O-GlcNAc Modification Alters the Chaperone Activity of HSP27 Charcot-Marie-Tooth Type 2 (CMT2) Variants in a Mutation-Selective Fashion.

Increased O-GlcNAc is a common feature of cellular stress, and the upregulation of this dynamic modification is associated with improved survival under these conditions. Likewise, the heat shock proteins are also increased under stress and prevent protein misfolding and aggregation. We previously linked these two phenomena by demonstrating that O-GlcNAc directly increases the chaperone of certain small heat shock proteins, including HSP27. Here, we examine this linkage further by exploring the potential function of O-GlcNAc on mutants of HSP27 that cause a heritable neuropathy called Charcot-Marie-Tooth type 2 (CMT2) disease. Using synthetic protein chemistry, we prepared five of these mutants bearing an O-GlcNAc at the major site of modification. Upon subsequent biochemical analysis of these proteins, we found that O-GlcNAc has different effects, depending on the location of the individual mutants. We believe that this has important implications for O-GlcNAc and other PTMs in the context of polymorphisms or diseases with high levels of protein mutation.

HSP27 Inhibitory Activity against Caspase-3 Cleavage and Activation by Caspase-9 Is Enhanced by Chaperone O-GlcNAc Modification in Vitro.

One of the O-GlcNAc modifications is the protection of cells against a variety of stressors that result in cell death. Previous experiments have focused on the overall ability of O-GlcNAc to prevent protein aggregation under stress as well as its ability to affect stress-response signaling pathways. Less attention has been paid to the potential role for O-GlcNAc in the direct inhibition of a major cell-death pathway, apoptosis. Apoptosis involves the sequential activation of caspase proteases, including the transfer of cell-stress information from initiator caspase-9 to effector caspase-3. Cells have multiple mechanisms to slow the apoptotic cascade, including heat shock protein HSP27, which can directly inhibit the activation of caspase-3 by caspase-9. We have previously shown that O-GlcNAc modification increases the chaperone activity of HSP27 against amyloid aggregation, raising the question as to whether this modification may play important roles in other facets of HSP27 biology. Here, we use protein chemistry to generate different versions of O-GlcNAc modified HSP27 and demonstrate that the modification enhances this antiapoptotic function of the chaperone, at least in an in vitro context. These results provide additional molecular insight into how O-GlcNAc functions as a mediator of cellular stress with important implications for human diseases like cancer and neurodegeneration.

Synthesis of O-GlcNAcylated small heat shock proteins.

A protein's structure and function often depend not only on its primary sequence, but also the presence or absence of any number of non-coded posttranslational modifications. Complicating their study is the fact that the physiological consequences of these modifications are context-, protein-, and site-dependent, and there exist no purely biological techniques to unambiguously study their effects. To this end, protein semisynthesis has become an invaluable chemical biology tool to specifically install non-coded or non-native moieties onto proteins in vitro using synthetic and/or recombinant polypeptides. Here, we describe two facets of protein semisynthesis (solid-phase peptide synthesis and expressed protein ligation) and their use in generating site-specifically glycosylated small heat shock proteins for functional studies. The procedures herein require limited specialized equipment, employ mild reaction conditions, and can be extended to myriad other proteins, modifications, and contexts.

Methods for Studying Site-Specific O-GlcNAc Modifications: Successes, Limitations, and Important Future Goals.

O-GlcNAcylation is a dynamic post-translational modification which affects myriad proteins, cellular functions, and disease states. Its presence or absence modulates protein function via differential protein- and site-specific mechanisms, necessitating innovative techniques to probe the modification in highly selective manners. To this end, a variety of biological and chemical methods have been developed to study specific O-GlcNAc modification events both in vitro and in vivo, each with their own respective strengths and shortcomings. Together, they comprise a potent chemical biology toolbox for the analysis of O-GlcNAcylation (and, in theory, other post-translational modifications) while highlighting the need and space for more facile, generalizable, and biologically authentic techniques.

O-GlcNAcylation of High Mobility Group Box 1 (HMGB1) Alters Its DNA Binding and DNA Damage Processing Activities.

