Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.ISSN:2399-364

Human Breast Fibroblasts Inhibit Growth of the MCF10AT Xenograft Model of Proliferative Breast Disease

Stromal fibroblasts are important for normal breast homeostasis and regulation of epithelial growth; however, this regulatory function is altered during carcinogenesis. To study the role of fibroblasts in the development of breast cancer, fibroblasts derived from normal breast (NAFs) were incorporated into the MCF10AT xenograft model of progressive proliferative breast disease. The persistence of human NAFs in xenografts was established by intracellular labeling and tyramide-coupled fluorescent in situ hybridization. Overall, the number of MCF10AT epithelial structures was decreased, and the rate of epithelial cell apoptosis was increased in xenografts containing NAFs. However, these changes were primarily in low-grade epithelial structures, corresponding to normal or mildly hyperplastic ductal epithelium. The level and rate of apoptosis of high-grade epithelial structures, corresponding to in situ and invasive carcinoma, were not consistently altered by NAFs. In addition, there was variability in the growth-inhibitory capacity of NAFs derived from different individuals. NAFs induced changes in the morphology of high-grade MCF10AT structures and in xenograft stroma, including the composition of extracellular matrix, and increased angiogenesis and lymphocytic infiltration. These findings imply that NAFs can inhibit the growth of normal and hyperplastic epi-thelium but are less able to regulate the more transformed epithelial cells that arise during carcino-genesis