Exploring the Roles of Post-Translational Modifications in the

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Exploring the Roles of Post-Translational Modifications in the Pathogenesis of Parkinson’s Disease Using Synthetic and Semisynthetic Modified #-Synuclein Huai Chen, Yufen Zhao, Yong-Xiang Chen, and Yan-Mei Li ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00447 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 12, 2019

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ACS Chemical Neuroscience

Exploring the Roles of Post-Translational Modifications in the Pathogenesis of Parkinson’s Disease Using Synthetic and Semisynthetic Modified α-Synuclein Huai Chen†, Yu-Fen Zhao†, Yong-Xiang Chen†, Yan-Mei Li*,†,‡,§ †Key

Lab of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua

University, Beijing 100084, P.R. China ‡Beijing §Center

Institute for Brain Disorders, Beijing 100069, P.R. China

for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, P.R. China.

*Corresponding

Author: E-mail: [email protected]

Abstract: Alpha-synuclein (α-syn), a small soluble protein containing 140 amino acids, is associated with the recycling pool of synaptic vesicles in presynaptic terminals. The misfolding and aggregation of α-syn is closely related to a group of neurodegenerative diseases, including Parkinson’s disease (PD), which is one of the most common progressive neurodegenerative diseases. Varieties of the posttranslational modifications (PTMs) of α-syn, including phosphorylation, ubiquitination and glycosylation, have been detected in soluble and aggregated α-syn in vivo. These PTMs can have either positive or negative effects on α-syn aggregation and toxicity, which may play critical roles in PD pathogenesis. Herein, we review the advances in synthetic and semisynthetic chemistry to generate homogeneous α-syn variants with site-specific modifications. Using these modified α-syn, we gain insight into the consequences of PTMs on α-syn aggregation and other biophysical properties, which can help elucidate the role of PTMs in the pathogenesis of PD and develop potential therapies to PD. Keywords: α-synuclein, Parkinson’s disease, posttranslational modifications, protein synthesis, protein semisynthesis Introduction Parkinson’s disease (PD) is considered the second most common progressive neurodegenerative disease.1 The main clinical manifestations of PD in patients include resting tremor, muscular rigidity, bradykinesia, and postural disorders.2 The neuropathological feature of PD is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, which results in a significant reduction in striatal dopamine. The precise mechanism of the relatively selective dopaminergic neuron degeneration is still unclear; however, a common hallmark, which can be found in most PD brain specimens, is the presence of insoluble inclusions in dopaminergic neurons. These inclusions are called Lewy bodies (LBs) or Lewy neurites (NTs), of which the main component is the aggregated alpha-synuclein (α-syn) protein.3 The pathogenic role of α-syn is supported by the genetic data. Multiplications of the SNCA gene (which encodes α-syn) and several point mutations of this gene (e.g., A30P, E46K and A53T) contribute to dominant familial Parkinsonism.4-9 In addition, some genetic polymorphisms in SNCA are the major risk factors for sporadic PD.10 The abnormal aggregation of α-syn is thought to play a central role in triggering the death of dopaminergic neurons in both familial and sporadic PD.11 Although the endogenous physiological functions of α-syn remain inconclusive, α-syn clearly localizes to presynaptic terminals and is considered to be associated with the recycling pool of synaptic vesicles.2,12-15 Moreover, it is reported that the knockdown or overexpression of α-syn

