The Reentry Helix Is Potentially Involved in Cholesterol Sensing of the ABCG1 Transporter Protein.

ABCG1 has been proposed to play a role in HDL-dependent cellular sterol regulation; however, details of the interaction between the transporter and its potential sterol substrates have not been revealed. In the present work, we explored the effect of numerous sterol compounds on the two isoforms of ABCG1 and ABCG4 and made efforts to identify the molecular motifs in ABCG1 that are involved in the interaction with cholesterol. The functional readouts used include ABCG1-mediated ATPase activity and ABCG1-induced apoptosis. We found that both ABCG1 isoforms and ABCG4 interact with several sterol compounds; however, they have selective sensitivities to sterols. Mutational analysis of potential cholesterol-interacting motifs in ABCG1 revealed altered ABCG1 functions when F571, L626, or Y586 were mutated. L430A and Y660A substitutions had no functional consequence, whereas Y655A completely abolished the ABCG1-mediated functions. Detailed structural analysis of ABCG1 demonstrated that the mutations modulating ABCG1 functions are positioned either in the so-called reentry helix (G-loop/TM5b,c) (Y586) or in its close proximity (F571 and L626). Cholesterol molecules resolved in the structure of ABCG1 are also located close to Y586. Based on the experimental observations and structural considerations, we propose an essential role for the reentry helix in cholesterol sensing in ABCG1.

MiR-202-3p Inhibits Foam Cell Formation and is Associated with Coronary Heart Disease Risk in a Chinese Population.

A previous study and a gene-annotation enrichment analysis for potential targets of the microRNA miR-202-3p both suggest that this microRNA might be implicated in cardiovascular and metabolic diseases. In the present study, the role of miR-202-3p in the pathogenesis of coronary heart disease (CHD) was explored. We conduct a case-control study to detect the expression levels of miR-202-3p in peripheral blood cells and found that miR-202-3p expression was significantly higher in CHD cases than in controls (P < 0.001). miR-202-3p levels were negatively correlated with platelet distribution width (r = -0.348, P = 0.002) and mean platelet volume (r = -0.29, P = 0.01). Further functional analyses suggested that stimulation with oxidized low-density lipoprotein (ox-LDL) induced miR-202-3p expression, and that this microRNA suppressed the formation of ox-LDL-induced macrophage foam cells derived from THP-1 cells in a feedback manner. In addition, miR-202-3p overexpression modulated the expression of several key genes involved in foam cell formation, including that of ABCG4, NCEH1I, and SCARB2. In summary, miR-202-3p was associated with CHD, exerting a protective role against CHD by feedback suppression of ox-LDL-induced macrophage foam cell formation.

Doxorubicin induces prostate cancer drug resistance by upregulation of ABCG4 through GSH depletion and CREB activation: Relevance of statins in chemosensitization.

Multidrug resistance mediated by ATP-binding cassette (ABC) transporters remains a major impediment to cancer chemotherapy. In the present study, we documented that doxorubicin (Dox) or cisplatin-induced prostate cancer (PCa) chemoresistance is predominantly mediated by the induction of ABCG4 in androgen-independent PCa cells. Treatment of DU-145 or PC-3 cells with Dox significantly enhanced the expression of ABCG4 that resulted in the efflux of intracellular Dox. However, incubation of cells with ABCG4 short hairpin RNA resulted in a significant accumulation of Dox and sensitized cells to Dox-induced cytotoxicity. Interestingly, simvastatin synergistically potentiated Dox-induced cytotoxicity by inhibiting ABCG4 in DU-145 and DU-145 Doxres cells. Mechanistically, ABCG4 expression was regulated redox-dependently by intracellular glutathione (GSH) levels. Treatment of cells with N-acetylcysteine or simvastatin restored Dox-induced depletion of GSH levels that in turn inhibited ABCG4 levels. In addition, a reduction in GSH levels by Dox caused a nuclear factor-κB dependent enhancement of c-Myc expression, which led to cAMP-regulatory element-binding protein (CREB) activation. Furthermore, chromatin immunoprecipitation experiments revealed that Dox-induced CREB activation transcriptionally upregulates ABCG4 expression. These results were further confirmed in an in vivo PCa xenograft mice model. Combination of simvastatin and Dox significantly regressed the tumor growth and size with no noticeable Dox-induced cardiotoxic side effects. Intriguingly, DU-145 cells with stably depleted ABCG4 levels not only significantly delayed the development of the tumor but also greatly sensitized the tumor to a low dose of Dox that resulted in complete tumor regression. Collectively, this data reinforces a novel function of ABCG4 in Dox-mediated chemoresistance, and as a potential therapeutic target in drug-induced PCa chemoresistance.

