The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca(2+) handling

Mutations 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

A Regulator Role for the ATP-Binding Cassette Subfamily C Member 6 Transporter in HepG2 Cells: Effect on the Dynamics of Cell-Cell and Cell-Matrix Interactions

There is growing evidence that various ATP-binding cassette (ABC) transporters contribute to the growth and development of tumors, but relatively little is known about how the ABC transporter family behaves in hepatocellular carcinoma (HCC), one of the most common cancers worldwide. Cellular model studies have shown that ABCC6, which belongs to the ABC subfamily C (ABCC), plays a role in the cytoskeleton rearrangement and migration of HepG2 hepatocarcinoma cells, thus highlighting its role in cancer biology. Deep knowledge on the molecular mechanisms underlying the observed results could provide therapeutic insights into the tumors in which ABCC6 is modulated. In this study, differential expression levels of mRNA transcripts between ABCC6-silenced HepG2 and control groups were measured, and subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Real-Time PCR and Western blot analyses confirmed bioinformatics; functional studies support the molecular mechanisms underlying the observed effects. The results provide valuable information on the dysregulation of fundamental cellular processes, such as the focal adhesion pathway, which allowed us to obtain detailed information on the active role that the down-regulation of ABCC6 could play in the biology of liver tumors, as it is involved not only in cell migration but also in cell adhesion and invasion

New heteroaryl carbamates: synthesis and biological screening in vitro and in mammalian cells of wild-type and mutant HIV-protease inhibitors

New heteroaryl HIV-protease inhibitors bearing a carbamoyl spacer were synthesized in few steps and high yield, from commercially available homochiral epoxides. Different substitution patterns were introduced onto a given isopropanoyl-sulfonamide core that can have either H or benzyl group. The in vitro inhibition activity against recombinant protease showed a general beneficial effect of both carbamoyl moiety and the benzyl group, ranging the IC50 values between 11 and 0.6 nM. In particular, benzofuryl and indolyl derivatives showed IC50 values among the best for such structurally simple inhibitors. Docking analysis allowed to identify the favorable situation of such derivatives in terms of number of interactions in the active site, supporting the experimental results. The inhibition activity was also confirmed in HEK293 mammalian cells and was maintained against protease mutants. Furthermore, the metabolic stability was comparable with that of the commercially available inhibitors

Antioxidant and Proapoptotic Activities of Sclerocarya birrea [(A. Rich.) Hochst.] Methanolic Root Extract on the Hepatocellular Carcinoma Cell Line HepG2

The main goal of this study was to characterize the in vitro antioxidant activity and the apoptotic potential of S. birrea methanolic root extract (MRE). Among four tested extracts, obtained with different solvents, MRE showed the highest content of polyphenols, flavonoids, and tannins together with antioxidant activities tested with superoxide, nitric oxide, ABTS, and beta-carotene bleaching assays. Moreover, the cytotoxic effect of MRE was evaluated on the hepatocarcinoma cell line HepG2. In these cells, MRE treatment induced apoptosis and generated reactive oxygen species (ROS) in dose-dependent manner. The cytotoxic effect promoted by MRE was prevented by pretreatment of HepG2 cells with N-acetyl-L-cysteine (NAC), suggesting that oxidative stress was pivotal in MRE-mediated cell death. Moreover, we showed that the MRE treatment induced the mitochondrial membrane depolarization and the cytochrome c release from mitochondria into the cytosol. It suggests that the apoptosis occurred in a mitochondrial-dependent pathway. Interestingly, MRE showed a sensibly lower cytotoxicity, associated with a low increase of ROS, in normal human dermal fibroblasts compared to HepG2 cells. It is suggested that the methanolic root extract of S. Birrea is able to selectively increase intracellular ROS levels in cancer cells, promoting cell death

The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane

Hepatitis B x antigen up-regulates the liver expression of URG7 that contributes to sustain chronic virus infection and to increase the risk for hepatocellular carcinoma by its anti-apoptotic activity. We have investigated the subcellular localization of URG7 expressed in HepG2 cells and determined its membrane topology by glycosylation mapping in vitro. The results demonstrate that URG7 is N-glycosylated and located to the endoplasmic reticulum membrane with an Nlumen–Ccytosol orientation. The results imply that the anti-apoptotic effect of URG7 could arise from the C-terminal cytosolic tail binding a pro-apoptotic signaling factor and retaining it to the endoplasmic reticulum membrane

PEGylated Liposomes Loaded with Carbamate Inhibitor ANP0903 Trigger Apoptosis by Enhancing ER Stress in HepG2 Cancer Cells

