Energy-dependent Mechanism Research Articles

An amphipathic trans-acting phosphorothioate RNA element delivers an uncharged phosphorodiamidate morpholino sequence in mdx mouse myotubes.

An efficient method for the delivery of uncharged polyA-tailed phosphorodiamidate morpholino sequences (PMO) in mammalian cells consists of employing a synthetic 8-mer amphipathic trans-acting poly-2'-O-methyluridylic thiophosphate triester element (2'-OMeUtaPS) as a transfection reagent. Unlike the dTtaPS DNA-based element, this RNA element is potent at delivering polyA-tailed PMO sequences to HeLa pLuc 705 cells or to myotube muscle cells. However, much like dTtaPS, the 2'-OMeUtaPS-mediated internalization of PMO sequences occurs through an energy-dependent mechanism; macropinocytosis appears to be the predominant endocytic pathway used for cellular uptake. The transfected PMO sequences induce alternate splicing of either the pre-mRNA encoding luciferase in HeLa pLuc 705 cells or the excision of exon 23 from the pre-mRNA encoding dystrophin in myotube muscle cells of the mdx mouse model of muscular dystrophy with an efficiency comparable to that of commercial cationic lipid reagents but without detrimental cytotoxicity.

A multi-functional PEGylated gold(iii) compound: potent anti-cancer properties and self-assembly into nanostructures for drug co-delivery.

Gold(iii) porphyrin-PEG conjugates [Au(TPP-COO-PEG5000-OCH3)]Cl (1) and [Au(TPP-CONH-PEG5000-OCH3)]Cl (2) have been synthesized and characterized. Based on the amphiphilic character of the conjugates, they were found to undergo self-assembly into nanostructures with size 120-200 nm and this did not require the presence of other surfactants or components for nano-assembly, unlike most conventional drug nano-formulations. With a readily hydrolyzable ester linkage, chemotherapeutic [Au(TPP-COOH)]+ exhibited triggered release from the conjugate 1 in acidic buffer solution as well as in vitro and in vivo without the formation of toxic side products. The nanostructures of 1 showed higher cellular uptake into cancer cells compared to non-tumorigenic cells, owing to their energy-dependent uptake mechanism. This, together with a generally higher metabolic rate and more acidic nature of cancer cells which can lead to faster hydrolysis of the ester bond, afforded 1 with excellent selectivity in killing cancer cells compared with non-tumorigenic cells in vitro. This was corroborated by fluorescence microscopy imaging and flow cytometric analysis of co-culture model of colon cancer (HCT116) and normal colon (NCM460) cells. In vivo experiments showed that treatment of nude mice bearing HCT116 xenografts with 1 resulted in significant inhibition of tumor growth and, more importantly, minimal systemic toxicity as revealed by histopathological analysis of tissue sections and blood biochemisty. The latter is explained by a lower accumulation of 1 in organs of treated mice at its effective dosage, as compared to that of other gold(iii) porphyrin complexes. Co-assembly of 1 and doxorubicin resulted in encapsulation of doxorubicin by the nanostructures of 1. The nanocomposites demonstrated a strong synergism on killing cancer cells and could overcome efflux pump-mediated drug-resistance in a doxorubicin-resistant ovarian cancer cell line (A2780adr) which was found in cells incubated with doxorubicin alone. Also, the nanocomposites accumulated more slowly in non-tumorigenic cells, resulting in a lower toxicity toward non-tumorigenic cells. These results indicate the potential application of 1 not only as an anti-cancer agent but also as a nanoscale drug carrier for chemotherapy.

ABCG2/BCRP: Specific and Nonspecific Modulators.

Multidrug resistance (MDR) in cancer cells is the development of resistance to a variety of structurally and functionally nonrelated anticancer drugs. This phenomenon has become a major obstacle to cancer chemotherapy seriously affecting the clinical outcome. MDR is associated with increased drug efflux from cells mediated by an energy-dependent mechanism involving the ATP-binding cassette (ABC) transporters, mainly P-glycoprotein (ABCB1), the MDR-associated protein-1 (ABCC1), and the breast cancer resistance protein (ABCG2). The first two transporters have been widely studied already and reviews summarized the results. The ABCG2 protein has been a subject of intense study since its discovery as its overexpression has been detected in resistant cell lines in numerous types of human cancers. To date, a long list of modulators of ABCG2 exists and continues to increase. However, little is known about the clinical consequences of ABCG2 modulation. This makes the design of novel, potent, and nontoxic inhibitors of this efflux protein a major challenge to reverse MDR and thereby increase the success of chemotherapy. The aim of the present review is to describe and highlight specific and nonspecific modulators of ABCG2 reported to date based on the selectivity of the compounds, as many of them are effective against one or more ABC transport proteins.

A shell-crosslinked polymeric micelle system for pH/redox dual stimuli-triggered DOX on-demand release and enhanced antitumor activity

Based on targeted amphiphilic block copolymer N-acetyl glucosamine-poly (styrene-alt-maleic anhydride)58-b-polystyrene130 (NAG-P(St-alt-MA)58-b-PSt130), a pH/redox dual-triggered shell-crosslinked polymeric micelle system was constructed. The shell-crosslinked micelles (CLM) were prepared by post-crosslinking method to regulate drug release kinetics using cystamine as linkers between carboxy groups of the shell. Compared with non-crosslinked micelles (NCLM), CLM showed spherical shapes with little increased mean diameter of 102.40±0.54nm, low polydispersity index (PDI) of 0.19±0.36, enlarged zeta potential value from −41.46±0.99 to −9.31±0.50mV, indicating the successful modification of disulfide bonds in shell. In vitro drug release study clearly exhibited a pH and redox dual-sensitive drug release profile with significantly accelerated drug release under pH 5.0 and 10mM GSH conditions (46.84% in 96h) without burst release. Both CLM and NCLM showed quite different release profiles between physiological (pH 7.4) and tumoral microenvironment (pH 5.0), effectively avoiding the premature drug leakage and realizing on-demand drug release. The MTT assay implied that CLM presented a time- and concentration-dependent manner to inhibit proliferation of A549 and MCF-7 cells and much lower IC50 values in comparison with that of NCLM after 72h incubation. Both FCM and CLSM results showed that CLM displayed much higher cellular uptake efficiency and anti-tumor activities than NCLM and free DOX. CLM and NCLM could be internalized by energy-dependent endocytosis mechanism due to similar surface properties. Overall, this dual-stimuli triggered micelle system provided a promising tumor-responsive platform for cancer therapy.

SiO2 encapsulated nanofluorophor: Photophysical properties, aggregation induced emission and its application for cell mitochondria imaging

An organic fluorescent 1,8-naphthalimide derivative (NI-1) was designed and synthesized. Its photophysical properties were systematically investigated by a series of spectroscopic and theoretical methods. The twisted intramolecular charge transfer (TICT) was proved within NI-1 in various solutions. After adding H2O into its CH3CN solution, the aggregation induced emission (AIE) of NI-1 occurred, during which the enhanced green-yellow fluorescence and quantum yield up to 0.62 were monitored. In addition, SiO2 was used to coat on the surface of NI-1 to fabricate the composite nano structure (NI-1@SiO2). This nanomaterial not only qualifies AIE-active characteristic, but also exhibits remarkable anti-photobleaching ability and good biocompatibility. The NI-1@SiO2 can go through the cell membrane and specifically light up the mitochondria. The cellular uptake mechanism studies revealed that the energy-dependent cell uptake mechanism was responsible for the NI-1@SiO2 entering cells. The NI-1@SiO2 could be utilized as an efficient AIE-active fluorescent material and mitochondria imaging reagent.

Nanomicelles loaded with doxorubicin and curcumin for alleviating multidrug resistance in lung cancer.

PurposeA new type of polymeric micelle (PM) was assembled using a polyethylene glycol (PEG)-linked (PEGylated) amphiphilic copolymer and d-tocopheryl PEG1000 succinate (TPGS1000). The micelles were used to deliver doxorubicin (DOX) and curcumin (CUR) for alleviating multidrug resistance (MDR) in lung cancer cells while enhancing the therapeutic efficacy of DOX.MethodsMicelles loaded with DOX and CUR were assembled using a film-forming technique. Micelles were used to treat A549/Adr cells to find out whether micelles had the ability to reverse the MDR of A549/Adr cells. Some investigations were conducted using tumor-bearing mice to assess whether these micelles had enhanced antitumor efficacy as compared to DOX alone or the combination of DOX and CUR.ResultsSome micelles (DOX + CUR)–PMs had a small average size of about 17 nm and showed definite ability to deliver both DOX and CUR into DOX-resistant A549/Adr cells. The PMs had high cytotoxicity toward A549/Adr cells when the applied equivalent DOX dose was 1 µg/mL or higher. The cellular uptake of (DOX + CUR)–PMs into A549/Adr cells was found to be associated with an energy-dependent, caveolae-mediated, and clathrin-independent mechanism. (DOX + CUR)–PMs helped to prolong the circulation of DOX or CUR as compared to the individual administration of DOX or CUR, and they exhibited high inhibiting efficiency against the growth of tumors and were able to reduce the side effects of DOX.ConclusionTPGS1000 and CUR could synergistically reverse DOX-resistance of A549/Adr cells. In vivo examinations confirmed that the micelles had the capability to increase the plasma concentration of DOX or CUR, as well as to prolong their respective blood circulation. These micelles were able to significantly inhibit tumor growth in Lewis lung carcinoma tumor-bearing mice while reducing the side effects of DOX. The micelles showed potential in the treatment of lung cancer.