The Adaptor Protein Alix is Involved in the Interaction Between the Ubiquitin Ligase NEDD4-1 and its Targets, ABCG1 and ABCG4.

Several ATP-Binding Cassette (ABC) transporters, including ABCG1 and the related ABCG4, are essential regulators of cellular lipid homeostasis. ABCG1 is expressed ubiquitously and is functional in the context of atherosclerosis. However, ABCG4 is expressed almost exclusively in brain and has been linked to Alzheimer's disease (AD). These transporters are highly regulated post-translationally by E3 ubiquitin ligases, with the ligase NEDD4-1 (Neural precursor cell-expressed developmentally downregulated gene 4) implicated in their protein stability. In this study, we investigated interacting partners of ABCG1 using peptide-mass spectrometry and identified the potential adaptor protein, Alix (apoptosis-linked gene 2-interacting protein X). In this paper, we hypothesized and investigated whether Alix could facilitate the interaction between NEDD4-1 and the ABC transporters. We showed that Alix and NEDD4-1 proteins were co-expressed in several commonly used cell lines. Knockdown of Alix in cells overexpressing ABCG1 or ABCG4 increased transporter protein expression while co-immunoprecipitation experiments showed interaction between NEDD4-1, Alix, and ABC transporters. In summary, we provide evidence that Alix serves as a co-factor for the interaction between the E3-ubiquitin ligase NEDD4-1 and the ABC transporter targets, ABCG1 and ABCG4.

Behavioral and Metabolic Effects of ABCG4 KO in the APPswe,Ind (J9) Mouse Model of Alzheimer's Disease.

The pathogenesis of Alzheimer's disease (AD) is complex and involves an imbalance between production and clearance of amyloid-ß peptides (Aß), resulting in accumulation of Aß in senile plaques. Hypercholesterolemia is a major risk factor for developing AD, with cholesterol shown to accumulate in senile plaques and increase production of Aß. ABCG4 is a member of the ATP-binding cassette transporters predominantly expressed in the CNS and has been suggested to play a role in cholesterol and Aß efflux from the brain. In this study, we bred Abcg4 knockout (KO) with the APPSwe,Ind (J9) mouse model of AD to test the hypothesis that loss of Abcg4 would exacerbate the AD phenotype. Unexpectedly, no differences were observed in novel object recognition (NOR) and novel object placement (NOP) behavioral tests, or on histologic examinations of brain tissues for senile plaque numbers. Furthermore, clearance of radiolabeled Aß from the brains did not differ between Abcg4 KO and control mice. Metabolic testing by indirect calorimetry, glucose tolerance test (GTT), and insulin tolerance test (ITT) were also mostly similar between groups with only a few mild metabolic differences noted. Overall, these data suggest that the loss of ABCG4 did not exacerbate the AD phenotype.

Behavioral and metabolic and effects of ABCG4 KO in the APPswe,Ind (J9) mouse model of Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is complex and involves an imbalance between production and clearance of amyloid-ß peptides (Aß), resulting in accumulation of Aß in senile plaques. Hypercholesterolemia is a major risk factor for developing AD, with cholesterol shown to accumulate in senile plaques and increase production of Aß. ABCG4 is a member of the ATP-binding cassette transporters predominantly expressed in the CNS, and has been suggested to play a role in cholesterol and Aß efflux from the brain. In this study, we bred Abcg4 knockout (KO) with the APPSwe,Ind (J9) mouse model of AD to test the hypothesis that loss of Abcg4 would exacerbate the AD phenotype. Unexpectedly, no differences were observed in Novel object recognition (NOR) and Novel object placement (NOP) behavioral tests, or on histologic examinations of brain tissues for senile plaque numbers. Furthermore, clearance of radiolabeled Aß from the brains did not differ between Abcg4 KO and control mice. Metabolic testing by indirect calorimetry, glucose tolerance test (GTT) and insulin tolerance test (ITT), were also mostly similar between groups with only a few mild metabolic differences noted. Overall these data suggest that the loss of ABCG4 did not exacerbate the AD phenotype.

