Using a ubiquitin ligase as an unfolded protein sensor.
A significant fraction of all proteins are misfolded and must be degraded. The ubiquitin-proteasome pathway provides an essential protein quality control function necessary for normal cellular homeostasis. Substrate specificity is mediated by proteins called ubiquitin ligases. In the endoplasmic reticulum (ER) a specialized pathway, the endoplasmic reticulum associated degradation (ERAD) pathway provides means to eliminate misfolded proteins from the ER. One marker used by the ER to identify misfolded glycoproteins is the presence of a high-mannose (Man5-8GlcNAc2) glycan. Recently, FBXO2 was shown to bind high mannose glycans and participate in ERAD. Using glycan arrays, immobilized glycoprotein pulldowns, and glycan competition assays we demonstrate that FBXO2 preferentially binds unfolded glycoproteins. Using recombinant, bacterially expressed GST-FBXO2 as an unfolded protein sensor we demonstrate it can be used to monitor increases in misfolded glycoproteins after physiological or pharmaceutical stressors.
Kansas City University of Medicine and Biosciences, Kansas City, MO 64106, USA.
Adam
A
Mallinger
Hsiang M
HM
Wen
Geoffrey M
GM
Dankle
Kevin A
KA
Glenn
D000818:Animals; D015374:Biosensing Techniques; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D044783:F-Box Proteins / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D058767:Protein Unfolding; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013312:Stress, Physiological; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
D018797:Cell Cycle Proteins; D044783:F-Box Proteins; C485508:FBXO2 protein, human; D006023:Glycoproteins; D009419:Nerve Tissue Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; C058642:mannosyl(5)-N-acetyl(2)-glucose; D044767:Ubiquitin-Protein Ligases