TAP-Dependent and -Independent Peptide Import into Dendritic Cell Phagosomes.

Cross-presentation of phagocytosed Ags by MHC class I (MHC-I) molecules is thought to involve transport of cytosolic peptides into dendritic cell phagosomes, mediated by TAP transporters recruited from the endoplasmic reticulum. However, because pure and tightly sealed phagosomes are difficult to obtain, direct evidence for peptide transport into phagosomes has remained limited. Moreover, the parameters determining peptide uptake by, and survival in, phagosomes remain little characterized. In this study, we monitored peptide import into phagosomes by flow cytometry using two types of fluorescent reporter peptides, one of which directly bound to intraphagosomal beads. We observed that a peptide with high TAP affinity is imported into phagosomes in a TAP- and ATP-dependent manner, as expected. However, surprisingly, import of the OVA peptide SIINFEKL, a CD8+ T cell epitope frequently used to study cross-presentation, is ATP-dependent but substantially TAP-independent. The half-life of both reporter peptides is shortened by enhanced phagosome maturation triggered by TLR signaling. Conversely, formation of complexes with MHC-I molecules enhances peptide accumulation in phagosomes. Collectively, these results confirm that TAP can import peptides into phagosomes, but they suggest that some peptides, including the popular SIINFEKL, can enter phagosomes also via a second unknown energy-dependent mechanism. Therefore, the frequently reported TAP dependence of cross-presentation of phagocytosed OVA may principally reflect a requirement for recycling MHC-I molecules rather than SIINFEKL import into phagosomes via TAP.

Dietary supplementation with coenzyme Q10 in poor responder patients undergoing IVF-ICSI Treatment

Ovarian aging and age-related reduction in live birth rate reflects a decline in response to ovarian stimulation, reduced embryo quality and pregnancy rate (PR), and an increased incidence of miscarriages and fetal aneuploidy. This is attributed to a high rate of aneuploidy and follicle depletion, probably due to energy-dependent mechanism as mitocondrial mutations in follicular cells increase with age and oxidative phosphorylation an ATP production in the follicle is also impaired in older women. Coenzyme Q10 (CoQ10) is a crucial substrate in the electron transport chain of mitochondria., and has been shown to decrease in several tissues with aging. The aim of this study was to asses if dietary supplementation of mitochondrial nutrients such as C0Q10 may improve mitochondrial energy production for the maturing oocyte and developing embryo, thereby improving ovarian response and reproductive outcome in poor responders. Prospective randomized controlled study. Seventy eight poor responders (36-40 years of age) in a previous IVF cycle according to Bologna criteria were randomized to Group 1 : 600 mg Co Q10 twice a day for 12 weeks and Group 2: no treatment for 12 weeks. Mean age was 37.8 yrs. in Group 1 and 37.2 yrs in group 2. Anti-Mullerian hormone (AMH) (0.67 +- 0.26vs 0.7 +-0.28) and FSH values (12 +- 2.11 vs 11.6 +- 1.89) were comparable. The primary outcomes were number of oocytes retrieved, number of good quality embryos obtained, pregnancy rate and live birth. Both groups were comparable in terms of age, BMI, ovarian reserve (AMH and FSH) and length of treatment. There were no significant differences in the number of MII oocytes retrieved (1.82 ± 0.82 vs. 1.87 ± 0.76; p=0.77), implantation rate (26.2% vs. 21.4%; p=0.75) and clinical pregnancy rate (fetal heartbeat at 7 weeks) (15.4% vs. 12.8%; p=0.64) No significant differences in outcome were detected between the CoQ10 and no treatment groups. However, the sample size of this study may be underpowered to detect a significant difference.

