Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of α-synuclein post-translational modifications.

The Parkinson's disease associated protein α-synuclein (αS) has been found to contain numerous post-translational modifications (PTMs), in both physiological and pathological states. One PTM site of particular interest is serine 87, which is subject to both O-linked ß-N-acetylglucosamine (gS) modification and phosphorylation (pS), with αS-pS87 enriched in Parkinson's disease. An often-overlooked aspect of these PTMs is their effect on the membrane-binding properties of αS, which are important to its role in regulating neurotransmitter release. Here, we show how one can study these effects by synthesizing αS constructs containing authentic PTMs and labels for single molecule fluorescence correlation spectroscopy measurements. We synthesize αS-gS87 and αS-pS87 by combining native chemical ligation with genetic code expansion approaches. We introduce the fluorophore by a click reaction with a non-canonical amino acid. Beyond the specific problem of PTM effects on αS, our studies highlight the value of this combination of methods for multiply modifying proteins.

α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson's disease.

A compelling link is emerging between the posttranslational modification O-GlcNAc and protein aggregation. A prime example is α-synuclein, which forms toxic aggregates that are associated with neurodegeneration in Parkinson's and related diseases. α-Synuclein has been shown to be O-GlcNAcylated at nine different positions in in vivo proteomics experiments from mouse and human tissues. This raises the possibility that O-GlcNAc may alter the aggregation of this protein and could be both an important biological mediator of neurodegeneration and also a therapeutic target. Here, we expand upon our previous research in this area through the chemical synthesis of six site-specifically O-GlcNAcylated variants of α-synuclein. We then use a variety of biochemical experiments to show that O-GlcNAc in general inhibits the aggregation of α-synuclein but can also alter the structure of α-synuclein aggregates in site-specific ways. Additionally, an α-synuclein protein bearing three O-GlcNAc modifications can inhibit the aggregation of unmodified protein. Primary cell culture experiments also show that several of the O-GlcNAc sites inhibit the toxicity of extracellular α-synuclein fibers that are likely culprits in the spread of Parkinson's disease. We also demonstrate that O-GlcNAcylation can inhibit the aggregation of an aggressive mutant of α-synuclein, indicating that therapies currently in development that increase this modification might be applied in animal models that rely on this mutant. Finally, we also show that the pan-selective antibody for O-GlcNAc does not generally recognize this modification on α-synuclein, potentially explaining why it remains understudied. These results support further development of O-GlcNAcylation tools and therapeutic strategies in neurodegenerative diseases.

In Vivo Experimental and Analytical Studies for Bevacizumab Diffusion Coefficient Measurement in the Rabbit Vitreous Humor.

In order to measure the effective diffusion coefficient D of Bevacizumab (Avastin, Genentech) in the vitreous humor, a new technique is developed based on the "contour method" and in vivo optical coherence tomography measurements. After injection of Bevacizumab-fluorescein conjugated compound solution into the rabbit eye, the contours of drug concentration distribution at the subsurface of injection were tracked over time. The 2D contours were extrapolated to 3D contours using reasonable assumptions and a numerically integrated analytical model was developed for the theoretical contours for the irregularly shaped drug distribution in the experimental result. By floating the diffusion coefficient, different theoretical contours were constructed and the least-squares best fit to the experimental contours was performed at each time point to get the best fit solution. The approach generated consistent diffusion coefficient values based on the experiments on four rabbit eyes over a period of 3 h each, which gave D = 1.2 ± 0.6 × 10 - 6 cm 2 / s , and the corresponding theoretical contours matched well with the experimental contours. The quantitative measurement of concentration using optical coherence tomography and fluorescein labeling gives a new approach for the "noncontact" in vivo drug distribution measurement within vitreous.

Ubiquitination Can Change the Structure of the α-Synuclein Amyloid Fiber in a Site Selective Fashion.

Toxic amyloid aggregates are a feature of many neurodegenerative diseases. A number of biochemical and structural studies have demonstrated that not all amyloids of a given protein are equivalent but rather that an aggregating protein can form different amyloid structures or polymorphisms. Different polymorphisms can also induce different amounts of pathology and toxicity in cells and in mice, suggesting that the structural differences may play important roles in disease. However, the features that cause the formation of polymorphisms in vivo are still being uncovered. Posttranslational modifications on several amyloid forming proteins, including the Parkinson's disease causing protein α-synuclein, may be one such cause. Here, we explore whether ubiquitination can induce structural changes in α-synuclein aggregates in vitro. We used protein chemistry to first synthesize ubiquitinated analogues at three different positions using disulfide linkages. After aggregation, these linkages can be reversed, allowing us to make relative comparisons between the structures using a proteinase K assay. We find that, while ubiquitination at residue 6, 23, or 96 inhibits α-synuclein aggregation, only modification at residue 96 causes an alteration in the aggregate structure, providing further evidence that posttranslational modifications may be an important feature in amyloid polymorphism formation.

O-GlcNAc modification inhibits the calpain-mediated cleavage of α-synuclein.

The major protein associated with Parkinson's disease (PD) is α-synuclein, as it can form toxic amyloid-aggregates that are a hallmark of many neurodegenerative diseases. α-Synuclein is a substrate for several different posttranslational modifications (PTMs) that have the potential to affect its biological functions and/or aggregation. However, the biophysical effects of many of these modifications remain to be established. One such modification is the addition of the monosaccharide N-acetyl-glucosamine, O-GlcNAc, which has been found on several α-synuclein serine and threonine residues in vivo. We have previously used synthetic protein chemistry to generate α-synuclein bearing two of these physiologically relevant O-GlcNAcylation events at threonine 72 and serine 87 and demonstrated that both of these modifications inhibit α-synuclein aggregation. Here, we use the same synthetic protein methodology to demonstrate that these same O-GlcNAc modifications also inhibit the cleavage of α-synuclein by the protease calpain. This further supports a role for O-GlcNAcylation in the modulation of α-synuclein biology, as proteolysis has been shown to potentially affect both protein aggregation and degradation.

