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O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics.

ABSTRACT: 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 them, 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.

SUBMITTER: Gamage K

PROVIDER: S-EPMC11342298 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics.

Gamage Kasun K Wang Binyou B Hard Eldon R ER Van Thong T Galesic Ana A Phillips George R GR Pratt Matthew M Lapidus Lisa J LJ

ACS chemical neuroscience 20240731 16

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 them, 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 c ...[more]

PMID: 39082221

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Project description:Aggregation of neuronal protein α-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of α-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to α-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such α-synuclein sequence extension on α-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of α-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of α-synuclein. To address these questions, we prepared mutants of α-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type α-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of α-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of α-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).

Dataset Information

O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics.

Publications

O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics.

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