Spermine cycling in mitochondria is mediated by adenine nucleotide translocase activity: mechanism and pathophysiological implications.

Spermine, besides to be transported in mitochondria by an energy dependent electrophoretic mechanism, can be also released by two different mechanisms. The first one is induced in deenergizing conditions by FCCP or antimycin A and it is mediated by an electroneutral exchange spermine protons. The second one takes place in energizing conditions during the activity of the adenine nucleotide translocase and is mediated by an electroneutral symport mechanism involving the efflux in co-transport of spermine and phosphate and the exchange of exogenous ADP with endogenous ATP. The triggering of this mechanism permits an alternating cycling of spermine across the mitochondrial membrane, that is spermine is transported or released by energized mitochondria in the absence or presence of ATP synthesis, respectively. The physiological implications of this cycling of spermine are related to the induction or prevention of mitochondrial permeability transition and, consequently, on apoptosis or its prevention.

An Amphipathic trans-Acting Phosphorothioate DNA Element Delivers Uncharged PNA and PMO Nucleic Acid Sequences in Mammalian Cells.

An innovative approach to the delivery of uncharged peptide nucleic acids (PNAs) and phosphorodiamidate morpholino (PMO) oligomers in mammalian cells is described and consists of extending the sequence of those oligomers with a short PNA-polyA or PMO-polyA tail. Recognition of the polyA-tailed PNA or PMO oligomers by an amphipathic trans-acting polythymidylic thiophosphate triester element (dTtaPS) results in efficient internalization of those oligomers in several cell lines. The authors' findings indicate that cellular uptake of the oligomers occurs through an energy-dependent mechanism and macropinocytosis appears to be the predominant endocytic pathway used for internalization. The functionality of the internalized oligomers is demonstrated by alternate splicing of the pre-mRNA encoding luciferase in HeLa pLuc 705 cells. Amphipathic phosphorothioate DNA elements may represent a unique class of cellular transporters for robust delivery of uncharged nucleic acid sequences in live mammalian cells. © 2016 by John Wiley & Sons, Inc.

Mechanistic Analysis of Cellular Internalization of a Cell- and Skin-Penetrating Peptide

Keratinocytes are a targeted cell-type for treating skin disease. However, delivering drugs into the skin and keratinocytes remains challenging due to transport barriers posed by both the stratum corneum and cell membrane. Nonetheless, SPACE™ peptide has been shown effective in delivering a number of macromolecules into the skin and cells. Accordingly, we sought to explore the mechanism of SPACE™ peptide internalization under conditions relevant for skin delivery. Mechanistic analysis was performed using live cell confocal microscopy. Potential binding targets were assessed by Western blot and mass spectrometry. Experimental data suggest an energy-dependent mechanism for the cellular entry of SPACE™ peptide into human epidermal keratinocytes. SPACE™ peptide associates with the cell membrane within seconds and then is internalized rapidly. Co-incubation with heparin sulfate or serum suggests that association with the cell membrane may be specific in nature. Western blot and mass spectrometry studies are consistent with earlier findings that keratin may play an important role in SPACE™ peptide internalization. This work highlights the potential for delivering drug into the cytoplasm of keratinocytes for the treatment of skin disease through topical application. Further, it highlights an ongoing need for studies involving cell-penetrating peptides under clinically relevant conditions. SPACE™ peptide has been shown to enhance delivery of a wide variety of macromolecules into the skin and cells. However, the mechanism of cell entry of SPACE™ peptide under conditions relevant for skin delivery is not understood. Accordingly, we sought to explore the mechanism of SPACE™ peptide internalization in keratinocytes under conditions relevant for skin delivery. A better understanding of the dominating pathways and major molecular players involved in SPACE™ peptide internalization under clinically relevant conditions will aid the development and translation of SPACE™ peptide-based technologies for treating skin disease.