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results in deficiencies in synaptic transmission.16-19 Therefore, it is widely believed that α-syn may relate to the transport and release of synaptic vesicles, synaptic function and plasticity, as well as the regulation of neurotransmitter release. The abnormal aggregation of α-syn contributes to the imbalance between the degradation and production of the protein, causing the inhibition of neurotransmitter exocytosis and the death of dopaminergic neurons in PD patients.13 α-Syn is a small, soluble protein with 140 amino acids (14 kDa). The sequence of α-syn can be divided into three different domains: an N-terminal domain (residues 1-60) with lysine-rich, repetitive segments, which is crucial for modulating its interactions with membranes; a nonamyloid-β component (NAC) domain (residues 61-95) with a highly hydrophobic motif, which is indispensable for α-syn aggregation; and an acidic C-terminal domain (residues 96-140), which is associated with its nuclear localization and interactions with small molecules, proteins and metals (Figure 1).12,20 The native state of α-syn is still controversial. Some studies have reported that α-syn exists as a soluble, helical, folded tetramer purified from mammalian cells,21,22 while others have shown that α-syn predominantly exists in cytosol as stable unfolded monomers and adopts an αhelical conformation when binding to cellular membranes.23,24 These studies suggest that α-syn may exist in a balance between different conformational and/or oligomeric states. The imbalance of these states can cause abnormal levels of α-syn, which might induce the formation and/or accumulation of toxic α-syn oligomers and fibrils.25 Several factors have been shown to have an impact on α-syn aggregation, including genetic mutations, oxidation stress, metal ions and posttranslational modifications (PTMs).26-30 A variety of PTMs of α-syn have been identified both in vivo and in LBs isolated from PD patients, including phosphorylation, nitration, ubiquitination, and glycosylation (Figure 1).31-35 In particular, α-syn phosphorylated at residue S129 was found to be the major constituent of LBs in PD brains, which suggested that PTMs might play a crucial role in α-syn aggregation.32,36 Therefore, it is indispensable to understand the effects of PTMs on α-syn aggregation and toxicity in order to reveal the pathogenesis of PD and develop effective diagnostic, preventative and therapeutic strategies. However, it is challenging to obtain homogeneous, structurally defined α-syn with PTMs from E. coli expression or specimen purification. Profit from the development of native chemical ligation (NCL) and expressed protein ligation (EPL), chemical synthesis or semisynthesis of proteins with PTMs has become an executable work.37,38 NCL ， which was developed by Kent et al, involves a chemoselective ligation reaction between a C-terminal peptide thioester on one peptide and an N-terminal cysteine on the other peptide.37 The first step of the reaction is the reversible interpeptide thioester bond formation, which is followed by a spontaneous S to N acyl shift to result in a native amide bond. Both the peptide segments can be prepared by solid-phase peptide synthesis (SPPS), and NCL can dramatically increase the size of polypeptides. More importantly, building blocks with modifications such as phosphorylated serine and O-GlcNAcylated threonine can be used in SPPS to synthesize peptides with PTMs and, finally, to generate proteins of interest with site-specific PTMs after NCL. Although NCL has been proven to be a powerful technique for the total synthesis of small proteins, it can hardly be used to synthesize larger proteins beyond ~15 kDa. To overcome the size limitation, Muir et al developed a general method, EPL, in which they carried out the ligation reaction between a recombinant protein thioester and a synthetic peptide with N-terminal cysteine.38 Adopting an intein-chitin binding domain fusion protein expression system, the protein thioester can be generated


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by thiol-dependent autocatalytic, intein-mediated cleavage. EPL allows us to synthesize larger proteins with PTMs at specific sites. A number of proteins with PTMs have been synthesized by NCL or EPL for structural and biological studies.39-43 In this review, we will focus on the synthetic strategies of α-syn with different PTMs and the consequences of these PTMs on biochemical, structural, toxic and aggregation properties of the protein. A better understanding of the molecular impact will help to elucidate the roles of these PTMs in the pathogenesis of PD.

Figure 1: Schematic representation of α-syn, in which the locations of the main PTMs are shown. Phosphorylation Phosphorylation is the most widely studied PTMs of α-syn. Phosphorylation at S129 (pS129) is regarded as one of the main disease-related α-syn PTMs. It has been reported that more than 90% of α-syn in LBs is phosphorylated, compared with only approximately 4% under physiological conditions.32,36 This suggests that pS129 may play an important role in the pathogenesis of PD. In addition to S129, it was found that other residues within α-syn, including Y39, S87, Y125, could also be phosphorylated in vivo.44-48 It is clear that phosphorylation influences the aggregation and toxicity of α-syn,49 but it is still controversial whether phosphorylation has promotive or obstructive effects on aggregation and toxicity.50 Multiple studies on α-syn phosphorylation have shown quite different, and even opposite, results because different biological models were used in these studies, and the phosphorylated α-syn variants might not be site-specific and homogeneous.48,51-53 Moreover, utilizing aspartic acid or glutamic acid, which is extensively used as a phosphorylation mimic in biological research, to simulate the phosphorylation of α-syn showed different aggregation properties compared with α-syn phosphorylated by phosphokinases.54 This finding implies that using genetic mutation to mimic α-syn phosphorylation is not accurate due to its structural sensitivity. Taken together, these results strongly highlight the significance of obtaining homogeneously phosphorylated α-syn variants for elucidating the role of this modification in PD pathogenesis. In 2012, the Lashuel lab used a protein semisynthesis strategy to obtain α-syn with phosphorylation at Y125 (pY125) (Figure 2A).55 They prepared the pY125 α-syn from a recombinant protein thioester (residues 1-106) using intein-mediated thioester formation and a synthetic peptide (residues A107C-140) using a phosphorylated tyrosine building block. An NCL reaction was carried out between the two fragments, followed by a desulfurization reaction in one pot to achieve full-length pY125 α-syn. The pY125 α-syn showed a virtually identical binding