Liver cancer is one of the most common causes of cancer death worldwide. In recent years, substantial progress has been made in the development of systemic therapies, but there is still the need for new drugs and technologies that can increase the survival and quality of life of patients. The present investigation reports the development of a liposomal formulation of a carbamate molecule, reported as ANP0903, previously tested as an inhibitor of HIV-1 protease and now evaluated for its ability to induce cytotoxicity in hepatocellular carcinoma cell lines. PEGylated liposomes were prepared and characterized. Small, oligolamellar vesicles were produced, as demonstrated by light scattering results and TEM images. The physical stability of the vesicles in biological fluids was demonstrated in vitro, alongside the stability during storage. An enhanced cellular uptake was verified in HepG2 cells treated with liposomal ANP0903, resulting in a greater cytotoxicity. Several biological assays were performed to elucidate the molecular mechanisms explaining the proapoptotic effect of ANP0903. Our results allow us to hypothesize that the cytotoxic action in tumor cells is probably due to the inhibition of the proteasome, resulting in an increase in the amount of ubiquitinated proteins within the cells, which in turn triggers activation of autophagy and apoptosis processes, resulting in cell death. The proposed liposomal formulation represents a promising approach to deliver a novel antitumor agent to cancer cells and enhance its activity

Biochemical Characterization and NMR Study of the Region E748-A785 of the Human Protein MRP6/ABCC6

Multidrug-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

Synthetic and natural compounds regulate activity of ABCC6 transporter

SIB 2019 Synthetic and natural compounds regulate activity of ABCC6 transporter DEPARTMENT OF SCIENCES- UNIVERSITY OF BASILICATA, Potenza, Italy *CORRESPONDING AUTHOR: [email protected] ATP-binding cassette transporters (ABC transporters) are members of a superfamily consisting of 49 proteins that transport a wide variety of substrates across extra- and intracellular membranes by using energy released from ATP hydrolysis [1]. Mutations in ABC genes underlie at least 19 human genetic disorders [2]. Many transporters are involved in the multidrug resistance, that is the failure due to the reduction of cellular accumulation of drugs. Many ABC transporters inhibitors have been proposed as molecules able to reverse multidrug resistance in cancer cells [3]. ABCC6 is a member of ABCC family poorly involved in drug resistance whose mutations are associated to the Pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by a progressive ectopic calcification [4]. Although the substrate transported is not known, ABCC6 has been shown to promote ATP release [5]. In this study we evaluate the effects of both probenecid, an uricosuric drug and quercetin, a nonterpenoid secondary metabolite on HepG2 cells. Both are known broad spectrum inhibitors of ABC transporters. The observed effects are all reversed by the addition of ATP suggesting the involvement of the ABCC6 protein. FUNDED BY INBIOMED PROJECT (MIUR, ARS01_01081) [1] S. Wilkens. (2015) F1000Prime Rep. 7, 14. [2] V. Vasiliou, K. Vasiliou, DW. Nebert (2009) Hum Genomics. 3(3):281-90. [3] F. Cuviello, F. Bisaccia, D. Spinelli, et al. (2019) Eur J Med Chem 164 1-7. [4] F. Martinelli, F. Cuviello, MC Pace et al. (2018) Cells. Front. Mol. Biosci., 5:75. [5] RS Jansen, S. Duijst, S. Mahakena, et al. . (2014) Arterioscler. Thromb. Vasc. Biol. 34, 1985–198

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

Is the Pseudoxanthoma Elasticum (PXE) a purinergic disease?

Mutations in the human ABCC6 gene, a member of ABCs protein superfamily that encodes MRP6 protein which is involved in multi-drug resistance phenomena [1], cause Pseudoxanthoma Elasticum (PXE), a multisystemic disorder characterized by progressive ectopic calcification of the mineralized elastic fibers in dermal, ocular and vascular tissues [2]. MRP6 is mainly present in liver and in kidney, especially in the basolateral plasma membrane of hepatocytes, and it was found that MRP6 is involved in the release of ATP out of the cell. ATP is immediately hydrolyzated in adenosine and in PPi (a mineralization inhibitor) by ENPP1 and CD73 proteins [3]. In order to characterize the role of ABCC6 in hepatic cells and in the physiopathology of PXE, ABCC6 gene expression was silenced in HepG2 cells. In HepG2 cells with silenced ABCC6, a variation of expression in genes involved in mineralization processes and a downregulation of NT5E gene have been observed [2]. In this work the effects of an inhibitor of ABC proteins activity in HepG2 cells have been evaluated. The results obtained show that, in the presence of this inhibitor, both CD73 and MRP6 protein levels are downregulated, while TNAP protein level is unchanged. In order to characterize the effects of the inhibition of purinergic pathway due to MRP6 low activity, the HepG2 cells were treated with this inhibitor in the presence and in the absence of adenosine. The results obtained show that there is a variation of genes involved in mineralization processes regulated by the purinergic pathway, such as NT5E and TNAP