mRNA escape from stress granule sequestration is dictated by localization to the endoplasmic reticulum.

In mammalian cells, cytotoxic stress triggers several signaling cascades that converge in the phosphorylation of translation initiation factor 2alpha, shuttling of nuclear RNA-binding proteins such as TIA-1 to the cytoplasm, and aggregation of most cellular mRNAs into TIA-1-containing stress granules (SGs). As a result, protein synthesis is greatly impaired. Here we describe different dynamics of endogenous transcripts according to their cellular location, in response to stress. While cytosolic mRNAs aggregate into SGs, endoplasmic reticulum (ER) -bound transcripts escape sequestration. This has been specifically demonstrated using the multidrug resistance transporter gene (MDR1) as a model and showing that chimeric RNA constructs can be directed to the cytosol or tethered to the ER depending on the nature of the chimera, in response to stress. In addition, polysome profile analyses indicate that, on stress, ribosomes do not disengage from ER-associated transcripts (puromycin insensitive) and recover their translation status faster than SG-targeted cytosolic mRNAs once the stress is lifted. These findings have important implications for cell survival given that many membrane proteins, which are translated at the ER, have important roles in detoxification.

ABCA1 expression in carotid atherosclerotic plaques.

BACKGROUND AND PURPOSE: The ATP-binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux from cells, a key process in reverse cholesterol transport. Whereas previous investigations focused on mutations causing impaired ABCA1 function, we assessed the role of ABCA1 in human carotid atherosclerotic disease. METHODS: We compared the mRNA and protein levels of ABCA1, and one of its key regulators, the liver X receptor alpha (LXRalpha), between minimally and grossly atherosclerotic arterial tissue. We established ABCA1 and LXRalpha gene expression by real-time quantitative polymerase chain reaction in 10 control and 18 atherosclerotic specimens. Presence of ABCA1 protein was assessed by immunoblotting. To determine whether differences observed at a local level were reflected in the systemic circulation, we measured ABCA1 mRNA in leukocytes of 10 patients undergoing carotid endarterectomy and 10 controls without phenotypic atherosclerosis. RESULTS: ABCA1 and LXRalpha gene expression were significantly elevated in atherosclerotic plaques (P<0.0001 and 0.03, respectively). The increased mRNA levels of ABCA1 and LXRalpha were correlated in atherosclerotic tissue (r=0.85; P<0.0001). ABCA1 protein expression was significantly reduced in plaques compared with control tissues (P<0.0001). There were no differences in leukocyte ABCA1 mRNA expression (P=0.67). CONCLUSIONS: ABCA1 gene and protein are expressed in minimally atherosclerotic human arteries. Despite significant upregulation of ABCA1 mRNA, possibly mediated via LXRalpha, ABCA1 protein is markedly reduced in advanced carotid atherosclerotic lesions. No differences in leukocyte ABCA1 expression were found, suggesting the plaque microenvironment may contribute to the differential ABCA1 expression. We propose that the decreased level of ABCA1 protein is a key factor in the development of atherosclerotic lesions.

Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1.

Overexpression of P-glycoprotein, encoded by the MDR1 gene, confers multidrug resistance (MDR) on cancer cells and is a frequent impediment to successful chemotherapy. Recent developments in the use of small interfering RNAs to inhibit specific protein expression have highlighted their potential use as therapeutic agents. We have expressed two different short hairpin RNAs from stably integrated plasmids in doxorubicin-resistant K562 leukaemic cells. The MDR1-targeted RNA interference (RNAi) resulted in decreased MDR1 mRNA, abolished P-glycoprotein expression, and completely reversed the MDR phenotype to that of the drug-sensitive K562 parental line. This study demonstrates that MDR, which is solely due to overexpression of P-glycoprotein, can be reversed by RNAi. These target sequences can in the future be integrated into gene therapy vectors with potential clinical application.

Leukocyte ABCA1 gene expression is associated with fasting glucose concentration in normoglycemic men.

Adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) mediates the efflux of cholesterol to apolipoprotein A1, a process necessary for high-density lipoprotein (HDL) formation and reverse cholesterol transport. In patients with Tangier disease, mutations in ABCA1 result in low circulating HDL-cholesterol and predisposition to coronary heart disease (CHD). ABCA1 gene expression is decreased in diabetic mice. In humans, glycated hemoglobin (HbA(1c)) predicted future CHD events, even within the normal range. We hypothesised that leukocyte ABCA1 gene expression would be inversely associated with indices of glycemia in normoglycemic men. Fasting blood samples were taken from 32 healthy, nonsmoking, normoglycemic men (age 23 to 46 years). ABCA1, peroxisome proliferator-activated receptor gamma (PPARgamma), and liver X receptor alpha (LXRalpha) gene expressions in circulating leukocytes were measured using TaqMan technology. Significant inverse associations between ABCA1 gene expression and both fasting glucose concentration (r = -0.49, P =.008) and age (r = -0.39, P =.043) were found. There was no association with HbA(1c) (r = -0.23, P =.238) or HDL-cholesterol concentration (r = 0.02, P =.904). In a multiple regression model, fasting glucose remained a significant independent predictor (P =.037), whereas age did not (P =.226). Mechanisms underlying the association were explored; there were no significant associations between fasting glucose concentration and leukocyte PPARgamma gene expression, or between fasting glucose concentration and leukocyte LXRalpha gene expression. This is the first demonstration of an association between ABCA1 gene expression and fasting glucose concentration in vivo.

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: application to single virus-like particle entry into a cell.

We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane.

Functional analysis of candidate ABC transporter proteins for sitosterol transport.

Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance protein (Bcrp; Abcg2) and the bile salt export pump (Bsep; Abcb11) was assessed using several assays. Unexpectedly, none of the candidate proteins mediated significant sitosterol transport. This has implications for the pathology of sitosterolemia. In addition, the data suggest that otherwise broad-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols.

The vinblastine binding site adopts high- and low-affinity conformations during a transport cycle of P-glycoprotein.

Conceptually one may envisage that substrate binding sites on the ABC transporter P-gp cycle between high- and low-affinity conformations in response to signals arising from nucleotide hydrolysis to effect active transport. A radioligand binding assay was used to characterize the interaction of [3H]vinblastine with P-gp and determine how drug binding site parameters are altered during a catalytic cycle of P-gp. In the absence of nucleotide, we show that [3H]vinblastine interacts with a single class of binding site with high affinity (K(d) = 80 +/- 18 nM). In the presence of the nonhydrolyzable ATP analogue AMP-PNP, the drug binding site was in a low-affinity conformation, manifest by a 9-fold increase in K(d) (K(d) = 731 +/- 20 nM). There was no alteration in the binding capacity, reflecting a complete shift in the high-affinity site to a low-affinity form. The posthydrolytic (Mg-ADP-V(i) bound) form of P-gp also exhibited low-affinity substrate binding (K(d) = 446 +/- 57 nM). Restoration of the high-affinity drug binding site conformation (K(d) = 131 +/- 32 nM) did not occur until release of phosphate from the posthydrolysis P-gp-Mg-ADP-P(i). complex. Our results suggest that alteration of the affinity of the vinblastine binding site involves only one nucleotide binding domain per transport cycle. The binding of ATP provides the signal to instigate this change, while release of phosphate post-ATP hydrolysis returns the transporter to its original state to complete the cycle.

Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein.

1. Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P-glycoprotein. Wild-type (wt) and the mutant (cys-less) forms of P-gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies. 2. No significant differences were observed in substrate ([(3)H]-azidopine) binding to wt or cys-less P-gp. Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace [(3)H]-azidopine from the wt or cys-less P-gp. These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions. 3. The Michaelis-Menten characteristics of basal ATP hydrolysis of the two isoforms of P-gp were identical. The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the K(m) was unchanged in cys-less P-gp compared to control. However, cys-less P-gp displayed lower overall maximal ATPase activity (62%), a decreased K(m) and a lower degree of stimulation (76%) in the presence of the modulator nicardipine. 4. Therefore, the serine to cysteine mutations in P-gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways. The wt and cys-less P-gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys-less P-gp as a template for future cysteine-directed structure/function analysis.

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter.

For effective gene therapy of chronic disease, persistent transgene expression at therapeutic levels is required. Clinical studies of airway gene transfer in patients with cystic fibrosis (CF) have resulted in short-lived transgene expression. We used intra-nasal dosing of naked plasmid DNA to the murine lung as a model for investigating the duration of airway gene transfer from a series of reporter expression plasmids. Transgene expression was transient when mediated by the viral promoters CMV, RSV and SV40, falling to less than 10% of peak expression after 2 weeks, although the presence of the adenoviral E4ORF3 gene in cis, resulted in extended duration of reporter activity from the CMV promoter. Transient expression from these promoters was not due to loss of the vector as determined by quantitative TaqMan PCR analysis. However, use of the promoters from the human polybiquitin C (UbC) and the elongation factor 1alpha (EF1alpha) genes resulted in persistent gene expression in the mouse lung. The UbC promoter directed high-level reporter activity which was maintained for up to 8 weeks and was still detectable 6 months after a single administration. Such persistent airway transgene expression from a nonviral vector without the concomitant expression of a potential antigen has not been reported previously. Thus, despite the persistence of vector DNA in vivo, attenuation of promoter function may lead to silencing of transgene expression and careful selection of promoter sequences is recommended for in vivo gene transfer.

