11 research outputs found
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O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.
Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation
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
Recommended from our members
O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.
Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation