Differential functional rescue of Lys513 and Lys516 processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain–domain interactions of the transporter

AbstractMultidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys513 and Lys516 in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km. In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km. Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys513 and Lys516 participate in different interdomain interactions crucial for the proper folding and assembly of MRP1

Nuclear interactions of topoisomerase II α and β with phospholipid scramblase 1

DNA topoisomerase (topo) II modulates DNA topology and is essential for cell division. There are two isoforms of topo II (α and β) that have limited functional redundancy, although their catalytic mechanisms appear the same. Using their COOH-terminal domains (CTDs) in yeast two-hybrid analysis, we have identified phospholipid scramblase 1 (PLSCR1) as a binding partner of both topo II α and β. Although predominantly a plasma membrane protein involved in phosphatidylserine externalization, PLSCR1 can also be imported into the nucleus where it may have a tumour suppressor function. The interactions of PLSCR1 and topo II were confirmed by pull-down assays with topo II α and β CTD fusion proteins and endogenous PLSCR1, and by co-immunoprecipitation of endogenous PLSCR1 and topo II α and β from HeLa cell nuclear extracts. PLSCR1 also increased the decatenation activity of human topo IIα. A conserved basic sequence in the CTD of topo IIα was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIα amino acids 1430-1441. These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Fabrication of psoralen-loaded lipid-polymer hybrid nanoparticles and their reversal effect on drug resistance of cancer cells

In the present study, a lipid-polymer hybrid drug carrier system was developed to encapsulate psoralen (PSO), a multidrug resistance reversal agent and traditional Chinese medicine. Emphasis was focused the parameters that influence physicochemical characteristics, and then the drug release profile, stability, cytotoxicity and drug resistance reversal effect of the lipid-polymer hybrid nanoparticles (LPNs) were investigated. It was found that various formulation parameters affected NP size, drug loading (DL) and release characteristics. Hydrophilic 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)2000 increased the ζ potential and thus the stability of the NPs, but also enlarged their diameter. The amount of PSO influenced their DL and encapsulation efficiency, but did not show any effect on drug release kinetics. Next, the stability of the LPNs in different media and their storage characteristics were assessed. Finally, the cytotoxicity and multidrug resistance reversal effect was studied in the K562 and HepG2 cell lines. The analysis of half maximal inhibitory concentration values demonstrated that combination therapy with doxorubicin (DOX) and PSO-loaded LPNs (P-LPNs) was 14- and 23-fold more effective than a single-dose DOX treatment in resistant K562 and HepG2 cells, respectively, and 2.2- and 2.1-fold more effective than a single-dose combination regimen of DOX and PSO in solution, respectively. These data indicate that the LPNs have superior properties compared with a combination therapy in solution.This study was supported by the National Natural Science Foundation of China (grant no. 81273707), the Ministry of Education in the New Century Excellent Talents (grant no. NECT-12-0677), the Natural Science Foundation of Guangdong (grant nos. S2013010012880 and 2016A030311037), the Science and Technology Program of Guangzhou (grant nos. 2014J4500005 and 201704030141), the Science Program of the Department of Education of Guangdong (grant nos. 2013KJCX0021 and 2015KGJHZ012), the Science and Technology Program of Guangdong (grant no. 2015A050502027), and the Special Project of International Scientific and Technological Cooperation in Guangzhou Development District (grant no. 2017GH16)