Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

The first hydrophobic region of the HPV16 E5 protein determines protein cellular location and facilitates anchorage-independent growth.

The human papillomavirus type 16 E5 protein (HPV16 E5) is 83 amino acids in length and contains three well-defined hydrophobic regions. The protein is expressed at very limited amounts in transfected cells and the absence of specific antibodies has strongly hampered functional analyses. To investigate the relationship between structure and function we have synthesized a codon-adapted version of the gene (hE5) and prepared a series of N-terminal and C-terminal deletions. Immunofluorescence analyses show colocaliation of the protein with calnexin, an ER marker, EEA-1, an early endosomes marker, and Lamp-2, a lysosomal marker. No major colocalization was found between hE5 and the Golgi marker 58 K. Whereas deletions at the C-terminal end of the protein do not greatly alter the localisation pattern, deletion of the first hydrophobic region results in loss of colocalisation with the ER, early endosomes and lysosomes. Further, we show that while the complete E5 protein confers to HaCaT cells the property to grow in an anchorage-independent manner, deletion of the first hydrophobic region results in loss of growth in soft agar. We conclude that the first hydrophobic region of the E5 protein largely determines the biological properties of the viral protein.