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Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse Liver

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application.

ABCC6 as a Target in Pseudoxanthoma Elasticum

The ABCC6 gene encodes an organic anion transporter protein, ABCC6/MRP6. Mutations in the gene cause a rare, recessive genetic disease, pseudoxanthoma elasticum, while the loss of one ABCC6 allele is a genetic risk factor in coronary artery disease. We review here the information available on gene structure, evolution as well as the present knowledge on its transcriptional regulation. We give a detailed description of the characteristics of the protein, and analyze the relationship between the distributions of missense disease-causing mutations in the predicted three-dimensional structure of the transporter, which suggests functional importance of the domain-domain interactions. Though neither the physiological function of the protein nor its role in the pathobiology of the diseases are known, a current hypothesis that ABCC6 may be involved in the efflux of one form of Vitamin K from the liver is discussed. Finally, we analyze potential strategies how the gene can be targeted on the transcriptional level to increase protein expression in order to compensate for reduced activity. In addition, pharmacologic correction of trafficking-defect mutants or suppression of stop codon mutations as potential future therapeutic interventions are also reviewed.

Clustering of disease-causing mutations on the domain-domain interfaces of ABCC6

Mutations in ABCC6 are responsible for pseudoxanthoma elasticum (PXE), a rare genetic disease affecting the elastic tissues of the body. ABCC6 encodes a 1503 amino acid long ABC transporter, ABCC6/MRP6. The functional link between the impaired activity of the protein and the disease is not known. We have built a homology model of this transporter, and analyzed the distribution of the known 119 missense PXE-associated mutations within the predicted structure. Significant clustering of the missense mutations has been found at complex domain-domain interfaces: at the transmission interface that involves four intracellular loops and the two ABC domains as well as at the ABC-ABC interacting surfaces. The mutations affecting these regions are 2.75 and 3.53-fold more frequent than the average mutational rate along the transporter protein sequence. These data provide a genetic proof of the importance of these domain-domain interactions in the ABCC6 transporter.

Analysis of Pseudoxanthoma Elasticum-Causing Missense Mutants of ABCC6 in vivo; Pharmacological Correction of the Mislocalized Proteins

Mutations in the ABCC6 gene cause soft tissue calcification in pseudoxanthoma elasticum (PXE) and in some patients generalized arterial calcification of infancy (GACI). PXE is characterized by late-onset and progressive mineralization of elastic fibers in dermal, ocular and cardiovascular tissues. GACI patients present a more severe, often prenatal arterial calcification. We have tested ten frequent disease-causing ABCC6 missense mutants for the transport activity using Sf9 cells, characterized the subcellular localization in MDCKII cells and in mouse liver, and tested the phenotypic rescue in zebrafish. We aimed at identifying mutants with preserved transport activity but with improper plasma membrane localization for rescue by the chemical chaperone 4-phenylbutyrate (4-PBA). Seven of the mutants were transport-competent but mislocalized in mouse liver. The observed divergence in cellular localization of mutants in MDCKII cells vs. mouse liver underlined the limitations of this two-dimensional in vitro cell system. The functionality of ABCC6 mutants was tested in zebrafish and minimal rescue of the morpholino-induced phenotype was found. However, 4-PBA, a drug approved for clinical use, restored the plasma membrane localization of four ABCC6 mutants (R1114P, S1121W, Q1347H, R1314W), suggesting that allele-specific therapy may be useful for selected patients with PXE and GACI.

Effects of excess UV-B irradiation on the antioxidant defence mechanisms in wheat (Triticum aestivum L) seedlings

Summary In the present experiments, changes in the antioxidant defence systems of wheat ( Triticum aestivum L. cv. Tiszataj) as affected by excess UV-B irradiation were investigated. Wheat seedlings were grown hydroponically in phytotrons for 11 days. In one of the phytotrons 2.5 Wm −2 excess UV-B irradiation was applied and samples were taken daily. Catalase, guaiacol peroxidase, superoxide dismutase (SOD) and glutathione reductase (GR) activities were measured and changes in the level of glutathione disulfide (GSSG) were determined by HPLC technique. It was found that catalase, guaiacol peroxidase and SOD activities decreased, while GSSG level and GR activity increased under excess UV-B irradiation. In plant responses, the alarm and adaptation phases were recognised. During the alarm reaction, inhibition of stem and leaf elongation, accumulation of flavonoid pigments, decreased antioxidant enzyme activities, increased GSSG level and triggering of GR activity were observed, while normalisation of growth, disappearance of stem pigmentation, levelling of antioxidant enzyme activities, high GR activity and decreased level of GSSG were found as parts of adaptation to UV-B irradiation. Afier 3 days of UV-B treatment, the activities of Ca 2+ -dependent protein kinases (52 and 65 kDa) increased, suggesting that they may participate in the UV-B signal transduction pathway.

ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: Relevance to pathomechanisms of pseudoxanthoma elasticum

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6(-/-) mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6(-/-) animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms of pseudoxanthoma elasticum.

Environmental stress and the biological clock in plants: Changes of rhythmic behavior of carbohydrates, antioxidant enzymes and stomatal resistance by salinity

Summary In this report, the influence of environmental stress as effector on rhythmic phenomena in plants is discussed based on data from literature and from own studies. In our experiments, the effects of NaCl salinity (0, 50, 100 and 150 mM) on the levels of total soluble carbohydrates and on the activities of the antioxidant enzymes, superoxide dismutase and glutathione reductase, were followed as the function of time in young winter wheat ( Triticum aestivum L.) plants grown under controlled conditions. Salinity effects on stomatal movements of the halophytic Aster tripolium L. were also investigated. Periodicity was calculated by Fourier analysis. The results indicate that the fluctuations in the above functions were modulated by the stressor suggesting the connection between environmental stress and biological rhythmicity. On the basis of these observations and literature data we hypothesize that the stressor may modify and desynchronize the biochemical and physiological oscillations by acting either on the input (from receptor till oscillator) or on the output (from the oscillator till the oscillating component) pathways. In these terms, adaptation means re-setting of the new circadian and/or ultradian rhythms of biochemical/physiological processes and the restoration of their concerted action results in maximal resistance for the organism under the new environmental conditions.

Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations.

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4α which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain–domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy.

Predictable difficulty or difficulty to predict

Predicting the functional consequence of a given amino acid replacement, that is, the difference between the wild type protein and its nonsynonymous single nucleotide polymorph variants (nsSNPs) is a great challenge. This is especially true in the case of large membrane proteins, like ABC transporters. Kelly et al. has published such an ambitious study in a recent issue of Protein Science focusing on nsSNP in structurally conserved segments within the nucleotide binding domains (NBDs) of ABC transporters supposed to be involved in interdomain communication.1 With the aid of structural rationalization the authors predicted the impact of 40 nsSNPs found in seven clinically important human ABC transporters using a bivalent scoring: disease or neutral. We have performed a deep search of the available literature to find published data on the 40 nsSNPs to delineate them with the predictions. We have incorporated only data from original peer-reviewed publications into our dataset, and our hits were checked in various gene-specific databases too [BioBase and ABCC6 Database]. Based on the available information, we categorized the published data on a given nsSNP as (i) “disease-associated” when the publication clearly demonstrates that the variant segregates with the disease but is absent from at least 200 alleles of unrelated, nonaffected individuals; or (ii) in vitro experimental evidence when the specific variant was heterologously expressed and its (transport) function and/or folding-stability was investigated (Table I). We considered functional differences only if the level of expression of the given nsSNP variant was comparable to the wild type expressed in the same system. Our results, in comparison with the predictions of Kelly et al. are summarized in Table I. The left five columns of the table is taken from the publication of Kelly et al. On the right we list our findings and give the related primary reference. In several cases more than one independent studies were found showing the same result. To keep the list of references short, we indicated only one reference, if more publications were found we added +n (where n is the number of additional independent publications). Table I Predicted and Published Phenotype of nsSNPs in Human ABC Transporters We found the published data on 19 of the 40 ABC nsSNPs. Of these, 16 nsSNPs had been tested in in vitro experiments and in seven examples the given nsSNP resulted in impaired function and/or folding. In one case the published nsSNP generates a new splice site (exon skipping), what affects the mRNA maturation therefore the function or folding of the protein with the given nsSNP can not be concluded (ABCB11, E1186K). One of the nsSNPs was characterized solely by its association to a genetic disease (ABCC6, A1291T associated to pseudoxanthoma elasticum). One additional nsSNP was found as homozygous nsSNP in several healthy individuals therefore it was categorized as neutral, on the basis of this human genetic data (ABCC6, R1268Q). There are two nsSNPs with both published genetic association and in vitro experimental characterization in our data set (ABCB11 V444A associated to intrahepatic cholestasis of pregnancy; ABCG2, Q141K associated to gout). Our conclusions on the nsSNPs are listed in column “Published phenotype” of Table I.; for the sake of simplicity we kept the bivalent terminology of Kelly et al.: disease or neutral. Comparing the predicted phenotype of the Kellys paper with the phenotype extracted from the published data reveals misprediction in roughly half of the cases (10/19) thus demonstrating the inherent difficulties of rationalizing and predicting the functional impact of snSNPs.