Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression.

The ABC transporter ABCC4 is recognized as an ATP-dependent exporter of endogenous substances as well as an increasing variety of anionic chemotherapeutics. A loss-of-function variant of zebrafish Abcc4 was identified with a single amino acid substitution in the cytoplasmic loop T804M. Because this substituted amino acid is highly conserved among ABCC4 orthologs and is located in cytoplasmic loop 3 (CL3), we investigated the impact of this mutation on human and zebrafish Abcc4 expression. We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. To understand the molecular basis for the localization defect, we developed a homology model of zebrafish Abcc4. The homology model suggested that the bulky methionine substitution disrupted side-chain contacts. Molecular dynamic simulations of a fragment of human or zebrafish CL3 containing a methionine substitution indicated altered helicity coupled with reduced thermal stability. Trifluoroethanol challenge coupled with circular dichroism revealed that the methionine substitution disrupted the ability of this fragment of CL3 to readily form an α-helix. Furthermore, expression and plasma membrane localization of these mutant ABCC4/Abcc4 proteins are mostly rescued by growing cells at subphysiological temperatures. Because the cystic fibrosis transmembrane conductance regulator (ABCC7) is closely related to ABCC4, we extended this by engineering certain pathogenic CFTR-CL3 mutations, and we showed they destabilized human and zebrafish ABCC4. Altogether, our studies provide the first evidence for a conserved domain in CL3 of ABCC4 that is crucial in ensuring its proper plasma membrane localization.

ATP-dependent mitochondrial porphyrin importer ABCB6 protects against phenylhydrazine toxicity.

Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient (Abcb6(+/-)) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, Abcb6(-/-) mice derived from these stem cells appeared phenotypically normal. We hypothesized that other ATP-dependent and/or -independent mechanisms conserve porphyrins. Here, we demonstrate that Abcb6(-/-) mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in up-regulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in Abcb6(-/-) mice, possibly because of sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer, and loss of Abcb6 produces up-regulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g. phenylhydrazine stress), these adaptations appear inadequate, which suggests that under these conditions Abcb6 is important for optimal survival.

Deregulated hepatic metabolism exacerbates impaired testosterone production in Mrp4-deficient mice.

The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, an intracellular messenger formed when the luteinizing hormone (LH) receptor is activated. Because Mrp4 regulates cAMP, we compared testosterone levels in Mrp4(-/-) and Mrp4(+/+) mice. Young Mrp4(-/-) mice had significantly impaired gametogenesis, reduced testicular testosterone, and disruption of Leydig cell cAMP homeostasis. Both young and adult mice had impaired testosterone production. In Mrp4(-/-) primary Leydig cells treated with LH, intracellular cAMP production was impaired and cAMP-response element-binding protein (CREB) phosphorylation was strongly attenuated. Notably, expression of CREB target genes that regulate testosterone biosynthesis was reduced in Mrp4(-/-) Leydig cells in vivo. Therefore, Mrp4 is required for normal Leydig cell testosterone production. However, adult Mrp4(-/-) mice are fertile, with a normal circulating testosterone concentration. The difference is that in 3-week-old Mrp4(-/-) mice, disruption of gonadal testosterone production up-regulates hepatic Cyp2b10, a known testosterone-metabolizing enzyme. Therefore, defective testicular testosterone production de-regulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. These findings have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and are reported to alter androgen production.

Abcb11 deficiency induces cholestasis coupled to impaired ß-fatty acid oxidation in mice.

