1,720,981 research outputs found
Biochemical Characterization and NMR Study of the Region E748-A785 of the Human Protein MRP6/ABCC6
No full textMultidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family
which couple ATP hydrolysis with the transport of molecules across biological membranes. MRP6 topology presents
three transmembrane domains and two nucleotide-binding domains (NBDs). The protein is structurally and functionally
poorly characterized. Mutations in ABCC6 gene cause Pseudoxanthoma elasticum, a recessive genetic disorder affecting
the elastic tissues. Most mutations have been found in NBDs that are critical for ATP binding and hydrolysis.
With the aim to better characterize MRP6, we have performed a preliminary study on the fragment E748-A785 of MRP6-
NBD1, with the wild type sequence and the R765Q mutation found in PXE affected patients. CD and NMR spectroscopy
show the presence of helical structures in both peptides. Fluorescence experiments demonstrate that peptides bind ATP.
The NMR structure of the mutated peptide is compared with the corresponding region of the MRP6-NBD1 modeled structure
using as a template the X-ray structure of MRP1-NBD1. The finding that both wild type and mutated peptide present
the same structure and similar affinity for ATP suggests that the onset of PXE symptoms is a consequence of the different
type of interactions involving residue 765 R/Q inside the protein
Evidence for different cadmium detoxification system in Saccharomyces Cerevisiae.
No full textThe yeast genome contains 30 ABC proteins. Of these proteins 22 are predicted to contain multiple membrane spans and are thus considered to be true ABC transporters, while the remaining 8 presumably carry out non-transport functions in the cell. Phylogenetic analyses established the existence of six ABC subfamilies. Member of ABCC subfamily also called MRP are involved in the efflux of xenobiotics in eukaryotic cells. Five members of the ABCC subfamily are full length (Ycf1p, Bpt1p, Ybt1, Nft1p, and Vmr1p), one is short (Yor1p). While Yor1p localizes to the plasma membrane the others localized to the vacuolar membrane (1). In many cases yeast ABCCs exhibit overlapping substrate specificity. There is not a critical functional distinction between full-length and short ABCCs. On glucose medium the main transporter of cadmium-GS conjugates into the vacuole is the Yeast Cadmium Factor, Ycf1p (2), while on ethanol/glycerol medium Vmr1p mediates cadmium detoxification (3).
In this study, experiments carried out in presence of L-Buthionine-sulfoximine (BSO), a specific inhibitor of γglutamylcysteine synthetase, on Saccharomyces Cerevisiae wild type and Vmr1p- deleted strain, suggested that on ethanol/glycerol medium the Vmr1p mediates cadmium detoxification but not through formation of Cd[GS]2 complexes.
1. Paumi C, et al. (2009) Microbiol.Mol.Biol.Rev 73, 577
2. Li Z, Szczypka M, Lu Y, Thiele D & Rea P. J Biol Chem (1996) 271: 6509–6517.
3. D Wawrzycka, I Sobczak, G Bartosz, T Bocer, S Ułaszewski1 & Andr ́e Goffeau. FEMS Yeast Res. 2010;10(7):828-38
Dysregulation of gene expression in ABCC6 knockdown HepG2 cells
Full text linkABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a β-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes
Expression, Purification and Structural Characterization of Up-Regulated Gene 7 Encoded Protein
No full textUp-Regulated Gene 7 (URG7) is a host gene up-regulated in HBV infected hepatocytes that has been suggested to have an anti-apoptotic activity mediated by caspases 3 and 8 and an endoplasmic reticulum localization. Here we report the structural characterization of the encoded protein URG7 by circular dichroism and fluorescence spectroscopy in different solvent media: phosphate buffer and two membrane-mimetic solvents, i.e. 2,2,2-trifluoroethanol (TFE) and SDS micelles. In all solvents URG7 contains substantial amounts of secondary structures. To obtain information about the structural organization and stability of URG7, its thermal denaturation in a membrane environment was studied and intermediate states of thermal unfolding were observed. Furthermore, fluorescence results in SDS micelles could be compatible with different environments for the four tryptophan residues in URG7. Preliminary NMR data indicate that URG7 in TFE solution is quite flexible and not well folded. These data are the first structural information on URG7 and might provide an insight into its structure-function relationships
The NBD domains of the MRP6/ABCC6
No full textMultidrug resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC energy-dependent efflux pumps family which members share many characteristic structural features, including two membrane-spanning domains and two nucleotide-binding domains (NBD1 and NBD2) that function cooperatively but not equally bind and hydrolyze ATP [1]. In this family MRP6 plays an important physiological role as demonstrated by the fact that mutations in this gene cause Pseudoxanthoma elasticum (PXE) in humans, a recessive genetic disorder affecting connective tissues characterized by progressive mineralization of elastic fibers [2]. In the present study, NBD1 and NBD2 of MRP6 has been expressed in Escherichia coli, purified and structurally and functionally characterized. The CD spectra demonstrate the presence of -helices, -strands and turns. Both polypeptides are shown to be biologically active. NBD2 binds ATP with an affinity similar to NBD1 but the ATPase activity is significantly different in the isolated NBDs. The mixture of NBD2 and NBD1 exhibited an activity similar to the NBD2 alone, indicating that NBD1 and NBD2 form a heterodimer with the latter limiting ATP hydrolysis. These findings suggest that NBD1 has a higher tendency to form an active homodimer, which is also supported by in silico analysis of energy-minimized dimers of the homology models of the two domains.
