Expression, Purification and Structural Characterization of Up-Regulated Gene 7 Encoded Protein

Up-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

Structural characterization of the L0 cytoplasmic loop of human multidrug resistance protein 6 (MRP6)

ABCC6 is a member of the C subfamily of ATP-binding cassette transporters whose mutations are correlated to Pseudoxanthoma elasticum, an autosomal recessive, progressive disorder characterized by ectopic mineralization and fragmentation of elastic fibers. Structural studies of the entire protein have been hindered by its large size, membrane association, and domain complexity. Studies previously performed have contributed to shed light on the structure and function of the nucleotide binding domains and of the N-terminal region. Here we report the expression in E. coli of the polypeptide E205-G279 contained in the cytoplasmic L0 loop. For the first time structural studies in solution were performed. Far-UV CD spectra showed that L0 is structured, assuming predominantly α-helix in TFE solution and turns in phosphate buffer. Fluorescence spectra indicated some flexibility of the regions containing aromatic residues. 1H NMR spectroscopy identified three helical regions separated by more flexible regions

New insight in cadmium detoxification system in Saccharomyces Cerevisiae

ATP-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

Extracellular ATP Regulates CD73 and ABCC6 Expression in HepG2 Cells

The ATP-binding cassette sub-family C member 6 transporter (ABCC6) is an ATP dependent transporter mainly found in the basolateral plasma membrane of hepatic and kidney cells. Mutations in ABCC6 gene were associated to the Pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by a progressive ectopic calcification of elastic fibers in dermal, ocular, and vascular tissues. It is reported that the over-expression of ABCC6 in HEK293 cells results in the cellular efflux of ATP and other nucleoside triphosphates, which in turn are rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since PPi is an inhibitor of mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the ectopic mineralization, a typical feature of PXE. In the extracellular environment, ATP is converted, not only into pyrophosphate, but also into AMP by an ectonucleosidase, which in turn is transformed into adenosine and phosphate. ABCC6 protein is thus involved in the production of extracellular adenosine and therefore it could have a role in the activation of the purinergic system. In the liver, purinergic signaling has been shown to regulate key basic cellular functions. Our previous studies showed that in ABCC6 knockdown HepG2 cells the expression of some genes, related with the calcification processes, is dysregulated. In this study, experiments have been carried out in order to verify if ABCC6, besides supplying the pyrophosphate required to prevent the mineralization of soft tissues, also plays a role in the activation of the purinergic system. For this purpose, the transport activity of ABCC6 was blocked with Probenecid and the expression of ABCC6 and NT5E was analyzed with real time PCR and western blotting. The results of this study showed that both proteins are downregulated in the presence of Probenecid and upregulated in the presence of adenosine or ATP

The P-glycoprotein inhibitor diltiazem-like 8-(4-chlorophenyl)-5- methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4] thiazin-3-one inhibits esterase activity and H3 histone acetylation

With the aim to reduce multidrug resistance several molecules were synthesized and tested for their ability to inhibit ATP-binding cassette (ABC) proteins, which are responsible for drugs transport out from cells. The compound 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo [3,4-c][1,4]thiazin-3-one namely 2c, is structurally related to the myocardial-calcium-channel-modulator diltiazem and is considered one of the most efficient P-glycoprotein inhibitors, able to induce apoptosis at low concentrations of doxorubicin in multidrug resistant ovarian cells. In this study experiments were carried out to evaluate other biological activities of compound 2c. We verified the ability of 2c to inhibit ABC transporters do not involved in drug resistance and considering the inhibitory effect of diltiazem on recombinant human carboxylesterase, we observed its inhibitory effect on the esterase activity. Our findings demonstrated that 2c exhibits broad-spectrum activity as ABC transporters inhibitor being able to inhibit ABCC6, a protein belonging to the ABC family although poorly involved in drug resistance. 2c also inhibits cell esterase activity, acetylcholine esterase activity in vitro and cell histone H3 acetylation according to its structural homology with some known HAT inhibitors. The results obtained provide new knowledge on the biological activities of 2c and represent useful information when it is used as an inhibitor of drug resistance

Expression of VDAC (Voltage-Dependent Anion Channel) isoforms in hematological cancers

Voltage-Dependent Anion Channels (VDACs), also known as mitochondrial porins, are proteins located in the mitochondrial outer membrane (OMM). In humans there are three VDAC isoforms namely VDAC1, VDAC2 and VDAC3, encoded by three different genes with high level of homology. VDACs form pores through OMM and are involved in mitochondrial metabolic and energetic functions and in apoptotic cell death [1]. VDACs exchange metabolites between cytoplasm and mitochondria and serve as anchor point for mitochondria-interacting proteins [2-3]; in particular hexokinase II (HKII) binds to VDAC in cancer cells and provides both metabolic advantage and anti-apoptotic activity [4]. With the aim to evaluate if VDACs are useful prognostic markers for hematological cancers, we analyzed, by Real time PCR, expression levels of VDACs genes in bone marrow cells derived from patients with Acute Myeloid Leukemia (AML), Multiple Myeloma (MM) at the outset of the disease and in bone marrow cells derived from healthy subjects. The results found show that patients with VDAC2 and/or VDAC1 isoforms up-regulated had poor prognosis. Moreover, VDAC3 was down regulated in all patients with poor prognosis and HKII was up-regulated in almost all samples. Although these results have been observed on a limited number of samples, they suggest that VDAC1 and VDAC2 have anti-apoptotic activity in hematological cancers and could explain cancer cells survival in patients with poor prognosis. If these results will be confirmed on a larger number of patients, expression level of VDACs could be used as prognostic marker of hematological cancers. [1] V. Shoshan-Barmatz, et al (2010) VDAC, a multi-functional mitochondrial protein regulating cell life and death. Mol. Aspects Med. 31, 227-85. [2] A. Messina, et al (2012) VDAC isoforms from mammals. Biochim Biophys Acta, 1818, 1466-1476. [3] S. Reina, et al. (2010) Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell. FEBS Letters 584, 2837-44