Protein O-GlcNAcylation is an essential and dynamic regulator of myriad cellular processes, including DNA replication and repair. Proteomic studies have identified the multifunctional nuclear protein HMGB1 as O-GlcNAcylated, providing a potential link between this modification and DNA damage responses. Here, we verify the protein's endogenous modification at S100 and S107 and found that the major modification site is S100, a residue that can potentially influence HMGB1-DNA interactions. Using synthetic protein chemistry, we generated site-specifically O-GlcNAc-modified HMGB1 at S100 and characterized biochemically the effect of the sugar modification on its DNA binding activity. We found that O-GlcNAc alters HMGB1 binding to linear, nucleosomal, supercoiled, cruciform, and interstrand cross-linked damaged DNA, generally resulting in enhanced oligomerization on these DNA structures. Using cell-free extracts, we also found that O-GlcNAc reduces the ability of HMGB1 to facilitate DNA repair, resulting in error-prone processing of damaged DNA. Our results expand our understanding of the molecular consequences of O-GlcNAc and how it affects protein-DNA interfaces. Importantly, our work may also support a link between upregulated O-GlcNAc levels and increased rates of mutations in certain cancer states.

Consequences of post-translational modifications on amyloid proteins as revealed by protein semisynthesis.

Alterations to the global levels of certain types of post-translational modifications (PTMs) are commonly observed in neurodegenerative diseases. The net influence of these PTM changes to the progression of these diseases can be deduced from cellular and animal studies. However, at the molecular level, how one PTM influences a given protein is not uniform and cannot be easily generalized from systemic observations, thus requiring protein-specific interrogations. Given that protein aggregation is a shared pathological hallmark in neurodegeneration, it is important to understand how these PTMs affect the behavior of amyloid-forming proteins. For this purpose, protein semisynthesis techniques, largely via native chemical and expressed protein ligation, have been widely used. These approaches have thus far led to our increased understanding of the site-specific consequences of certain PTMs to amyloidogenic proteins' endogenous function, their propensity for aggregation, and the structural variations these PTMs induce toward the aggregates formed.

O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity.

A major role for the intracellular post-translational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area focused on O-GlcNAc modification of the amyloid-forming proteins themselves. Here we used synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we found that O-GlcNAc increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aß(1-42). Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Finally, we found that, although O-GlcNAc levels are globally reduced in Alzheimer's disease brains, the modification of relevant sHSPs is either maintained or increased, which suggests a mechanism to maintain these potentially protective O-GlcNAc modifications. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.

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.

O-GlcNAcylated peptides and proteins for structural and functional studies.

O-GlcNAcylation is an enzymatic post-translational modification occurring in hundreds of protein substrates. This modification occurs through the addition of the monosaccharide N-acetylglucosamine to serine and threonine residues on intracellular proteins in the cytosol, nucleus, and mitochondria. As a highly dynamic form of modification, changes in O-GlcNAc levels coincide with alterations in metabolic state, the presence of stressors, and cellular health. At the protein level, the consequences of the sugar modification can vary, thus necessitating biochemical investigations on protein-specific and site-specific effects. To this end, enzymatic and chemical methods to 'encode' the modification have been developed and the utilization of these synthetic glycopeptides and glycoproteins has since been instrumental in the discovery of the mechanisms by which O-GlcNAcylation can affect a diverse array of biological processes.

Traceless native chemical ligation of lipid-modified peptide surfactants by mixed micelle formation.

Biology utilizes multiple strategies, including sequestration in lipid vesicles, to raise the rate and specificity of chemical reactions through increases in effective molarity of reactants. We show that micelle-assisted reaction can facilitate native chemical ligations (NCLs) between a peptide-thioester - in which the thioester leaving group contains a lipid-like alkyl chain - and a Cys-peptide modified by a lipid-like moiety. Hydrophobic lipid modification of each peptide segment promotes the formation of mixed micelles, bringing the reacting peptides into close proximity and increasing the reaction rate. The approach enables the rapid synthesis of polypeptides using low concentrations of reactants without the need for thiol catalysts. After NCL, the lipid moiety is removed to yield an unmodified ligation product. This micelle-based methodology facilitates the generation of natural peptides, like Magainin 2, and the derivatization of the protein Ubiquitin. Formation of mixed micelles from lipid-modified reactants shows promise for accelerating chemical reactions in a traceless manner.

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.