Ulla Henriksen

Identification of Intra- and Intermolecular Disulfide Bridges in the Multidrug Resistance Transporter ABCG2 *

ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.

Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2

The ATP binding cassette (ABC) half-transporter ABCG2 (MXR/BCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. The mutation did not affect the predicted oligomerization properties of the transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABCG2-K86M. We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. This is consistent with the requirement for at least two active NBDs for transporter activity and suggests that the transporter is a functional dimer. Finally, we analyzed targeting of ABCG2-wt and ABCG2-K86M and observed that they localize to two distinct subcellular compartments: ABCG2-wt targets the cell surface whereas ABCG2-K86M is targeted to the Golgi apparatus followed by retrieval to the endoplasmic reticulum. This suggests an as yet unknown role of the NBDs in assisting proper surface targeting of ABC transporters.

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

The PSD‐95/Discs‐large/ZO‐1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C‐terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N‐terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain‐dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative α‐helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C‐terminus transferred to the single transmembrane protein Tac or the dopamine transporter C‐terminus transferred to Tac. In contrast, transfer of the GluR2 C‐terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain‐dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N‐terminal myristoylation site produced BAR domain‐dependent, but ligand‐independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain‐dependent and linker‐dependent mechanism. Moreover, they suggest that unmasking of the BAR domain’s membrane‐binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment.

Aryl azides as photolabels. Retention of iodine during photochemical ring expansion of an iodinated tyrosine derivative

Abstract In contrast to the photochemical behaviour of iodinated aromatic azides with iodine and azide in the same ring, iodinated N-(4-azidobenzoyl)tyrosine readily undergoes singlet photochemistry with ring expansion and trapping of nucleophiles while retaining iodine substituents in the phenol nucleus.

Profiling of Multiple Signal Pathway Activities by Multiplexing Antibody and GFP-Based Translocation Assays

Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFkappaB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca(2+) sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.

PHOTOCHEMISTRY OF QUINOLYLMETHYLISOTHIORONIUM SALTS. GUANINE SELECTIVE DNA PHOTOCLEAVAGE REAGENTS

Quinolylmethylisothioronium salts (1a and 4a) cleave DNA upon irradiation. The cleavage is more than 10‐fold enhanced by piperidine treatment and subsequently shows a high preference for guanines. Photolysis of 1a, 2a and 4a in water at Λ > 300 nm resulted in photoheterolysis. Irradiation of 1a in 2‐propanol gave only products from photohomolysis, whereas irradiation of 1a in methanol and 2a and 4a in 2‐propanol resulted in products from both photoheterolysis and photohomolysis. Quantum yields for the disappearance of 1a in water and 2‐propanol were determined. The presence or absence of oxygen had no effect in water, whereas oxidation products were observed upon irradiation in methanol and 2‐propanol in the presence of oxygen. The guanine specific DNA photoreaction is proposed to take place by alkylation at N7 via the quinolylmethyl carbocation and thus to represent a photoalkylation.

Azidobenzoyl-, azidoacridinyl-, diazocyclopentadienyl-carbonyl- and 8-propyloxypsoralen photobiotinylation reagents. Syntheses and photoreactions with DNA and protein

Abstract A series of novel reagents is described for the photobiotinylation of DNA and protein. The reagents consist of a photoactive ligand (photoprobe) tethered to biotin via a polymethylene or azopolymethylene linker. The photoprobes are 4-azidobenzamido, 2-methoxy-6-azido-9-acridinylamino, 2-diazocyclopentadienylcarboxamido, 4-azido-2-nitroanilino and 8-(3-aminopropyloxy)psoralen. The reagents were irradiated with long-wavelength UV light in the presence of DNA or protein and the biotinylation was monitored by blotting to nitrocellulose and staining with avidin—alkaline phosphatase conjugate. The highest DNA photolabelling was obtained with reagents containing the 4-azidobenzamido or psoralen ligands with an amino group in the linker. The 4-azidobenzamido reagent also exhibited the highest protein labelling. Some of these new photobiotinylation reagents are superior to existing ones.

Facile Synthesis of N-(1-Alkenyl) Derivatives of 2,4-Pyrimidinediones

Abstract N-(1-alkenyl) derivatives of 2,4-pyrimidinediones (6–9) were prepared in a one pot synthesis from aldehydes and the nucleobases using trimethylsilyl trifluoromethanesulfonate (TfOTMS) as coupling reagent. Presilylation of the above nucleobases, and N 6-benzoyladenine, with excess N,O-bis(trimethylsilyl)acetamide (BSA) followed by addition of one mol eq. TfOTMS yielded the N-(1-trimethylsilyloxyalkyl) derivatives 1–5.

Preparation of N-(alk-1-enyl) nucleobase compounds by Horner and Horner–Wadsworth–Emmons reactions

A series of new N9-(alk-1-enyl)adenines and N1-(alk-1-enyl)thymines has been prepared by Horner reactions of the new phosphine oxides N9-(diphenylphosphorylmethyl)adenine and N1-(diphenylphosphorylmethyl)thymine derivatives 13a–c, or Horner–Wadsworth–Emmons reactions of the corresponding new phosphonates 14a–b, with benzaldehyde and various ketones. Yields were highest for the Horner reactions (10–79%), and were limited by steric hindrance, enolization of the ketones, and decomposition of some of the N1-(alk-1-enyl)thymines in the presence of excess base (NaH). Butanal gave mixtures of products under Horner conditions, probably because aldol reactions intervened.

Improved synthesis of oligonucleotides with an allylic backbone. Oligonucleotides containing acyclic, achiral nucleoside analogues: N-1 or N-9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases

An improved phosphoramidite method is described to prepare oligonucleotides modified with the acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed that phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation. The phosphordiester dimers were found to be stable towards capping and oxidation, but were somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides hybridized to complementary DNA and RNA, although with reduced affinity (DeltaT(m) per modification -1 to -5 degrees C).