Expression of some ATP-binding cassette transporters in Acute Myeloid Leukemia.

Hematopoietic cells express ATP binding cassette (ABC) transporters in relation to different degrees of differentiation. One of the known multidrug resistance mechanisms in acute myeloid leukemia (AML) is the overexpression of efflux pumps belonging to the superfamily of ABC transporters such as ABCB1, ABCG2 and ABCC1. Although several studies were carried out to correlate ABC transporters expression with drug resistance, little is known about their role as markers of diagnosis and progression of the disease. For this purpose we investigated the expression, by real-time PCR, of some ABC genes in bone marrow samples of AML patients at diagnosis and after induction therapy. At diagnosis, ABCG2 was always down-regulated, while an up regulated trend for ABCC1 was observed. After therapy the examined genes showed a different expression trend and approached the values of healthy subjects suggesting that this event could be considered as a marker of AML regression. The expression levels of some ABC transporters such as ABCC6, seems to be related to gender, age and to the presence of FLT3/ITD gene mutation

Expression of ABCC6 gene in Acute Myeloid Leukemia

Multidrug resistance (MDR) is a major impediment to the successful treatment of acute myeloid leukemia (AML). One of the known MDR mechanisms is the over expression of efflux pumps belonging to the superfamily of ABC transporters, such as P-glycoprotein (ABCB1), BCRP (ABCG2) and MRP1 (ABCC1) [1]. At present, little is known about the clinical relevance of other ABC-transporters in AML. However it was observed that patients with acute lymphoblastic leukemia presenting high MRPs expression, including MRP6, have unfavorable prognosis [2]. In this study, we investigated the expression of ABCB1, ABCC1, ABCG2 and ABCC6 genes in six healthy controls and in thirteen patients with AML, at diagnosis, after chemotherapy, at refractory disease and at relapse. Real-time PCR results showed that at diagnosis, compared to healthy subjects, all patients, except one, presented at least one among ABCB1/ABCC1/ABCC6 genes up-regulated; instead ABCG2 was always down-regulated. Moreover, we interestingly observed that three patients with poor prognosis exhibited higher level of ABCC6 mRNA after treatment compared to diagnosis and that in these patients also BCRP was up-regulated, sign of chemoresistance. We also observed that ninety percent of treated patients presented ABCG2 expression significantly higher after treatment than at diagnosis; thirty percent presented over expression of ABCB1 and only two patients showed ABCC1 up-regulation. In conclusion, our results showed a possible involvement of MRP6 in the development of MDR in AML and that BCRP has, more than MDR1 and MRP1, a relevant role in this mechanism. ABCC6 resulted up-regulated in thirty percent of treated AML patients, but further studies on a larger number of patients are necessary to establish if MRP6 may be involved in the treatment failure of AML and if co-expression of these ABC-transporters may have prognostic significance. REFERENCES 1. Brian C. Shaffer et al. Drug Resist Updat. 2012; 15(1-2): 62–69. 2. Plasschaert SL et al. Clin Cancer Res. 2005; 15;11(24 Pt 1):8661-8

Immunochemical Characterization of the Specific Sequence of URG7 Protein

URG7 is an anti-apoptotic protein which consists of 99 amino acid residues up regulated by antigen x during the HBV infection. The first 74 amino acids are identical to those of the multidrug resistance protein 6 (MRP6), while the amino acid residues from 75 to 99 are specific for URG7 protein. Immuno-informatics tools and secondary structure analysis were carried out to identify the antigenic properties of this URG7 sequence. The 75-99 peptide was synthesized by the solid-phase method, structurally characterized by CD spectroscopy and conjugated to a protein carrier. New Zealand white rabbits were immunized and sera were tested for anti-peptide specific antibodies by ELISA and western blot analysis. Finally ELISA test with human sera was performed. Rabbits immunized with the 75-99 peptide produce antibodies that recognize both the 75-99 peptide and the URG7 recombinant polypeptide. Moreover, both antigens allowed for the detection of the anti-URG7 antibodies in sera of healthy and HBV infected subjects although with a different discriminant threshold. Our data suggested that peptide ELISA assay against the specific sequence of the URG7 protein allows with good sensitivity and specificity for the detection of anti-URG7 antibodies in sera from HBV infected patients