VIGS as a strategy to reverse aphid wing induction by Y-satellite RNA of cucumber mosaic virus.

Y-satellite RNA (Y-sat) of cucumber mosaic virus upregulates the expression of the aphid ABCG4 gene, which promotes aphid wing formation. We used ABCG4 virus-induced gene silencing (VIGS) to prevent the wing-induction mechanism of Y-sat and thus inhibited aphid wing formation. Of the aphids on plants with VIGS of ABCG4, only about 30% had wings, and 60-70% of the winged aphids were small and likely impaired in flying ability. In addition, we showed that double-stranded RNAs (dsRNAs) and small RNAs were transferred from the plant to the aphid to adequately silence aphid genes. Supplying ABCG4 dsRNA by VIGS to aphids is thus a potential strategy to inhibit aphid wing formation.

Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis.

Ostrinia furnacalis is an important borer on maize. Long-term and large-scale planting of transgenic corn has led O. furnacalis evolving resistance and reducing the control effect. Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily G gene ABCG4 in O. furnacalis. In order to further determine the relationship between ABCG4 gene and the resistance to Cry1 toxins in O. furnacalis, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct ABCG4-KO knockout homozygous strain. Bioassay results indicated that an ABCG4-KO strain had a higher resistance to Cry1 proteins compared with a susceptible strain (ACB-BtS). The result indicates that the ABCG4 gene may act as a receptor of the Bt Cry1 toxin in O. furnacalis. Furthermore, the development time was significantly changed in the early stage ABCG4-KO larvae, and the population parameters were also significantly changed. In summary, our CRISPR/Cas9-mediated genome editing study presents evidence that ABCG4 gene is a functional receptor for Bt Cry1 toxins, laying the foundation for further clarification of the Bt resistance mechanism.

ABC-Transporter Mediated Sterol Export from Cells Using Radiolabeled Sterols.

Cholesterol export from cells to extracellular acceptors represents the first step of the reverse cholesterol transport process and is an essential part of the multifaceted pathway for cells to control their cholesterol levels. Malfunction of this pathway leads to cholesterol accumulation in cells such as macrophages, which can form the basis of conditions like atherosclerosis. A number of ATP-binding cassette (ABC) transporters, namely ABCA1, ABCA7, ABCG1, and ABCG4, play an essential role in this process. In this chapter, we describe methods utilizing radiolabeled sterols for measuring ABC-transporter mediated sterol export, utilizing endogenously expressed transporters as well as overexpression systems.

Age-Dependent Regulation of the Blood-Brain Barrier Influx/Efflux Equilibrium of Amyloid-ß Peptide in a Mouse Model of Alzheimer's Disease (3xTg-AD).

The involvement of transporters located at the blood-brain barrier (BBB) has been suggested in the control of cerebral Aß levels, and thereby in Alzheimer's disease (AD). However, little is known about the regulation of these transporters at the BBB in animal models of AD. In this study, we investigated the BBB expression of Aß influx (Rage) and efflux (Abcb1-Abcg2-Abcg4-Lrp-1) transporters and cholesterol transporter (Abca1) in 3-18-month-old 3xTg-AD and control mice. The age-dependent effect of BBB transporters regulation on the brain uptake clearance (Clup) of [3H]cholesterol and [3H]Aß1 - 40 was then evaluated in these mice, using the in situ brain perfusion technique. Our data suggest that transgenes expression led to the BBB increase in Aß influx receptor (Rage) and decrease in efflux receptor (Lrp-1). Our data also indicate that mice have mechanisms counteracting this increased net influx. Indeed, Abcg4 and Abca1 are up regulated in 3- and 3/6-month-old 3xTg-AD mice, respectively. Our data show that the balance between the BBB influx and efflux of Aß is maintained in 3 and 6-month-old 3xTg-AD mice, suggesting that Abcg4 and Abca1 control the efflux of Aß through the BBB by a direct (Abcg4) or indirect (Abca1) mechanism. At 18 months, the BBB Aß efflux is significantly increased in 3xTg-AD mice compared to controls. This could result from the significant up-regulation of both Abcg2 and Abcb1 in 3xTg-AD mice compared to control mice. Thus, age-dependent regulation of several Aß and cholesterol transporters at the BBB could ultimately limit the brain accumulation of Aß.