Significance
Epidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.
O-linked N-acetylglucosamine
(O-GlcNAc) is a prevalent
protein modification that plays fundamental roles in both cell physiology
and pathology. O-GlcNAc is catalyzed solely by O-GlcNAc transferase
(OGT). The study of protein O-GlcNAc function is limited by the lack
of tools to control OGT activity with spatiotemporal resolution in
cells. Here, we report light control of OGT activity in cells by replacing
a catalytically essential lysine residue with a genetically encoded
photocaged lysine. This enables the expression of a transiently inactivated
form of OGT, which can be rapidly reactivated by photo-decaging. We
demonstrate the activation of OGT activity by monitoring the time-dependent
increase of cellular O-GlcNAc and profile glycoproteins using mass-spectrometry-based
quantitative proteomics. We further apply this activation strategy
to control the morphological contraction of fibroblasts. Furthermore,
we achieved spatial activation of OGT activity predominantly in the
cytosol. Thus, our approach provides a valuable chemical tool to control
cellular O-GlcNAc with much needed spatiotemporal precision, which
aids in a better understanding of O-GlcNAc function.
N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, and it includes both protease digestion and peptide-N4–(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) F deglycosylation. Prolonged incubation time is generally required because of the limited digestion efficiency of the conventional in-solution digestion method. Here, we propose novel thermoresponsive magnetic fluid (TMF)-immobilized enzymes (trypsin or PNGase F) for ultrafast and highly efficient proteome digestion and deglycosylation. Unlike other magnetic material-immobilized enzymes, TMF-immobilized enzymes display a unique temperature-triggered magnetic response behavior. At room temperature, a TMF-immobilized enzyme completely dissolves in an aqueous solution and forms a homogeneous system with a protein/peptide sample for efficient digestion but cannot be separated by magnetic force because of its excellent water dispersity. Above its lower critical solution temperature (LCST), thermoflocculation of a TMF-immobilized enzyme allows it to be easily recovered by increasing the temperature and magnetic force. Taking advantage of the unique homogeneous reaction of a TMF-immobilized enzyme, both protein digestion and glycopeptide deglycosylation can be finished within 3 min, and the whole sample processing time can be reduced by more than 20 times. The application of a TMF-immobilized enzyme in large-scale profiling of protein N-glycosylation in urine samples led to the successful identification of 2,197 N-glycopeptides and further demonstrated the potential of this strategy for fast and high-throughput analysis of N-glycoproteome in clinical samples.
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