affinity to membrane compared with wild-type α-syn (WT α-syn). The NMR data showed that phosphorylation at Y125 had generally little alteration to the structure of monomeric α-syn. There was also no significant difference between the aggregation property of pY125 α-syn and WT α-syn shown by thioflavin T fluorescence (ThT fluorescence), transmission electron microscopy (TEM) and SDS-PAGE analysis. They also found that pY125 did not alter the subsequent phosphorylation of α-syn at S129 or S87 by polo-like kinase 3 (PLK3) or casein kinase 1 (CK1). Although this synthetic pY125 α-syn variant showed little difference compared with WT α-syn, it highlights the efficient and promising semisynthetic strategy that introduces single or multiple PTMs to the homogeneously modified α-syn. Semisynthesis of α-syn with phosphorylation at Y39 (pY39) was approached by the Lashuel and Eliezer labs using sequential NCL steps in one-pot manner (Figure 2B).56 They divided α-syn into three fragments: a recombinant protein (residues A56C-140), a synthetic phosphorylated peptide thioester with phosphorylation at Y39 (residues A30C-55), and a synthetic peptide thioester (residue 1-29). Two steps of NCL and the following desulfurization reaction were carried out to generate full-length pY39 α-syn. Although phosphorylation at Y39 had little or no effect on the global secondary structure of α-syn, it could decrease the helix-2 region of the N-terminal domain bound to lipid vesicles, which might influence the function of α-syn by affecting its interaction with synaptic vesicles and plasma membranes. In addition, although phosphorylation at Y39 did not promote α-syn aggregation, it changed the membrane-binding state and might influence the aggregation process in vivo. pY39 α-syn showed similar effects on membrane binding compared with the disease-associated G51D α-syn, which indicated that pY39 α-syn could promote the pathogenesis of PD. As pS129 α-syn is a major component in LBs in PD brains, it is considered the most relevant form of α-syn to PD pathogenesis. The Lashuel lab and Li lab adopted semisynthetic strategies to synthesize pS129 α-syn with a recombinant protein thioester (residues 1-106) and a synthetic peptide (residues A107C-140) phosphorylated at S129, respectively (Figure 2C).57,58 Li and coworkers found that monomeric pS129 α-syn and WT α-syn existed in indistinguishable random coil conformations predominantly by circular dichroism (CD). While binding to lipid vesicles, pS129 α-syn showed a weaker α-helical structure than that of WT α-syn, which indicated that phosphorylation at S129 might reduce the membrane binding affinity of α-syn. Meanwhile, a ThT assay was carried out to investigate the aggregation kinetic of pS129 α-syn. The data showed that phosphorylation at S129 promoted α-syn aggregation compared with WT α-syn. Notably, although PS fiber and WT fiber (fiber formed from pS129 α-syn and WT α-syn, respectively) had a similar morphology under TEM, the two fibers assembled into distinct conformations detected by X-ray diffraction. Moreover, the PS fiber displayed significantly higher cytotoxicity and less resistance to proteinase K than WT fiber, which strongly suggested that phosphorylation at S129 induced distinct strain formation of α-syn (called PS strain). WT and PS strains were shown to have the ability to induce the assembly of α-syn into aggregates and propagate their intrinsic structural features dutifully in vitro and in cells, respectively. This is the first demonstration that PTMs of α-syn can affect the aggregated strain formation, suggesting the important role of α-syn phosphorylated at S129 in the PD pathogenetic process.58


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Figure 2: Synthetic strategies to generate phosphorylated α-syn. Ubiquitination Ubiquitinated α-syn is another α-syn variant that was identified within LBs isolated from PD patients.59-61 It has been reported that α-syn can be directly ubiquitinated in LBs, and the major form of the ubiquitination is mono-ubiquitination, with partial polyubiquitination at several lysine residues.61,62 Therefore, revealing the consequences of ubiquitin (Ub) modifications to α-syn aggregation, toxicity and other properties will be helpful for drawing the complete picture of PD. However, the use of traditional biological methods, such as modifying α-syn with Ub ligases in vitro or in vivo, has resulted in heterogeneously ubiquitinated α-syn at distinct lysine sites and reaching contradictory conclusions for the impacts of ubiquitination on α-syn aggregation and toxicity.62-65 Thus, it is challenging to investigate the consequences of ubiquitination at specific lysine residues by enzymatic methods. In 2009, the Brik lab developed a strategy to synthesize site-specifically ubiquitinated peptides and proteins, which used a mercaptolysine to mediate the NCL reaction with a Ub thioester on its side-chain to form a native isopeptide bond (this reaction is called isopeptide chemical ligation or ICL), followed by a desulfurization reaction.66 This strategy provides an elegant and feasible approach to semisynthesize ubiquitinated proteins and polyubiquitin chains.67-69 The Lashuel and Brik labs cooperated to synthesize α-syn with mono-ubiquitination at lysine 6 (K6-Ub α-syn) using the strategy mentioned above (Figure 3A).70 They prepared the synthetic K6-Ub α-syn from a synthetic peptide thioester (residues 1-18) with an Acm-protected mercaptolysine building block instead of Lys6, a recombinant α-syn fragment (A19C-140) and a recombinant Ub thioester T7-Ub-SR. NCL reaction between the peptide thioester and the recombinantly expressed α-syn fragment was carried out to generate full-length α-syn. After Acm group deprotection, the Ub thioester was installed at Lys6 side-chain through a ligation reaction. Since there is no native cysteine residue in either α-syn or Ub, the synthetic protein underwent



