Regulatory Assessment of Casgevy for the Treatment of Transfusion-Dependent ß-Thalassemia and Sickle Cell Disease with Recurrent Vaso-Occlusive Crises.

Sickle cell disease (SCD) and transfusion-dependent ß-thalassemia (TDT) are hereditary haemoglobinopathies characterized by a reduction in functional ß-globin chains. Both conditions cause tiredness and increase susceptibility to infection, which can lead organ failure, significantly reducing life expectancy and typically requiring those affected to undergo regular erythrocyte transfusion. Recently, a novel therapeutic treatment for SCD and TDT was approved by the UK regulatory body (Medicines and Healthcare products Regulatory Agency; MHRA). Exagamglogene autotemcel (Casgevy) is the first licensed therapy globally to utilize CRIPSR/Cas9 technology and induces an increase in expression of γ-globin chains to compensate for the reduction in functional ß-globin. Casgevy represents a first-in-class therapeutic, and numerous considerations were made by the MHRA throughout its assessment of the medicine. These include, but are not limited to, the risk of tumorigenicity and off-target editing, a limited cohort size, the validity of proposed dosing and the conduction of only single-arm studies. The MHRA's analyses of the data to support the proposed indications are presented and discussed throughout this manuscript. Overall, the sponsors claims were considered well supported by their data, and Casgevy was licensed for the treatment of TDT or SCD in patients 12 years of age and older for whom hematopoietic stem cell (HSC) transplantation is appropriate, but a human leukocyte antigen-matched related HSC donor is not available.

Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic Metabolites.

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumour induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette (ABC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the GSH conjugate of the highly toxic monomethylarsonous acid (MMAIII), MMA(GS)2, and the major human urinary arsenic metabolite dimethylarsinic acid (DMAV). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M4I-10 epitope was first mapped to an octapeptide (411HVQDFTA418F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in HEK293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMAIII, monomethylarsonic acid, dimethylarsinous acid or DMAV], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)2 or DMAV, despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17ß-estradiol-17-(ß-D-glucuronide). Abcc4(+/+) mouse embryonic fibroblasts (MEFs) were ~3-fold more resistant to arsenate than Abcc4(-/-) MEFs; however, further characterization indicated this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to differences in human and mouse arsenic toxicokinetics. Significance Statement Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. The human ATP-binding cassette transporter hABCC4 is a high affinity transporter of toxicologically important arsenic metabolites. Here we used multiple cell models to demonstrate that mouse Abcc4 does not protect cells against, or transport, any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.

Addressing health equity for breastfeeding women: primaquine for Plasmodium vivax radical cure.

Plasmodium vivax malaria remains a global health challenge, with approximately 6.9 million estimated cases in 2022. The parasite has a dormant liver stage, the hypnozoite, which reactivates to cause repeated relapses over weeks, months, or years. These relapses erode patient health, contribute to the burden of malaria, and promote transmission. Radical cure to prevent relapses requires administration of an 8-aminoquinoline, either primaquine or tafenoquine. However, malaria treatment guidelines updated by the World Health Organization (WHO) in October 2023 restrict primaquine use for women breastfeeding children < 6 months of age, or women breastfeeding older children if their child is G6PD deficient or if the child's G6PD status is unknown. Primaquine restrictions assume that 8-aminoquinoline exposures in breast milk would be sufficient to cause haemolysis in the nursing infant should they be G6PD deficient. WHO recommendations for tafenoquine are awaited. Notably, the WHO recommends that infants are breastfed for the first 2 years of life, and exclusively until 6 months old. Repeated pregnancies, followed by extended breastfeeding leaves women in P. vivax endemic regions potentially vulnerable to relapses for many years. This puts women's health at risk, increases the malaria burden, and perpetuates transmission, hindering malaria control and elimination. The benefits of lifting restrictions on primaquine administration to breastfeeding women are significant, avoiding the adverse consequences of repeated episodes of acute malaria, such as severe anaemia. Recent data challenge the restriction of primaquine in breastfeeding women. Clinical pharmacokinetic data in breastfeeding infants ≥ 28 days old show that the exposure to primaquine is very low and less than 1% of the maternal exposure, indicating negligible risk to infants, irrespective of their G6PD status. Physiologically-based pharmacokinetic modelling complements the clinical data, predicting minimal primaquine exposure to infants and neonates via breast milk from early post-partum. This article summarizes the clinical and modelling evidence for a favourable benefit:risk evaluation of P. vivax radical cure with primaquine for breastfeeding women without the need for infant G6PD testing, supporting a change in policy. This adjustment to current treatment guidelines would support health equity in regard to effective interventions to protect women and their children, enhance malaria control strategies, and advance P. vivax elimination.

