Background O-Fucose is added to cysteine-rich domains called Thrombospondin type 1 repeats (TSRs) by Protein O-fucosyltransferase 2 (POFUT2) and is elongated with glucose by β3-glucosyltransferase (B3GLCT). Mutations in B3GLCT result in Peters Plus Syndrome (PPS), an autosomal recessive disorder characterized by eye and other developmental defects. Although 49 putative targets are known, the function of the disaccharide and its role in PPS remain unexplored. Results Here we show that while POFUT2 is required for secretion of all targets tested, B3GLCT only affects the secretion of a subset, consistent with the observation that B3GLCT mutant phenotypes in PPS patients are less severe than embryonic lethal phenotypes of Pofut2-null mice. O-Glycosylation occurs co-translationally, as TSRs fold. Mass spectral analysis reveals that TSRs from mature, secreted protein are stoichiometrically modified with the disaccharide, while TSRs from protein still folding in the ER are partially modified, suggesting that O-glycosylation marks folded TSRs and promotes ER exit. In vitro unfolding assays demonstrate that fucose and glucose stabilize folded TSRs in an additive manner. In vitro refolding assays under redox conditions showed that POFUT2 recognizes, glycosylates, and stabilizes the folded form of TSRs, resulting in a net acceleration of folding. Conclusions While known ER quality control machinery rely on identifying and tagging unfolded proteins, we find that POFUT2 and B3GLCT mediate a non-canonical ER quality control mechanism that recognizes folded TSRs and stabilizes them by glycosylation. Our findings provide a molecular basis for the defects observed in PPS and potential targets that contribute to the pathology.
Despite significant research, our understanding of the molecular mechanisms of Human Papilloma Virus (HPV) induced cancers remains incomplete. Majority of invasive cervical cancers are caused by high-risk HPV 16 and 18. Two potent HPV oncoproteins, E6 and E7, promote human malignancies by disrupting the activities of key regulators of cell proliferation and apoptosis. Recent investigations have identified hADA3, a transcriptional coactivator protein as a target of high-risk HPV16E6. However, the mechanism of degradation of hADA3 by E6 and its contribution in HPV induced carcinogenesis is poorly understood. Here, we showed that E6-mediated proteolysis of hADA3 is responsible for maintaining low levels of hADA3 in HPV-positive cervical cancer cell lines. We demonstrate that HPV16E6 targets hADA3 for ubiquitin-mediated degradation via E6AP ubiquitin ligase. We also show that hADA3 undergoes accelerated SUMOylation in the presence of HPV16E6. Our data represent the first evidence that hADA3 is posttranslationally modified by SUMOylation, which makes it unstable and establishes a link between SUMOylation and E6-mediated ubiquitination of hADA3. Furthermore, depletion of Ubc9 prevented rapid degradation of hADA3 in E6 expressing cervical cancer cells and overexpression of hADA3 resulted in suppression of proliferation and migration abilities of SiHa cells. Overall, this study underscores the importance of posttranslational modifications in HPV16E6-mediated downregulation of hADA3 thereby unveiling a novel mechanism by which HPV induces oncogenesis.
Fusion tags – amino acid sequences that are genetically coded to be expressed as attached moieties to a protein – have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB). The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli. Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based support through affinity binding, and the immobilized and free Strep-tag II CALB were compared to a commercial CALB. Following modification, the enzyme could be selectively purified from culture media with no observable non-specific binding. The catalytic efficiency of the purified fusion-tagged enzyme was significantly greater than that of the commercial CALB in its free form. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose support yielded a catalytically active enzyme; however, the kcat of the immobilized enzyme was significantly reduced compared to the free tagged enzyme. This work indicates that a C-terminus Strep-tag II fusion tag may be employed to improve the catalytic efficiency of free CALB, but may not be suitable for immobilized applications that employ binding of the enzyme to a Strep-Tactin-modified support.
To all the enlightened and saintly beings, Dhamma, Sangha, my revered Dhamma teachers (Satya Narayan Goenka, Shyam Sunder Taparia and Dr. Neena Lakhani), my Dhamma sister (Sumegha Kapoor Ohri), my parents, grandparents, brother, sister-in-law and niece whose countless blessings and unconditional support provided me an invaluable opportunity to work for my venerable professor Dr.
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