The bile salt export pump (BSEP) is an ATP-binding cassette transporter that serves as the primary system for removing bile salts from the liver. In humans, deficiency of BSEP, which is encoded by the ABCB11 gene, causes severe progressive cholestatic liver disease from early infancy. In previous studies of Abcb11 deficiency in mice generated on a mixed genetic background, the animals did not recapitulate the human disease. We reasoned that ABCB11 deficiency may cause unique changes in hepatic metabolism that are predictive of liver injury. To test this possibility, we first determined that Abcb11 knock-out (KO) C57BL/6J mice recapitulate human deficiency. Before the onset of cholestasis, Abcb11 KO mice have altered hepatic lipid metabolism coupled with reduced expression of genes important in mitochondrial fatty acid oxidation. This was associated with increased serum free-fatty acids, reduced total white adipose, and marked impairment of long-chain fatty acid ß-oxidation. Importantly, metabolomic analysis confirmed that Abcb11 KO mice have impaired mitochondrial fatty acid ß-oxidation with the elevated fatty acid metabolites phenylpropionylglycine and phenylacetylglycine. These metabolic changes precede cholestasis but may be of relevance to cholestatic disease progression because altered fatty acid metabolism can enhance reactive oxygen species that might exacerbate cholestatic liver damage.

Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity.

Purine nucleoside antimetabolites, such as clofarabine, are effective antileukemic agents. However, their effectiveness depends on an initial activation step in which they are monophosphorylated by deoxycytidine kinase (dCK). Some purine nucleoside antimetabolites and their monophosphate derivatives are exported by the ABC transporter ABCG2. Because clofarabine is a dCK substrate, and we show substantial variation in dCK and ABCG2 in myeloid leukemia, we hypothesized that the activity of dCK may modulate ABCG2-mediated resistance to clofarabine by regulating the formation of clofarabine monophosphate. We show that ABCG2 influence on clofarabine cytotoxicity was markedly influenced by dCK activity. When dCK expression was reduced by siRNA, clofarabine cytotoxicity was strongly reduced by enhanced ABCG2-mediated efflux. Conversely, dCK overexpression blunted ABCG2-mediated efflux of clofarabine by increasing the formation of clofarabine nucleotides. The use of an ABCG2 inhibitor confirmed that ABCG2 export of clofarabine is maximal when dCK levels are minimal. Analysis of intracellular clofarabine metabolites suggested that ABCG2 exported clofarabine more readily than clofarabine monophosphate. That ABCG2 primarily effluxes clofarabine, but not chlorfarabine-monophosphate, was confirmed by HPLC analysis of drug exported from ABCG2-overexpressing cells. Because the level and function of dCK and ABCG2 vary substantially among other types of cancer, these findings have important implications not only for clofarabine therapy but for purine nucleoside therapy in general. Therefore, we propose that addition of ABCG2 inhibitors would effectively increase the antitumor efficacy of purine nucleosides by blocking drug efflux that may be a significant mode of resistance when dCK levels are low.

Transporter-mediated protection against thiopurine-induced hematopoietic toxicity.

Thiopurines are effective immunosuppressants and anticancer agents, but intracellular accumulation of their active metabolites (6-thioguanine nucleotides, 6-TGN) causes dose-limiting hematopoietic toxicity. Thiopurine S-methyltransferase deficiency is known to exacerbate thiopurine toxicity. However, many patients are highly sensitive to thiopurines for unknown reasons. We show that multidrug-resistance protein 4 (Mrp4) is abundant in myeloid progenitors and tested the role of the Mrp4, an ATP transporter of monophosphorylated nucleosides, in this unexplained thiopurine sensitivity. Mrp4-deficient mice experienced Mrp4 gene dosage-dependent toxicity caused by accumulation of 6-TGNs in their myelopoietic cells. Therefore, Mrp4 protects against thiopurine-induced hematopoietic toxicity by actively exporting thiopurine nucleotides. We then identified a single-nucleotide polymorphism (SNP) in human MRP4 (rs3765534) that dramatically reduces MRP4 function by impairing its cell membrane localization. This SNP is common (>18%) in the Japanese population and indicates that the increased sensitivity of some Japanese patients to thiopurines may reflect the greater frequency of this MRP4 SNP.

Host cell cathepsins potentiate Moloney murine leukemia virus infection.

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.