Lfng and Dll3 cooperate to modulate protein interactions in cis and coordinate oscillatory Notch pathway activation in the segmentation clock.

In mammalian development, oscillatory activation of Notch signaling is required for segmentation clock function during somitogenesis. Notch activity oscillations are synchronized between neighboring cells in the presomitic mesoderm (PSM) and have a period that matches the rate of somite formation. Normal clock function requires cyclic expression of the Lunatic fringe (LFNG) glycosyltransferase, as well as expression of the inhibitory Notch ligand Delta-like 3 (DLL3). How these factors coordinate Notch activation in the clock is not well understood. Recent evidence suggests that LFNG can act in a signal-sending cell to influence Notch activity in the clock, raising the possibility that in this context, glycosylation of Notch pathway proteins by LFNG may affect ligand activity. Here we dissect the genetic interactions of Lfng and Dll3 specifically in the segmentation clock and observe distinctions in the skeletal and clock phenotypes of mutant embryos showing that paradoxically, loss of Dll3 is associated with strong reductions in Notch activity in the caudal PSM. The patterns of Notch activity in the PSM suggest that the loss of Dll3 is epistatic to the loss of Lfng in the segmentation clock, and we present direct evidence for the modification of several DLL1 and DLL3 EGF-repeats by LFNG. We further demonstrate that DLL3 expression in cells co-expressing DLL1 and NOTCH1 can potentiate a cell's signal-sending activity and that this effect is modulated by LFNG, suggesting a mechanism for coordinated regulation of oscillatory Notch activation in the clock by glycosylation and cis-inhibition.

A tale of two clocks: phosphorylation of NICD by CDKs links cell cycle and segmentation clock.

The Notch signaling pathway is tightly controlled via post-transcriptional regulatory mechanisms that promote or terminate pathway activity. In this issue, Carrieri et al [1] show that phosphorylation of the Notch intracellular domain (NICD) by cyclin-dependent kinases (CDKs) suppresses Notch activity by promoting NICD turnover. These findings link Notch pathway activity to the cell cycle, and the authors propose connections between this regulation and the segmentation clock that times embryonic somitogenesis.

The First Cytoplasmic Loop in the Core Structure of the ABCC1 (Multidrug Resistance Protein 1; MRP1) Transporter Contains Multiple Amino Acids Essential for Its Expression.

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly392, Tyr404, Arg405), the perpendicular coupling helix (Asn412, Arg415, Lys416), and the ascending α-helix (Glu422, Phe434) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu422 and Arg615 could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.

Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model.

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.

EMA Review of Daunorubicin and Cytarabine Encapsulated in Liposomes (Vyxeos, CPX-351) for the Treatment of Adults with Newly Diagnosed, Therapy-Related Acute Myeloid Leukemia or Acute Myeloid Leukemia with Myelodysplasia-Related Changes.

On June 28, 2018, the Committee for Medicinal Products for Human Use adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Vyxeos, intended for the treatment of acute myeloid leukemia (AML). Vyxeos was designated as an orphan medicinal product on January 11, 2012. The applicant for this medicinal product was Jazz Pharmaceuticals Ireland Limited. Vyxeos is a liposomal formulation of a fixed combination of daunorubicin and cytarabine, antineoplastic agents that inhibit topoisomerase II activity and also cause DNA damage. The strength of Vyxeos is 5 units/mL, where 1 unit equals 1.0 mg cytarabine plus 0.44 mg daunorubicin. The marketing authorization holder Jazz Pharmaceuticals had found that this was an optimal ratio for the efficacy of the product. Study CLTR0310-301, a phase III, multicenter, randomized, trial of Vyxeos (daunorubicin-cytarabine) liposome injection versus standard 3+7 daunorubicin and cytarabine in patients aged 60-75 years with untreated high-risk (secondary) AML, showed a statistically significant difference between the two groups in overall survival (OS) with a median OS of 9.56 months in the daunorubicin-cytarabine arm compared with 5.95 months for standard chemotherapy (hazard ratio, 0.69; 95% confidence interval, 0.52-0.90; one-sided p = .003). The most common side effects were hypersensitivity including rash, febrile neutropenia, edema, diarrhea/colitis, mucositis, fatigue, musculoskeletal pain, abdominal pain, decreased appetite, cough, headache, chills, arrhythmia, pyrexia, sleep disorders, and hypotension. IMPLICATIONS FOR PRACTICE: Vyxeos has demonstrated a clinically significant improvement in overall survival compared with the standard of care 7+3 in the proposed population of patients with newly diagnosed acute myeloid leukemia (AML) with myelodysplasia-related changes and therapy-related AML. This is remarkable given the very poor prognosis of these patients and their unmet medical need. Secondary endpoints support the primary outcome, in particular an increased rate of hematopoietic stem cell transplantation, which is potentially the only curative treatment in AML.

Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).

The human multidrug resistance protein 1 (hMRP1) transporter is implicated in cancer multidrug resistance as well as immune responses involving its physiologic substrate, glutathione (GSH)-conjugated leukotriene C4 (LTC4). LTC4 binds a bipartite site on hMRP1, which a recent cryoelectron microscopy structure of LTC4-bound bovine Mrp1 depicts as composed of a positively charged pocket and a hydrophobic (H) pocket that binds the GSH moiety and surrounds the fatty acid moiety, respectively, of LTC4. Here, we show that single Ala and Leu substitutions of H-pocket hMRP1-Met1093 have no effect on LTC4 binding or transport. Estrone 3-sulfate transport is also unaffected, but both hMRP1-Met1093 mutations eliminate estradiol glucuronide transport, demonstrating that these steroid conjugates have binding sites distinct from each other and from LTC4. To eliminate LTC4 transport by hMRP1, mutation of 3 H-pocket residues was required (W553/M1093/W1246A), indicating that H-pocket amino acids are key to the vastly different affinities of hMRP1 for LTC4vs. GSH alone. Unlike organic anion transport, hMRP1-mediated drug resistance was more diminished by Ala than Leu substitution of Met1093. Although our findings generally support a structure in which H-pocket residues bind the lipid tail of LTC4, their critical and differential role in the transport of conjugated estrogens and anticancer drugs remains unexplained.-Conseil, G., Arama-Chayoth, M., Tsfadia, Y., Cole, S. P. C. Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).

A review of clinical pharmacology deficiencies of European centralised drug marketing authorisation applications.

The aim of this observational review was to review trends in deficiencies in clinical pharmacology dossiers by analysing the frequency and characteristics of major objections (MOs) related to clinical pharmacokinetics and dose-exposure-response (DER) relationships in assessment reports for medicinal products submitted in centralised procedures to the European Medicines Agency (EMA). Initial Assessor (Day 120) assessment reports between 2013 and 2018 were reviewed MOs and characterised with regards to ATC code, orphan status, legal basis and type of molecule, major objection topic and if scientific advice had been sought during development. 23% of the 551 identified Day 120 assessments contained at least one major objection related to clinical pharmacology. Most common topics identified were related to the pharmacokinetics in the target populations, analytical methods, dose-exposure-response relationships, absorption, distribution, metabolism, excretion, comparative bioavailability, and bioequivalence issues. The importance of a robust clinical PK dossier in the assessment of marketing authorisation applications was highlighted by the high frequency of major objections. This review should provide valuable insights to ensure that aspects of bioanalytical methods, comparative bioavailability, PK in the target population and DER relationships are thoroughly addressed in future marketing authorisation applications.

Disruption of somitogenesis by a novel dominant allele of Lfng suggests important roles for protein processing and secretion.

Vertebrate somitogenesis is regulated by a segmentation clock. Clock-linked genes exhibit cyclic expression, with a periodicity matching the rate of somite production. In mice, lunatic fringe (Lfng) expression oscillates, and LFNG protein contributes to periodic repression of Notch signaling. We hypothesized that rapid LFNG turnover could be regulated by protein processing and secretion. Here, we describe a novel Lfng allele (Lfng(RLFNG)), replacing the N-terminal sequences of LFNG, which allow for protein processing and secretion, with the N-terminus of radical fringe (a Golgi-resident protein). This allele is predicted to prevent protein secretion without altering the activity of LFNG, thus increasing the intracellular half-life of the protein. This allele causes dominant skeletal and somite abnormalities that are distinct from those seen in Lfng loss-of-function embryos. Expression of clock-linked genes is perturbed and mature Hes7 transcripts are stabilized in the presomitic mesoderm of mutant mice, suggesting that both transcriptional and post-transcriptional regulation of clock components are perturbed by RLFNG expression. Contrasting phenotypes in the segmentation clock and somite patterning of mutant mice suggest that LFNG protein may have context-dependent effects on Notch activity.

The many roles of Notch signaling during vertebrate somitogenesis.