1. Slot AJ, Molinski SV, Cole SP. Mammalian multidrug-resistance proteins (MRPs). Essays Biochem. 2011;50(1):179-207.
2. Kavukcuoglu NB, Li Q, Pleshko N, Uitto J. Connective tissue mineralization in Abcc6-/- mice, a model for pseudoxanthoma elasticum. Matrix Biol. 2012;31(4):246-52
The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca(2+) handling
Full text linkMutations in the Lamin A/C gene (LMNA), which encodes A-type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co-segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease-causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP- (or mCherry)-tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK-CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry-R321X also induced impaired ER Ca(2+) handling, reduced capacitative Ca(2+) entry at the plasma membrane and abnormal nuclear Ca(2+) dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions
New insight in cadmium detoxification system in Saccharomyces Cerevisiae
No full textATP-binding cassette (ABC) proteins constitute one of the largest protein superfamilies, with more than 3000 members operating from bacteria to man. These proteins catalyze the ATP-dependent transport of different molecules across cellular membranes (1). Generally, ABCC subfamily members transport most substrates in the form of GSH conjugates and, in some cases, glucuronide or sulfate conjugates, rather than transporting the unmodified substrates themselves (2). Mutations in several members of the ABC transporter genes cause human diseases, including cystic fibrosis, pseudoxanthoma elasticum, and Dubin-Johnson syndrome (3). Because yeast harbors several homologues of mammalian ABC proteins with medical importance, understanding their molecular mechanisms and substrate interaction might help identifying new approaches aimed to overcome drug resistance or other ABC-mediated diseases.
The yeast genome contains 30 ABC proteins. Phylogenetic analyses established the existence of six ABC subfamilies (ABCB-ABCG using the mammalian nomenclature). Five members of ABCC subfamily (Ycf1p, Bpt1p, Ybt1, Nft1p and Vmr1p) are “full length” and one (Yor1p) is “short” . While Yor1p localizes to the plasma membrane the others localized to the vacuolar membrane (4). Vmr1 and Nft1p are the least characterized of the yeast ABCC transporters. Recent studies have shown that Vmr1p is specifically involved in multidrug resistance and contributes to cadmium resistance on ethanol/glycerol medium, while Ycf1p (Yeast Cadmium Factor) is the main transporter of GSH - cadmium on glucose medium (5).
In this study, experiments carried out on Saccharomyces Cerevisiae wild type and Vmr1p- deleted strain in presence of L-Buthionine-sulfoximine (BSO), a specific inhibitor of γglutamylcysteine synthetase, suggested that on ethanol/glycerol medium the Vmr1p mediates cadmium detoxification but not through formation of Cd[GS]2 complexes.
Also, fluorescence microscopy experiments of wild type, YCF1 and VMR1 cells, grown on respiratory medium containing monochlorobimane, suggest that, compared to Ycf1p, Vmr1p does not contribute to the vacuolar accumulation of monochlorobimane-GS by intact cells. This result leaves assume that Vmr1p does not transport glutathione S-coniugate.
References
1. Holland I., et al. (2003) ABC proteins from bacteria to men. Academic Press-Elsevier Science.
2. Cole S. P. and Deeley R. G. (2006) Trends Pharmacol. Sci. 27:438–446.
3. Dean M. et al. (2005) Methods Enzymol. 400:409–429.
4. Paumi C, et al. (2009) Microbiol.Mol.Biol.Rev 73, 577-593.
5. Wawrzycka D. et al. (2010) FEMS Yeast Res.;10(7):828-38
Structural and functional studies on multidrug resistance protein 6 (mrp6): new insights.
No full textA Comparative Study on Phytochemical Profiles and Biological Activities of Sclerocarya birrea (A.Rich.) Hochst Leaf and Bark Extracts
Full text linkSclerocarya birrea (A.Rich.) Hochst (Anacardiaceae) is a savannah tree that has long been used in sub-Saharan Africa as a medicinal remedy for numerous ailments. The purpose of this study was to increase the scientific knowledge about this plant by evaluating the total content of polyphenols, flavonoids, and tannins in the methanol extracts of the leaves and bark (MLE and MBE, respectively), as well as the in vitro antioxidant activity and biological activities of these extracts. Reported results show that MLE is rich in flavonoids (132.7 ± 10.4 mg of quercetin equivalents/g), whereas MBE has the highest content of tannins (949.5 ± 29.7 mg of tannic acid equivalents/g). The antioxidant activity was measured using four different in vitro tests: β-carotene bleaching (BCB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), O2-•, and nitric oxide (NO•) assays. In all cases, MBE was the most active compared to MLE and the standards used (Trolox and ascorbic acid). Furthermore, MBE and MLE were tested to evaluate their activity in HepG2 and fibroblast cell lines. A higher cytotoxic activity of MBE was evidenced and confirmed by more pronounced alterations in cell morphology. MBE induced cell death, triggering the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation, which led to a loss of mitochondrial membrane potential with subsequent cytochrome c release from the mitochondria into the cytosol. Moreover, MBE showed lower cytotoxicity in normal human dermal fibroblasts, suggesting its potential as a selective anticancer agent
- …