Post-translational Modification of α-Synuclein Modifies Monomer Dynamics and Aggregation Kinetics.

The intrinsically disordered protein α-Synuclein is identified as a major toxic aggregate in Parkinson's as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among these, O-GlcNAcylation of α-Synuclein has been observed to inhibit the aggregation propensity of the protein. Here we investigate the monomer dynamics of two O-GlcNAcylated α-Synucleins, α-Syn(gT72) and α-Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.

Comparison of N-Acetyl-Glucosamine to Other Monosaccharides Reveals Structural Differences for the Inhibition of α-Synuclein Aggregation.

O-GlcNAc modification of the microtubule associated protein tau and α-synuclein can directly inhibit the formation of the associated amyloid fibers associated with major classes of neurodegenerative diseases. However, the mechanism(s) by which this posttranslational modification (PTM) inhibit amyloid aggregation are still murky. One hypothesis is that O-GlcNAc simply acts as a polyhydroxylated steric impediment to the formation of amyloid oligomers and fibers. Here, we begin to test this hypothesis by comparing the effects of O-GlcNAc to other similar monosaccharides-glucose, N-acetyl-galactosamine (GalNAc), or mannose-on α-synuclein amyloid formation. Interestingly, we find that this quite reasonable hypothesis is not entirely correct. More specifically, we used four types of biochemical and biophysical assays to discover that the different sugars display different effects on the inhibition of amyloid formation, despite only small differences between the structures of the monosaccharides. These results further support a more detailed investigation into the mechanism of amyloid inhibition by O-GlcNAc and has potential implications for the evolution of N-acetyl-glucosamine as the monosaccharide of choice for widespread intracellular glycosylation.

Investigating the Effects of O-GlcNAc Modifications in Parkinson's Disease Using Semisynthetic α-Synuclein.

α-Synuclein is a small aggregation-prone protein associated with Parkinson's disease (PD). The protein's biochemical and biophysical properties can be heavily influenced by various types of posttranslational modification (PTMs) such as phosphorylation, ubiquitination, and glycosylation. To understand the site-specific effects of various PTMs have on the protein and its aggregation, obtaining a homogeneous sample of the protein of interest with the specific modification of interest is key. Expressed protein ligation (EPL) has emerged as robust tool for building synthetic proteins bearing site-specific modifications. Here, we outline our approach for building α-synuclein with site specific O-GlcNAc modifications, an intracellular subtype of glycosylation that has been linked to the inhibition of protein aggregation. More specifically, we provide specific protocols for the synthesis of α-synuclein bearing an O-GlcNAc modification at threonine 72, termed α-synuclein(gT72). However, this general approach utilizing two recombinant fragments and one synthetic peptide is applicable to other sites and types of modifications and should be transferable to various other protein targets, including aggregation prone proteins like tau and TDP-43.

O-GlcNAcylation of α-Synuclein at Serine 87 Reduces Aggregation without Affecting Membrane Binding.

The aggregation of neurodegenerative-disease associated proteins can be affected by many factors, including a variety of post-translational modifications. One such modification, O-GlcNAcylation, has been found on some of these aggregation prone proteins, including α-synuclein, the major protein that plays a causative role in synucleinopathies like Parkinson's disease. We previously used synthetic protein chemistry to prepare α-synuclein bearing a homogeneous O-GlcNAc modification at threonine 72 and showed that this modification inhibits protein aggregation. However, the effects of the other eight O-GlcNAcylation sites that have been identified were unknown. Here, we use a similar synthetic strategy to investigate the consequences of this modification at one of these sites, serine 87. We show that O-GlcNAcylation at this site also inhibits α-synuclein aggregation but to a lesser extent than that for the same modification at threonine 72. However, we also find that this modification does not affect the membrane-binding properties of α-synuclein, which differentiates it from phosphorylation at the same site. These results further support the development of therapies that can elevate O-GlcNAcylation of α-synuclein to slow the progression of Parkinson's disease.

Synthesis of a Bis-thio-acetone (BTA) Analogue of the Lysine Isopeptide Bond and its Application to Investigate the Effects of Ubiquitination and SUMOylation on α-Synuclein Aggregation and Toxicity.

The reversible modification of protein by the small protein ubiquitin and other ubiquitin-like modifiers plays important roles in virtually every key biological process in eukaryotic cells. The establishment of a range of chemical methods for the preparation of ubiquitinated proteins has enabled the site-specific interrogation of the consequences of these modifications. However, many of these techniques require significant levels of synthetic expertise, somewhat limiting their widespread application by the biological community. To overcome this issue, the creation of structural analogues of the ubiquitin-protein linkage that can be readily prepared with commercially available reagents and buffers is an important goal. Here we present the development of conditions for the facile synthesis of bis-thio-acetone (BTA) linkages of ubiquitinated proteins in high yields. Additionally, we apply this technique to the preparation of the aggregation prone protein α-synuclein bearing either ubiquitin or the small ubiquitin-like modifier (SUMO). With these proteins, we demonstrate that the BTA linkage recapitulates the previously published effects of either of these proteins on α-synuclein, suggesting that it is a good structural mimic. Notably, the BTA linkage is chemically and enzymatically stable, enabling us to study the consequences of site-specific ubiquitination and SUMOylation on the toxicity of α-synuclein in cell culture, which revealed modification and site-specific differences.