metal-free desulfurization to approach site-specifically ubiquitinated α-syn without any mutation. Taking advantage of the synthetic protein, the authors investigated the effects of the K6-Ub on the secondary structure, membrane binding and aggregation properties of α-syn. CD spectrum suggested that the conjugation of mono-Ub on K6 did not cause major alterations to the structure of α-syn in solution or change its membrane binding affinity with synthetic lipid vesicles. Whereas they demonstrated that ubiquitination at K6 significantly inhibited α-syn aggregation by ThT assay and TEM, which indicated a protective role of ubiquitination at K6 in PD. In addition, K6-Ub did not affect the phosphorylation of α-syn at either S87 or S129 by kinases. The typical function of ubiquitination is to target the substrate proteins for proteasomal degradation.71 However, the most common form of ubiquitination found in cells is K48-linked polyUb chains that mediate the degradation process.72-74 In 2012, Ciechanover and coworkers reported that a mono-Ub moiety conjugated to a small protein (up to ~150 residues) was sufficient to mediate degradation by proteasome.75 This finding implied that ubiquitination of α-syn may help to remove the excess α-syn and its aggregates in vivo. In this same study, a synthetic protein, K12-Ub α-syn, was prepared by the same strategy mentioned above. The hypothesis was confirmed by the results of in vitro studies showing that K12-Ub α-syn was efficiently degraded by proteasomes, while WT α-syn was stable. K48-linked di- and tetra-Ub chains on the side chain of K12 in α-syn were also been semisynthesized by the same labs through NCL, multiple steps of ICL and desulfurization.76 Polyubiquitinated α-syn variants were shown to be more resistant to deubiquitinases and more sensitive to proteasomal degradation than mono-ubiquitinated α-syn. Moreover, tetra-ubiquitination at K12 could induce the formation of non-amyloid-like aggregates but largely blocked the fibrillization of α-syn in vitro. These findings support the hypothesis that ubiquitination is not necessary for α-syn fibril and LB formation. Mono- or polyubiquitination of α-syn may occur after the formation of α-syn fibrils, and stimulate cellular responses to dissociate the α-syn aggregates and/or target their degradation by the proteasome to promote clearance of α-syn. Although the ICL is an elegant strategy to produce mono- or polyubiquitinated protein without any mutation or structural change, the synthetic challenge of the chemistry makes it difficult to generate ubiquitinated α-syn variants at all potential ubiquitination sites. To overcome the obstacles, the Pratt lab took advantage of a strategy termed disulfide-directed ubiquitination, in which they used disulfide linkage to simulate the native Ub-lysine isopeptide bond (Figure 3B).77 This technology allowed them to facilely prepare site-specifically ubiquitinated α-syn analogs at all nine known modification sites. These analogs were generated from two parts: an Ub protein with a Cterminal thiol, which was recombinantly expressed in E. coli as a fusion to the Gyr intein followed by thiolysis with cysteamine, and a recombinant full-length α-syn, in which the target site was mutated to cysteine. The thiol at the C-terminal of Ub was activated by 2,2’-dithiobis(5nitropyridine) (DTNP), and then the resulting mixed disulfide product Ub-DTNP was incubated with α-syn mutants to generate the disulfide-directed ubiquitinated α-syn analogs (K#C-Ub α-syn). With all the ubiquitinated α-syn analogs in hand, the effects of Ub at different residues on α-syn aggregation were investigated by ThT assay and TEM. Notably, the data strongly suggested that the effects of Ub on α-syn aggregation were differential and site-dependent. K10C-Ub and K23C-Ub α-syn readily formed fibrils, with a decreased rate of fibril formation compared with WT α-syn. Ubiquitination at K6, K12, and K21 presented a moderate inhibition of fibril formation. This was consistent with the previous result that K6-Ub inhibited α-syn aggregation. K32C-Ub, K34C-Ub, K43C-Ub and K96C-Ub α-syn showed no fibril formation, which indicated a strong inhibitory effect


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of ubiquitination at these sites on α-syn aggregation. Taken together, these data can explain the contradictory results using heterogeneously ubiquitinated forms of α-syn generated through enzymatic methods, which may ubiquitinate α-syn at nonspecific lysine sites. The results also highly emphasize the importance of obtaining homogeneous α-syn with PTMs for the investigation of their biophysical and biochemical properties and elucidation of their roles in PD pathogenesis.