The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence.

In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

The multidrug resistance P-glycoprotein. Oligomeric state and intramolecular interactions.

The human multidrug resistance P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of transporters, is frequently responsible for the failure of chemotherapy by virtue of its ability to export hydrophobic cytotoxic drugs from cells. Elucidating the inter- and intramolecular interactions of this protein is critical to understanding its cellular function and mechanism of action. Toward this end, we have used both biochemical and genetic techniques to probe potential oligomerization interactions of P-gp. Differentially epitope-tagged P-gp molecules did not co-immunoprecipitate when co-expressed in HEK293 cells or when co-translated in vitro, demonstrating that P-gp is monomeric in both the presence and absence of detergents. The two cytoplasmic domains of P-gp did not interact with each other in vivo when co-expressed as gene fusions in yeast. In contrast, the homologous domains of the transporter associated with antigen processing (TAP), which reside on separate polypeptides and must form a heterodimeric transporter (TAP1/TAP2), did interact in this system, suggesting a role for these domains in TAP dimerization. Implications for understanding the subunit organization of ABC transporters are discussed.

ABC transporters: physiology, structure and mechanism--an overview.

ABC transporters form one of the largest of all protein families with a diversity of physiological functions. In Escherichia coli almost 5% of the genome is occupied by genes encoding components of these transporters, and there are examples in all species from microbes to man. In this overview, the importance of studies on bacteria in elucidating many basic principles pertaining to ABC transporters is emphasised. The family is described and a general overview of the structure and function of these transporters is presented.

Multidrug transport by ATP binding cassette transporters: a proposed two-cylinder engine mechanism.

The elevated expression of ATP binding cassette (ABC) multidrug transporters in multidrug-resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. This review summarizes current insights into the mechanism(s) by which ATP hydrolysis is coupled to drug transport in bacterial LmrA and its human homolog P-glycoprotein. In addition, the relevance of these insights for other ABC transporters will be discussed.

Molecular basis of multidrug transport by ATP-binding cassette transporters: a proposed two-cylinder engine model.

ATP-binding cassette multidrug transporters are probably present in all living cells, and are able to export a variety of structurally unrelated compounds at the expense of ATP hydrolysis. The elevated expression of these proteins in multidrug resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. Insights into the structural elements in drug molecules and transport proteins that are required for this interaction are now beginning to emerge. However, much remains to be learned about the nature and number of drug binding sites in the transporters, and the mechanism(s) by which ATP hydrolysis is coupled to changes in affinity and/or accessibility of drug binding sites. This review summarizes recent advances in answering these questions for the human multidrug resistance P-glycoprotein and its prokaryotic homolog LmrA. The relevance of these findings for other ATP-binding cassette transporters will be discussed.

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS.

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.

The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants.

The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin. Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown. We show that StpA exhibits similar DNA-binding activities to H-NS. Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively. It has previously been reported that expression of stpA is derepressed in an hns mutant. We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells. Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.

Human ClC-3 is not the swelling-activated chloride channel involved in cell volume regulation.

Volume regulation is essential for normal cell function. A key component of the cells' response to volume changes is the activation of a channel, which elicits characteristic chloride currents (I(Cl, Swell)). The molecular identity of this channel has been controversial. Most recently, ClC-3, a protein highly homologous to the ClC-4 and ClC-5 channel proteins, has been proposed as being responsible for I(Cl, Swell). Subsequently, however, other reports have suggested that ClC-3 may generate chloride currents with characteristics clearly distinct from I(Cl, Swell). Significantly different tissue distributions for ClC-3 have also been reported, and it has been suggested that two isoforms of ClC-3 may be expressed with differing functions. In this study we generated a series of cell lines expressing variants of ClC-3 to rigorously address the question of whether or not ClC-3 is responsible for I(Cl, Swell). The data demonstrate that ClC-3 is not responsible for I(Cl, Swell) and has no role in regulatory volume decrease, furthermore, ClC-3 is not activated by intracellular calcium and fails to elicit chloride currents under any conditions tested. Expression of ClC-3 was shown to be relatively tissue-specific, with high levels in the central nervous system and kidney, and in contrast to previous reports, is essentially absent from heart. This distribution is also inconsistent with the previous proposed role in cell volume regulation.