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Balana AT, Mahul-Mellier AL, Nguyen BA, Horvath M, Javed A, Hard ER, Jasiqi Y, Singh P, Afrin S, Pedretti R, Singh V, Lee VM, Luk KC, Saelices L, Lashuel HA, Pratt MR. O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology. Nature chemical biology 2024 38347213
Abstract:
Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer's and Parkinson's disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc-modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that posttranslational modifications, such as O-GlcNAc modification, of α-synuclein are key determinants of α-synuclein amyloid strains and pathogenicity.
O-GlcNAc proteins:
SYUA, SYUA
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Miller SA, Jeanne Dit Fouque K, Hard ER, Balana AT, Kaplan D, Voinov VG, Ridgeway ME, Park MA, Anderson GA, Pratt MR, Fernandez-Lima F. Top/Middle-Down Characterization of α-Synuclein Glycoforms. Analytical chemistry 2023 95(49) 38047498
Abstract:
α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.
O-GlcNAc proteins:
SYUA
Species: Homo sapiens
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Balana AT, Levine PM, Craven TW, Mukherjee S, Pedowitz NJ, Moon SP, Takahashi TT, Becker CFW, Baker D, Pratt MR. O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity. Nature chemistry 2021 13(5) 33723378
Abstract:
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 Aβ(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.
O-GlcNAc proteins:
CRYAB, HSPB1
Species: Homo sapiens
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Balana AT, Mukherjee A, Nagpal H, Moon SP, Fierz B, Vasquez KM, Pratt MR. O-GlcNAcylation of High Mobility Group Box 1 (HMGB1) Alters Its DNA Binding and DNA Damage Processing Activities. Journal of the American Chemical Society 2021 143(39) 34546745
Abstract:
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.
O-GlcNAc proteins: