BPB Reports

2019 - Vol. 2 No. 5

2019 - Vol. 2

Regular Article
A PRNP-Disrupted Human Neuroblastoma Cell Line and Its Stable Transformants Expressing Prion Protein Variants Vol.2, No.5, p.73-79
Yuko Okemoto-Nakamura , Isei Tanida , Toshiyuki Yamaji , Kentaro Hanada , Ken’ichi Hagiwara
Received: July 25, 2019
Accepted: September 11, 2019
Released: October 16, 2019
Abstract Full Text PDF[4M]

A key event in prion diseases is the conformational conversion of a cellular normal form of the prion protein (PrPC) to disease-associated conformers (PrPSc). Polymorphisms or mutations in the human prion protein gene (PRNP) are associated with acquired or hereditary human prion diseases. When human prion protein (PrP) variants are characterized by cDNA expression systems in cultured cells, the endogenous prion protein may affect the behavior of ectopically expressed prion variants. To eliminate this issue, we herein created a PRNP-disrupted mutant cell line from human neuroblastoma BE(2)-M17 cells. No morphological differences were observed between PRNP-null and parental cells. We stably introduced cDNAs encoding E196K-PrP and E200K-PrP (both of which are Creutzfeldt-Jakob disease (CJD)-related mutants), a non-pathogenic E219K-PrP, and wild-type control PrP into PRNP-null mutant cells using the PiggyBac system, and found that the glycosylation pattern of E196K-PrP differed from that of other PrPs. We also found that non-glycosylated PrPs for E196K- and E200K-PrPs had distinct electrophoretic mobilities from that of WT-PrP, and E219K-PrPs exhibited slightly different mobilities in polyacrylamide gel electrophoresis under endogenous PrP-null conditions, demonstrating that the PRNP-disrupted human neuroblastoma cell line serves as a useful tool for examining PrPs with mutations or polymorphisms.

Regular Article
Targeting of Wild-Type and Mutated Forms of Lysosome-Associated Membrane Protein-1 (LAMP-1) to Late Endosomes/Lysosomes Depends on Affinities of Their Cytoplasmic Tail Peptides with a Medium Subunit of Adaptor Protein Complex-3 (AP-3) Vol.2, No.5, p.67-72
Kenji Akasaki , Hiroshi Sakane , Yusuke Ohdoi , Kenta Okamoto , Kazutaka Nushida , Toshiyuki Hata
Received: September 02, 2019
Accepted: September 24, 2019
Released: October 11, 2019
Abstract Full Text PDF[3M]

Lysosome-associated membrane protein-1 (LAMP-1) is a type I membrane glycoprotein with a COOH-terminal cytoplasmic tail (CT) containing a lysosome-targeting signal of GYQTI382-COOH. This sequence is categorized as a tyrosine-based motif of GYXXΦ where Φ is a bulky hydrophobic amino acid residue. Lysosomal localization of LAMP-1 varies by changing the COOH-terminal amino acid residues. Adaptor protein (AP) complexes are reported to recognize the tyrosine-based signal peptide for efficient lysosomal transport of LAMP-1. In order to better understand the role of APs in lysosomal transport of LAMP-1, we have studied interactions of wild-type (WT) and mutated CTs of LAMP-1 with medium (μ) subunits of the four APs by a yeast two-hybrid (Y2H) system and subsequent computer-based molecular modeling. Among the μ subunits of AP-1, AP-2, AP-3 and AP-4, called μ1, μ2, μ3A, and μ4, respectively, the WT-CT significantly interacted with μ3Α in the Y2H system. The degree of interaction of the WT and mutated CTs with μ3A from the Y2H analyses correlated with that of their dissociation constants determined by computer-based molecular modeling, and also with that of the late endosomal and lysosomal amount of WT and the similar mutants of LAMP-1. These results reinforce the notion that AP-3 makes a major contribution to the vesicular transport of LAMP-1 to late endosomes and lysosomes via a direct intracellular route.

Regular Article
Advantage of a Co-expression System for Estimating Physiological Effects of Functional Interaction Between Cytochrome P450 3A4 and Uridine 5’-Diphospho-Glucuronosyltransferase 2B7 Vol.2, No.5, p.61-66
Yuu Miyauchi , Hideyuki Yamada , Yuji Ishii
Received: July 19, 2019
Accepted: September 15, 2019
Released: October 10, 2019
Abstract Full Text PDF[1M]

Cytochrome P450 (CYP, P450) and uridine 5’-diphospho-glucuronosyltransferase (UGT) play crucial roles in drug metabolism phase I and II, respectively. Our previous studies suggest that there are functional interactions between P450 and UGT. We previously established a co-expression system featuring CYP3A4 and UGT2B7 using baculovirus-infected insect cells. Commercial microsomes are available that individually express CYP3A4/NADPH P450 reductase (CPR) or UGT2B7. It would be much easier if we could evaluate the functional interaction of CYP3A4 and UGT2B7 by simply mixing the microsomes. To address this issue, we presently compared our established co-expression system with a simple microsome mixing system. Co-expressed UGT2B7 suppressed CYP3A4 activity. On the contrary, adding UGT2B7 microsomes to CYP3A4/CPR microsomes significantly enhanced CYP3A4 activity. The enhancement was systematic and strongly dependent on UGT2B7 microsomes, and was abrogated by detergent treatment. The collective results suggested that the enhancement of CYP3A4 activity resulted from a non-physiological interaction between CYP3A4 and UGT2B7, which were both expressed on different membranes. The phenomenon was distinguishable and hardly ever reflected the physiological interaction. This pitfall can be avoided by not using simple mixing procedures. In selecting experimental materials and methods depending on the subject of the study, a co-expression system should be applied in the analysis of functional P450-UGT interaction.