Figure 3. Synthetic strategies to generate ubiquitinated and SUMOylated α-syn. # means the site of modification. SUMOylation Small ubiquitin-like modifier (SUMO) proteins, which are members of the ubiquitin-like family, can also be conjugated to the amine of a lysine side-chain by isopeptide linkage similar to ubiquitin.78 SUMOylation is involved in a variety of cellular processes in living organisms, including intracellular localization, protein-protein interaction, as well as the protein degradation of its substrates.79 There are three mammalian isoforms of SUMO (SUMO1, 2/3); SUMO2 and SUMO3 share 97% sequence identity but only 47% to SUMO1.80 There are two potential SUMOylation sites in the α-syn sequence, VK96KD and GK102NE, according to the typical SUMOylation consensus motif: ψ-K-x-D/E, ψ = hydrophobic amino acid, x = any amino acid.81 It has been reported that aggregates from LBs of PD patients are immunoreactive to SUMO1, and



proteasome dysfunction can accelerate SUMOylated α-syn aggregate formation.82-85 However, other studies showed that SUMOylation could block α-syn aggregation, and mutations of the SUMOylation sites would promote α-syn aggregation both in vitro and in vivo using SUMOylated α-syn from a bacterial coexpression system.86-88 These contradictory data may result from the heterogeneous SUMOylated α-syn in the bacterial system. To investigate the effects of SUMOylation on α-syn aggregation, the Pratt lab used a disulfidedirected strategy to generate site-specifically SUMOylated α-syn at either K96 or K102 with either SUMO1 or SUMO3 (Figure 3C).89 They found that SUMOylation inhibited the aggregation of αsyn in site- and isoform-specific manners. In general, the SUMOylation of α-syn at K102 had a higher inhibitory effect than at K96. In addition, they also found that modification by SUMO1 had a higher inhibitory effect than SUMO3 at both SUMOylation sites. However, because of the labile nature of disulfide linkage, it was impossible to evaluate the effects of SUMOylation or ubiquitination on α-syn toxicity using disulfide-linked SUMOylated or Ubiquitinated α-syn by cell culture assays. The Pratt lab developed another strategy based on bisthio-acetone (BTA) linkage as the analogue of native isopeptide bond, which is more chemically and enzymatically stable than disulfide linkage (Figure 3D).90 They used this strategy to synthesize α-syn with ubiquitination at K6, K23, K43, or K96, and α-syn with SUMOylation at K96 or K102. Mostly consistent with the previous disulfide-linked α-syn, ubiquitination or SUMOylation at these sites inhibited α-syn aggregation, although to different degrees. Additionally, ubiquitination at the tested sites blocked toxicity of α-syn in the treatment of SH-SY5H cells. α-Syn with SUMOylation at K102 also showed nontoxicity to SH-SY5H cells, while SUMOylation at K96 was still toxic to cells with a little decrease compared with WT α-syn. However, there were somewhat different results between the disulfide-linked K23Ub α-syn and BTA-linked K23Ub α-syn under aggressive aggregation conditions. Different linkage analogues may therefore not be able to mimic the native isopeptide linkage in all tests. Thus, it is necessary to develop novel and facile methods to generate ubiquitinated or SUMOylated proteins with native linkage. On all accounts, these results provide more evidence that ubiquitination and SUMOylation may play protective roles in PD. The results suggest that the methods to increase the levels of ubiquitination and SUMOylation to α-syn may be potential therapies for PD. Glycosylation Monosaccharide N-acetyl-glucosamine modification (O-GlcNAcylation) is one type of glycosylation that occurs extensively in hundreds of proteins in living organisms. It has been reported that the O-GlcNAcylation may be linked to neurodegenerative diseases.91,92 For example, O-GlcNAcylated tau (an Alzheimer’s disease (AD) associated protein) shows a decreased tendency for aggregation, and increasing the global level of O-GlcNAcylation by a small molecule inhibitor of O-GlcNAcase in AD mice slows the pace of neurodegeneration.93 These results suggest that OGlcNAcylation may play a protective role in neurodegenerative diseases. α-Syn can be OGlcNAcylated in vivo at threonine 64 and 72 in mice and at serine 87 in humans.94 The location of O-GlcNAc at the NAC region of α-syn raises the possibility that O-GlcNAcylation may influence the aggregation of α-syn. However, the precise consequences of O-GlcNAcylation on α-syn aggregation and toxicity remain unknown. To investigate the effects of O-GlcNAc on α-syn properties, the Pratt lab used a protein semisynthesis strategy to obtain α-syn with O-GlcNAcylation at T72 (gT72 α-syn) with three


