MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD 0 ), which is connected to a P-glycoprotein-like core region (⌬MRP) by a cytoplasmic linker domain zero (L 0 ).It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD 0 and L 0 are not known. We synthesized [125 I]11-azidophenyl agosterol A ([ 125 I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C 932-1531) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C 4 . Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L 0 is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L 0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.Following exposure to a natural product chemotherapeutic agent, tumor cells often acquire resistance to several structurally and functionally unrelated drugs, so-called multidrug resistance (MDR).1 MDR is the major obstacle to successful cancer chemotherapy. Two membrane proteins, P-glycoprotein (Pgp) and the human multidrug resistance protein (MRP1) are frequently overexpressed in MDR cells (for reviews see Refs. 1-4). Although both MRP1 (190 kDa) and P-gp (170 kDa) are members of the family of ATP-binding cassette transporters (5), they share only 15% amino acid sequence identity (6). The amino acid sequence suggests that P-gp consists of two homologous halves and a variable linker region. Each half of the protein has six transmembrane segments and one nucleotide binding domain (NBD). MRP1 differs from P-gp by the presence of an extra N-terminal extension with five transmembrane segments (TMD 0 ), which is connected to the P-gp-like core (⌬MRP) by a cytoplasmic linker domain zero (L 0 ) (7, 8).The precise roles of TMD 0 and L 0 are unknown. Although MRP1 and P-gp both confer multidrug resistance by actively effluxing drugs from the cells (9, 10), there is compelling evidence that they function very differently in drug transport (6, 11). P-gp confers multidrug resistance by directly binding and transporting unmodified drugs (12, 13). MRP1, however, is an active transporter of amphiphilic conjugated organic anions, including a number of compounds conjugated with GSH, glucuronide, and sulfate. To date, leukotriene C 4 (LTC 4 ) is the best substrate for MRP1 (14). It has been reported that GSH plays a critical role in MRP1-mediated MDR (15). Reduction of GSH level in MRP1-expressing cells by buthionine su...

Therapeutic payload delivery to the central nervous system (CNS) remains a major challenge in gene therapy. Recent studies using function-driven evolution of adeno-associated virus (AAV) vectors have successfully identified engineered capsids with improved blood-brain barrier (BBB) penetration and CNS tropism in mouse. However, these strategies require transgenic animals and thus are limited to rodents. To address this issue, we developed a directed evolution approach based on recovery of capsid library RNA transcribed from CNS-restricted promoters. This RNA-driven screen platform, termed TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), was tested in the mouse with AAV9 peptide display libraries and showed rapid emergence of dominant sequences. Ten individual variants were characterized and showed up to 400-fold higher brain transduction over AAV9 following systemic administration. Our results demonstrate that the TRACER platform allows rapid selection of AAV capsids with robust BBB penetration and CNS tropism in non-transgenic animals.

Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing.

A systematic study on the structure and function of Glucose-6-phosphate dehydrogenase (G6PD) variations was carried out in China. A total of 155,879 participants were screened for G6PD deficiency by the G6PD/6PGD ratio method and 6,683 cases have been found. The prevalence of G6PD deficiency ranged from 0 to 17.4%. With informed consent, 1,004 cases from 11 ethnic-based groups were subjected to molecular analysis. Our results showed the followings: (1) The G6PD variants are consistent across traditional ethnic boundaries, but vary in frequencies across ethnic-based groups in Chinese population, (2) The G6PD variants in Chinese population are different from those in African, European, and Indian populations, (3) A novel G6PD-deficiency mutation, 274C-->T, has been found, and (4) Denaturing high performance liquid chromatography is of great advantage to detecting G6PD-deficient mutations for diagnosis and genetic counseling. Moreover, functional analysis of the human G6PD variants showed the following: (1) The charge property, polarity, pK-radical and side-chain radical of the substituting amino acid have an effect on G6PD activity, (2) The G6PDArg459 and Arg463 play important roles in anchoring NADP+ to the catalytic domain to maintain the enzymatic activity, and (3) The sequence from codon 459 to the carboxyl terminal is essential for the enzymatic function.

The structural determinants of nicotinic acetylcholine receptor (AChR) trafficking have yet to be fully elucidated. Hydrophobic residues occur within short motifs important for endoplasmic reticulum (ER) export or endocytotic trafficking. Hence, we tested whether highly conserved hydrophobic residues, primarily leucines, in the cytoplasmic domain of the ␣4␤2 AChR subunits were required for cell surface expression of ␣4␤2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the ␣4 AChR subunit attenuates cell surface expression of mutant ␣4␤2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the ␤2 AChR subunit abolishes cell surface expression of mutant ␣4␤2 AChRs. The hydrophobic nature of the leucine residue is a primary determinant of its function because mutation of L343 to another hydrophobic amino acid, phenylalanine, in the ␤2 AChR subunit only poorly inhibits trafficking of mutant ␣4␤2 AChR to the cell surface. All mutant ␣4␤2 AChRs exhibit high-affinity binding for [ 3 H]epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type ␣4␤2 AChRs colocalize with the Golgi marker giantin, whereas mutant ␣4␤2 AChRs fail to do so. The striking difference between mutant ␣4 versus mutant ␤2 AChR subunits on cell surface expression of mutant ␣4␤2 AChRs points to a cooperative or regulatory role for the ␣4 AChR subunit and an obligatory role for the ␤2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of ␣4␤2 AChR ER export.