The embryonic vertebrate body axis contains serially repeated elements, somites, which form sequentially by budding from a posterior tissue called the presomitic mesoderm (PSM). Somites are the embryonic precursors of the vertebrae, ribs and other adult structures. Many inherited human diseases are characterized by dysregulated somitogenesis, resulting in skeletal abnormalities that are evident at birth. Several of these conditions, including some cases of autosomal recessive familial spondylocostal dysostosis (SCDO), arise from mutations in the Notch signaling pathway, which has been demonstrated to be a key player in the regulation of somitogenesis. Here, we review the functional roles of the Notch pathway in vertebrate segmentation, focusing on its activities in a clock that times the formation of somites, as well as in the patterning and production of epithelial somites.

An Outward-Facing Aromatic Amino Acid Is Crucial for Signaling between the Membrane-Spanning and Nucleotide-Binding Domains of Multidrug Resistance Protein 1 (MRP1; ABCC1).

The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe583 in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe583 is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe583 side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.

Putative binding sites for mir-125 family miRNAs in the mouse Lfng 3'UTR affect transcript expression in the segmentation clock, but mir-125a-5p is dispensable for normal somitogenesis.

BACKGROUND: In vertebrate embryos, a "segmentation clock" times somitogenesis. Clock-linked genes, including Lunatic fringe (Lfng), exhibit cyclic expression in the presomitic mesoderm (PSM), with a period matching the rate of somite formation. The clock period varies widely across species, but the mechanisms that underlie this variability are not clear. The half-lives of clock components are proposed to influence the rate of clock oscillations, and are tightly regulated in the PSM. Interactions between Lfng and mir-125a-5p in the embryonic chicken PSM promote Lfng transcript instability, but the conservation of this mechanism in other vertebrates has not been tested. Here, we examine whether this interaction affects clock activity in a mammalian species. RESULTS: Mutation of mir-125 binding sites in the Lfng 3'UTR leads to persistent, nonoscillatory reporter transcript expression in the caudal-most mouse PSM, although dynamic transcript expression recovers in the central PSM. Despite this, expression of endogenous mir-125a-5p is dispensable for mouse somitogenesis. CONCLUSIONS: These results suggest that mir-125a sites in the Lfng 3' untranslated region influence transcript turnover in both mouse and chicken embryos, and support the existence of position-dependent regulatory mechanisms in the PSM. They further suggest the existence of compensatory mechanisms that can rescue the loss of mir-125a-5p in mice. Developmental Dynamics 246:740-748, 2017. © 2017 Wiley Periodicals, Inc.

Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future.

The human ATP-binding cassette transporter multidrug resistance protein 1 (MRP1), encoded by ABCC1, was initially identified because of its ability to confer multidrug resistance in lung cancer cells. It is now established that MRP1 plays a role in protecting certain tissues from xenobiotic insults and that it mediates the cellular efflux of the proinflammatory cysteinyl leukotriene C4 as well as a vast array of other endo- and xenobiotic organic anions. Many of these are glutathione (GSH) or glucuronide conjugates, the products of Phase II drug metabolism. MRP1 also plays a role in the cellular efflux of the reduced and oxidized forms of GSH and thus contributes to the many physiological and pathophysiological processes influenced by these small peptides, including oxidative stress. In this review, the pharmacological and physiological aspects of MRP1 are considered in the context of the current status and future prospects of pharmacological and genetic modulation of MRP1 activity.

Arsenic Triglutathione [As(GS)3] Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) Is Selectively Modified by Phosphorylation of Tyr920/Ser921 and Glycosylation of Asn19/Asn23.