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fragments: a recombinant protein thioester (residues 1-68), a synthetic glycopeptide thioester bearing an O-GlcNAc at T72 (residues A69C-75), and a recombinant protein (residues A76C-140) (Figure 4A).95 Two steps of NCL and a following desulfurization reaction were carried out to generate the full-length gT72 α-syn. Compared with WT α-syn, O-GlcNAcylation at T72 significantly blocked α-syn aggregation and inhibited α-syn toxicity to primary neurons and SHSY5Y cells. Meanwhile, O-GlcNAc at T72 did not affect the membrane binding affinity of α-syn. The aggregation kinetics of α-syn were delayed when mixing gT72 α-syn and WT α-syn together, which suggested that O-GlcNAcylation could prevent incorporation of α-syn monomers into aggregates. In addition, O-GlcNAcylation at T72 increased subsequent α-syn phosphorylation at S87 by CK1, while inhibiting phosphorylation at S129 by CK1, PLK3 or G protein-coupled receptor kinase 5. α-Syn O-GlcNAcylated at S87 (gS87 α-syn) was also generated by the Pratt lab using a similar semisynthetic strategy (Figure 4B).96 O-GlcNAcylation at S87 also showed an inhibitory effect on α-syn aggregation, although to a lesser extent compared with gT72 α-syn. Similar to gT72, α-syn, gS87 α-syn also had similar membrane binding affinity compared with WT α-syn, which indicated that O-GlcNAcylation at both S87 and T72 would not disrupt α-syn’s endogenous functions. Recently, the Pratt lab found that O-GlcNAcylation at both T72 and S87 blocked α-syn proteolysis by calpain, which cleaved α-syn in vitro and potentially in PD.97 Taken together, the properties of these two synthetic O-GlcNAcylated α-syn provide evidence to support the hypothesis that O-GlcNAcylation of α-syn may play protective or beneficial roles in PD. These data also imply us that increasing O-GlcNAcylation levels may be a potential therapeutic strategy for PD. This highlights the unique superiority of protein synthetic methods for elucidating the direct influence of PTMs on proteins.

Figure 4. Synthetic strategies to generate O-GlcNAcylated α-syn. Nitration



The nitration of tyrosine to form 3-nitrotyrosine (nY) is a consequence of elevated cytoplasmic environment oxidative stress, which is considered to contribute to neuronal damage and cell degeneration in neurodegenerative disorders.33,98 Nitrated α-syn has been detected in LBs, LNs and cytoplasmic inclusions in nigral neurons from PD patients and animal models.99 In addition, a mixture of nitrated α-syn variants has been proven to be more toxic to several cell lines than WT αsyn and induce the loss of dopaminergic neurons in rats.100,101 There are four tyrosine residues in αsyn at residues 39, 125, 133 and 136, and all of these tyrosine residues can be nitrated.33 However, chemical methods or enzyme-catalytic methods to induce nitration can not only result in heterogeneous mixtures of nonspecifically nitrated α-syn at different tyrosine sites but can also lead to the formation of tyrosine-tyrosine cross-linked dimers or higher oligomer species.102-105 These mixtures make it challenging to reveal the roles of nitration on α-syn properties at specific sites or certain combinations of nitration sites. To remove the obstacle, the Lashuel lab utilized semisynthetic chemistry combined with an optimized desulfurization condition to achieve α-syn nitrated at Y39 and Y125, respectively (nY39 α-syn and nY125 α-syn) (Figure 5A and B). nY39 α-syn was generated by one pot EPL with three fragments, and nY125 α-syn was generated with two fragments. With these two site-specifically nitrated α-syn varieties in hand, they found that nitration at either site caused the α-syn to form shorter and wider fibrils than the fibrils formed by WT α-syn. In addition, nitration at Y39 or Y125 resulted in a modest but significant reduction in affinity to membranes. They also found that nitration had no effect on α-syn phosphorylation at S129 by PLK3 in vitro. Nitration and dityrosine cross-linking affected the recognition by several anti-α-syn antibodies, which might be due to the conformational changes, epitope exposure and availability. However, the toxicity and other properties of site-specifically nitrated α-syn was not investigated to show whether nitration played a beneficial or detrimental role in PD. Therefore, further studies are needed to understand the effects of nitration on α-syn in PD pathogenesis better.

