The dynamic modification of nuclear and cytoplasmic proteins by the monosaccharide N-acetyl-glucosamine (GlcNAc) continues to emerge as an important regulator of many biological processes. Herein we describe the development of an alkynyl-modified GlcNAc analog (GlcNAlk) as a new chemical reporter of O-GlcNAc modification in living cells. This strategy is based on metabolic incorporation of reactive functionality into the GlcNAc biosynthetic pathway. When combined with the Cu(I)-catalyzed [3 þ 2] azidealkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent visualization of O-GlcNAc and affinity enrichment and identification of O-GlcNAc-modified proteins. Using ingel fluorescence detection, we characterized the metabolic fates of GlcNAlk and the previously reported azido analog, GlcNAz. We confirmed previous results that GlcNAz can be metabolically interconverted to GalNAz, whereas GlcNAlk does not, thereby yielding a more specific metabolic reporter of O-GlcNAc modification. We also used GlcNAlk, in combination with a biotin affinity tag, to identify 374 proteins, 279 of which were not previously reported, and we subsequently confirmed the enrichment of three previously uncharacterized proteins. Finally we confirmed the O-GlcNAc modification of the ubiquitin ligase NEDD4-1, the first reported glycosylation of this protein.click chemistry | bioorthogonal labeling | proteomics
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, whilst 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.
Metabolic chemical reporters (MCRs)
of glycosylation are analogues
of monosaccharides that contain bioorthogonal functionalities and
enable the direct visualization and identification of glycoproteins
from living cells. Each MCR was initially thought to report on specific
types of glycosylation. We and others have demonstrated that several
MCRs are metabolically transformed and enter multiple glycosylation
pathways. Therefore, the development of selective MCRs remains a key
unmet goal. We demonstrate here that 6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics
with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz),
and N-azidoacetyl-galactosamine (GalNAz) revealed
that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz
and GalNAz are incorporated into a combination of intracellular and
extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be
biosynthetically transformed into the corresponding UDP sugar-donor
by the canonical salvage-pathway that requires phosphorylation at
the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass
this roadblock through direct phosphorylation of its 1-hydroxyl by
the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together,
6AzGlcNAc enables the specific analysis of O-GlcNAcylated
proteins, and these results suggest that specific MCRs for other types
of glycosylation can be developed. Additionally, our data demonstrate
that cells are equipped with a somewhat unappreciated metabolic flexibility
with important implications for the biosynthesis of natural and unnatural
carbohydrates.
Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear, but may involve the covalent modification of proteins or DMF serving as a pro-drug that is converted to monomethyl fumarate (MMF). Here, we found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF-sensitivity of > 2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase C θ (PKCθ). Furthermore, DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology.
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