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ABCC1) is responsible for the cellular export of a chemically diverse array of xenobiotics and endogenous compounds. Arsenic, a human carcinogen, is a high-affinity MRP1 substrate as arsenic triglutathione [As(GS)3]. In this study, marked differences in As(GS)3 transport kinetics were observed between MRP1-enriched membrane vesicles prepared from human embryonic kidney 293 (HEK) (Km 3.8 µM and Vmax 307 pmol/mg per minute) and HeLa (Km 0.32 µM and Vmax 42 pmol/mg per minute) cells. Mutant MRP1 lacking N-linked glycosylation [Asn19/23/1006Gln; sugar-free (SF)-MRP1] expressed in either HEK293 or HeLa cells had low Km and Vmax values for As(GS)3, similar to HeLa wild-type (WT) MRP1. When prepared in the presence of phosphatase inhibitors, both WT- and SF-MRP1-enriched membrane vesicles had a high Km value for As(GS)3 (3-6 µM), regardless of the cell line. Kinetic parameters of As(GS)3 for HEK-Asn19/23Gln-MRP1 were similar to those of HeLa/HEK-SF-MRP1 and HeLa-WT-MRP1, whereas those of single glycosylation mutants were like those of HEK-WT-MRP1. Mutation of 19 potential MRP1 phosphorylation sites revealed that HEK-Tyr920Phe/Ser921Ala-MRP1 transported As(GS)3 like HeLa-WT-MRP1, whereas individual HEK-Tyr920Phe- and -Ser921Ala-MRP1 mutants were similar to HEK-WT-MRP1. Together, these results suggest that Asn19/Asn23 glycosylation and Tyr920/Ser921 phosphorylation are responsible for altering the kinetics of MRP1-mediated As(GS)3 transport. The kinetics of As(GS)3 transport by HEK-Asn19/23Gln/Tyr920Glu/Ser921Glu were similar to HEK-WT-MRP1, indicating that the phosphorylation-mimicking substitutions abrogated the influence of Asn19/23Gln glycosylation. Overall, these data suggest that cross-talk between MRP1 glycosylation and phosphorylation occurs and that phosphorylation of Tyr920 and Ser921 can switch MRP1 to a lower-affinity, higher-capacity As(GS)3 transporter, allowing arsenic detoxification over a broad concentration range.

N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity.

Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity.

Cysteinyl Leukotriene Receptor 1/2 Antagonists Nonselectively Modulate Organic Anion Transport by Multidrug Resistance Proteins (MRP1-4).

Active efflux of both drugs and organic anion metabolites is mediated by the multidrug resistance proteins (MRPs). MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), and MRP4 (ABCC4) have partially overlapping substrate specificities and all transport 17ß-estradiol 17-(ß-d-glucuronide) (E217ßG). The cysteinyl leukotriene receptor 1 (CysLT1R) antagonist MK-571 inhibits all four MRP homologs, but little is known about the modulatory effects of newer leukotriene modifiers (LTMs). Here we examined the effects of seven CysLT1R- and CysLT2R-selective LTMs on E217ßG uptake into MRP1-4-enriched inside-out membrane vesicles. Their effects on uptake of an additional physiologic solute were also measured for MRP1 [leukotriene C4 (LTC4)] and MRP4 [prostaglandin E2 (PGE2)]. The two CysLT2R-selective LTMs studied were generally more potent inhibitors than CysLT1R-selective LTMs, but neither class of antagonists showed any MRP selectivity. For E217ßG uptake, LTM IC50s ranged from 1.2 to 26.9 µM and were most comparable for MRP1 and MRP4. The LTM rank order inhibitory potencies for E217ßG versus LTC4 uptake by MRP1, and E217ßG versus PGE2 uptake by MRP4, were also similar. Three of four CysLT1R-selective LTMs also stimulated MRP2 (but not MRP3) transport and thus exerted a concentration-dependent biphasic effect on MRP2. The fourth CysLT1R antagonist, LY171883, only stimulated MRP2 (and MRP3) transport but none of the MRPs were stimulated by either CysLT2R-selective LTM. We conclude that, in contrast to their CysLTR selectivity, CysLTR antagonists show no MRP homolog selectivity, and data should be interpreted cautiously if obtained from LTMs in systems in which more than one MRP is present.

Collagen Fibril Ultrastructure in Mice Lacking Discoidin Domain Receptor 1.

The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson's trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.

Posterior skeletal development and the segmentation clock period are sensitive to Lfng dosage during somitogenesis.

The segmental structure of the axial skeleton is formed during somitogenesis. During this process, paired somites bud from the presomitic mesoderm (PSM), in a process regulated by a genetic clock called the segmentation clock. The Notch pathway and the Notch modulator Lunatic fringe (Lfng) play multiple roles during segmentation. Lfng oscillates in the posterior PSM as part of the segmentation clock, but is stably expressed in the anterior PSM during presomite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng(∆FCE)) exhibit abnormal anterior development but relatively normal posterior development. This suggests distinct requirements for segmentation clock activity during the formation of the anterior skeleton (primary body formation), compared to the posterior skeleton and tail (secondary body formation). To build on these findings, we created an allelic series that progressively lowers Lfng levels in the PSM. Interestingly, we find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, we find that while low levels of oscillatory Lfng in the posterior PSM are sufficient to support relatively normal posterior development, the period of the segmentation clock is increased when the amplitude of Lfng oscillations is low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels. Further, they suggest that modulation of the Notch signaling by Lfng affects the clock period during development.