Figure 5. Synthetic strategies to generate nitrated α-syn. Nα-acetylation N-terminal acetylation (Nα-acetylation) is one of the most common posttranslational


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modifications occurring in mammalian cells.107 Nα-acetylated α-syn can also be found both in soluble α-syn and in LBs from PD patients.36,108 However, most in vitro experiments are carried out with recombinant α-syn that do not have this modification. To investigate the effects of Nαacetylation on α-syn properties, the Lashuel lab used two strategies: one strategy relied on a semisynthetic approach based on EPL (Figure 6), and the other relied on the co-expression of α-syn and NatB (an enzyme that can catalyzes Nα-acetylation of proteins) to obtain Nα-acetylated α-syn for biophysical and biological studies.109 After a number of assays both in vitro and in vivo, including CD, ThT, NMR, TEM, and murine synaptosomal binding assays, they concluded that Nαacetylation had little or no effect on structural and aggregation properties of α-syn compared with WT α-syn. Nα-acetylated and WT α-syn shared similar properties in secondary structure, aggregation, membrane-binding affinity, oligomeric state in vitro and in HeLa cells. However, other studies using recombinant Nα-acetylation α-syn reported that Nα-acetylation had distinct impacts on the α-syn oligomer and aggregate formation, α-helicity in the N-terminal, Cu2+ binding affinity and membrane-binding affinity.110-114 Recently, Diao and coworkers used a similar EPL strategy to synthesize Nα-acetylated α-syn and found that Nα-acetylation could destabilize the oligomerization state of α-syn by blocking intermolecular hydrogen bonds.115 Therefore, the effects of Nαacetylation on α-syn remain controversial and uncertain. Further studies are needed for more reliable conclusions and a better understanding of the roles of Nα-acetylation both in the endogenous functions of α-syn and in PD pathogenesis.

Figure 6. The synthetic strategy to generate Nα-acetylated α-syn. Summary PD, as one of the most common progressive neurodegenerative diseases, has put increasing burdens on patients’ families and society. However, because the exact causes of PD remain uncertain, current treatments for PD cannot cure the disease. α-Syn is considered to be the protein most closely related to PD and is an important target in the research field of PD pathology. α-Syn has many kinds of PTMs in soluble proteins, LBs, LNs and cytoplasmic inclusions, which indicates that PTMs may play a critical role in α-syn endogenous functions and PD pathology.116 However, these studies, which mainly relied on regulating the PTM-related enzymes or expressing PTMmimetic α-syn mutants, cannot explore the direct consequences of the modifications on α-syn properties and may sometimes lead to contradictory results due to the resulting heterogeneous mixtures of nonspecifically modified α-syn variants.43,117 Several studies used α-syn mutants, such



Page 12 of 22

as serine to aspartic acid or glutamic acid, to mimic phosphorylated α-syn, but such mutants cannot always exhibit the real properties of native modified proteins.54 The chemical synthesis and semisynthesis of proteins provides a powerful strategy to generate site-specifically modified α-syn, including phosphorylation, ubiquitination, and glycosylation.43 The effects of these modifications on α-syn properties were demonstrated by in vitro and in vivo experiments (Table 1). From these results, we can see that pS129 α-syn, which is the main component of LBs, may play an initial, critical role in accelerating PD pathogenesis by decreasing membrane-binding affinity, promoting α-syn aggregation and inducing cytotoxic strain. Ubiquitination, SUMOylation and OGlcNAcylation of α-syn may play protective roles in PD by inhibiting the aggregation and cytotoxicity of α-syn. The inhibition extent is related to the modification sites and modification forms. Ubiquitination may occur after α-syn aggregates formation, and may induce cell responses to disaggregate the aggregates and promote the clearance of excrescent and aggregated α-syn. The relationship between PD and other PTMs on α-syn (phosphorylation at other sites, nitration and Nαacetylation of α-syn) is still unclear and controversial, needing further study. These results provide valuable and helpful evidence for revealing the role of PTMs in α-syn endogenous functions and PD pathogenesis and suggest the regulation of the related modification enzymes as a promising therapy for PD. Table 1. Table of α-syn with PTMs that have been synthesized, and their effects on the properties of α-syn synthetic modified

modified site

α-syn Phosphorylation

effects

Y125

refs

No difference in membrane binding, aggregation or

55

subsequent phosphorylation at other sites by kinase. Phosphorylation

Y39

Decreases the binding of the helix-2 region to lipid vesicles.

56

Phosphorylation

S129

Decreases membrane binding, promotes aggregation, and

58

induces the formation of a more cytotoxic strain. Mono-

K6 and K12 (isopeptide

Inhibition of aggregation to different degrees: K10 and K23

ubiquitination

linkage); K6, K10, K12,

form fibrils in lower kinetics; K6, K12, and K21 moderately

K21, K23, K32, K34,

inhibit fibril formation; K32, K34, K43, and K96 strongly

K43, and K96 (disulfide

inhibit fibril formation; and K6, K10, K12, K21, and K23

linkage or BTA linkage)

promote proteasome-dependent degradation. Inhibition of

70, 75, 77, 90

the toxicity to SH-SY5Y cells. Di- or tetra-

K12 (isopeptide linkage)

ubiquitination

Inhibition

of

aggregation.

proteasome-dependent

Stronger

degradation

promotion than

of

76

mono-

ubiquitination. SUMOylation

K96 and K102 (disulfide

Inhibition of aggregation. K102 has a more inhibitory effect

linkage or BTA linkage)

than K96. SUMO1 has a higher inhibitory effect than

89, 90

SUMO3. K102 blocks the toxicity to SH-SY5H cells. O-GlcNAcylation

T72

Inhibition of aggregation and cytotoxicity. No effect on

95, 97

membrane binding. Increases subsequent phosphorylation at S87, while inhibits subsequent phosphorylation at S129 by kinases. Inhibition of α-syn proteolysis by calpain. O-GlcNAcylation

S87

Inhibition of aggregation. No effect on membrane binding.


96, 97

Page 13 of 22 1 2 3 4 5 6 7 8 9 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


Inhibition α-syn proteolysis by calpain. Nitration

Y39, Y125

Induces shorter and wider aggregates. The results in a

106

modest reduction in membrane binding. Nα-acetylation

M1

No difference in membrane binding or aggregation.

109

Nα-acetylation

M1

Destabilizes

115

the

oligomerization

state

by

blocking

intermolecular hydrogen bonds

Despite the advances in investigating α-syn bearing PTMs by chemical synthesis and semisynthesis of proteins, there are still some issues that need to be addressed. First, due to the technical challenges, proteins with site-specific PTMs are often produced in small quantities (< 10 mg), which becomes an obstacle for deeper and wider studies of the modified proteins. It is necessary to develop novel methods to prepare modified proteins more rapidly and easily. Second, since the endogenous functions of α-syn and its PTMs are still unclear, further studies are needed to focus on revealing the endogenous roles of α-syn and its PTMs, and the initial causes of α-syn aggregation in PD brains. Additionally, many enzymes that are related to PTMs of α-syn have not been identified yet; thus, the synthetic peptides or proteins and their analogues that carry PTMs may be used as probes to identify the key enzymes by proteomics technologies. Finally, different PTMs have different effects on α-syn properties, including aggregation and toxicity. The cross talk of multiple modifications on α-syn protein has not been tested in depth due to the challenge of preparing multi-modified α-syn. Protein synthesis and semisynthesis chemistry provides an available strategy to generate α-syn containing multiple site-specific modifications. In summary, protein synthesis and semisynthesis chemistry has contributed insights into the role of α-syn modifications in PD and has the power to help us to understand the effects of PTMs on α-syn properties more thoroughly, thereby inspiring us to develop new strategies to treat PD. Author Contributions Y.M.L. conceptualized the review. H.C. wrote the manuscript. H.C., Y.F.Z., Y.X.C. and Y.M.L. critically reviewed and approved the final version of the manuscript. Acknowledgements This work was supported by National Key R&D Program of China (2018YFA0507600) and the National Natural Science Foundation of China (81661148047, 21472109). References (1) Goedert, M.; Spillantini, M. G.; Del Tredici, K.; Braak, H., 100 years of Lewy pathology. Nat. Rev. Neurol. 2013, 9, 13-24. (2) Auluck, P. K.; Caraveo, G.; Lindquist, S., Alpha-synuclein: membrane interactions and toxicity in Parkinson's disease. Annu. Rev. Cell Dev. Biol. 2010, 26, 211-233. (3) Spillantini, M. G.; Schmidt, M. L.; Lee, V. M. Y.; Trojanowski, J. Q.; Jakes, R.; Goedert, M., Alphasynuclein in Lewy bodies. Nature 1997, 388, 839-840. (4) Polymeropoulos, M. H.; Lavedan, C.; Leroy, E.; Ide, S. E.; Dehejia, A.; Dutra, A.; Pike, B.; Root, H.; Rubenstein, J.; Boyer, R.; Stenroos, E. S.; Chandrasekharappa, S.; Athanassiadou, A.; Papapetropoulos, T.; Johnson, W. G.; Lazzarini, A. M.; Duvoisin, R. C.; DiIorio, G.; Golbe, L. I.; Nussbaum, R. L., Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997, 276, 2045-2047.



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