 Non normalized ThioT fluorescence

Non normalized ThioT fluorescence Time (h) 9000 Time (h)

Intensity of aSyn (a.u) 74%

levels in pellet (AU) α Syn 8000

aSyn

+ WT HYPE + E234G HYPE

+ E234G/H363A HYPE bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Figure 6

A αSyn only B + WT-HYPE

C + E234G/H363A-HYPE D + E234G-HYPE

200nm bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dutta et al - Figure 7

Combination of all data (normalized) 1.5

1.0 ****

0.5

Membrane permeabilization (AU) 0.0 Normalized Permeabilization

aSyn onlyα Syn only HYPE + WT HYPE HYPE + WT + EG HYPE + HA HPYE + E234G HYPE + E234G/H363A + E234G/H363A bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dua et al., 2019– Supplementary Figure 1

A: n = 2 n = 3

10X:

100µm 100µm

20X:

50µm 50µm bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dua et al., 2019– Supplementary Figure 2

HYPE BiP Merge with DAPI

100µm bioRxiv preprint doi: https://doi.org/10.1101/525659; this version posted January 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Sanyal, Dua et al., 2019 – Supplementary Figure 3

3000

2500

2000 aSyn

1500 aSyn (WT HYPE) aSyn (E234G HYPE)

1000 aSyn (E234G/H363A HYPE) ThT Fluorescence (a.u) ThT

500

0 0 10 20 30 40 50 60 Time (h)