Carbon Nanostructures Interaction with Giant Unilamellar Vesicles as a Cellular Model

Carbon is a highly versatile element capable of forming a countless number of compounds among which exist different molecular allotropes. These allotropic forms are of great interest to the medical and technological industries. In particular, carbon nanotubes (CNTs) are of interest ought to their ability of penetrating the membrane of mammalian cells without causing any damage. The CNTs internalization is a controversial topic. It has been reported to occur by two different mechanisms: 1) An energy dependent mechanism, where the cell actively internalizes the CNTs via endocytosis and 2) A simple diffusion process, which happens without the active participation of the cell. Nevertheless, it is known very little about the physical and chemical interactions leading these processes.In the present work we analyze these interactions using giant unilamellar vesicles (GUVs) as a representative model of the basic structure of the cell. To assess the effect caused by the CNTs on the GUVs' membranes we implemented the electrodeformation method, which allows us to measure the mechanical properties of the membranes i.e. the bending stiffness and the stretching modulus. Using confocal microscopy we observed the interaction between the CNTs and the GUVs membranes.We found that the bending stiffness of the GUVs in presence of the CNTs incremented one order of magnitude and the elastic module augmented up to 50 times in comparison with the GUVs without CNTs. Confocal microscopy revealed the interaction of the CNTs with the membrane as well as the internalization of the nanotubes and the adhesion of vesicles in presence of CNTs.

Internalization of Carbon Nanotubes in Biological Membranes

The development of nanomaterials for biological and biotechnological applications is an area of research that holds great promise. In particular, carbon nanotubes (CNTs) are attracting an increasing level of attention since they hold unique optical and mechanical properties, have a large surface, diameters on the nanometric scale, and are capable to penetrate the membrane of mammalian cells without causing any damage while transporting small molecules as nucleic acids, peptides and proteins. Mechanisms have been independently reported to explain the internalization of CNT into cells: 1) an energy-dependent mechanism and 2) an energy-independent process. In order to elucidate the cellular uptake mechanism of CNTs, in this work we present a study of the internalization of CNTs with different surface properties into giant unilamellar vesicles (GUVs), which is a simple representation of the basic structure of the cellular membrane. This strategy allows us to follow the CNT internalization process by optical and confocal microscopy while applying controlled external fields.Thin multi-walled carbon nanotubes (thin-MWNTs) with hydrophobic (h-MWNTs) and hydrophilic (ox-MWNTs) surfaces were incubated with GUVs to elucidate if the mechanism of internalization, localization and effects on the membranes, are dependent on the type of carbon nanotubes functionalization.We observed that the ox-MWNT interact with the hydrophilic part of the GUVs and were localized mostly on the surface of the vesicles. However, h-MWNTs were able to interact with the membrane, forming different structures in the GUVs, like aggregates, rings of CNTs across their surface and the strangling of GUVs, which caused the union of 2 or more vesicles.Our observations reveal that functionalized thin-MWNTs are able to translocate across GUVs by an energy-independent process and their localization was different in each case, which suggest that their surface properties play a significant role in this process.

Polystyrene nanoparticles internalization in human gastric adenocarcinoma cells

The increase in the use of nanoparticles, as a promising tool for drug delivery or as a food additive, raises questions about their interaction with biological systems, especially in terms of evoked responses. In this work, we evaluated the kinetics of uptake of 44nm (NP44) and 100nm (NP100) unmodified polystyrene nanoparticles (PS-NPs) in gastric adenocarcinoma (AGS) cells, as well as the endocytic mechanism involved, and the effect on cell viability and gene expression of genes involved in cell cycle regulation and inflammation processes. We showed that NP44 accumulate rapidly and more efficiently in the cytoplasm of AGS compared to NP100; both PS-NPs showed an energy dependent mechanism of internalization and a clathrin-mediated endocytosis pathway. Dose response treatments revealed a non-linear curve. PS-NPs also affected cell viability, inflammatory gene expression and cell morphology. NP44 strongly induced an up-regulation of IL-6 and IL-8 genes, two of the most important cytokines involved in gastric pathologies. Our study suggests that parameters such as time, size and concentration of NPs must be taken carefully into consideration during the development of drug delivery systems based on NPs and for the management of nanoparticles associated risk factors.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

Contraction is crucial in maintaining the differentiated phenotype of myofibroblasts. Contraction is an energy-dependent mechanism that relies on the production of ATP by mitochondria and/or glycolysis. Although the role of mitochondrial biogenesis in the adaptive responses of skeletal muscle to exercise is well appreciated, mechanisms governing energetic adaptation of myofibroblasts are not well understood. Our study demonstrates induction of mitochondrial biogenesis and aerobic glycolysis in response to the differentiation-inducing factor transforming growth factor β1 (TGF-β1). This metabolic reprogramming is linked to the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition of p38 MAPK decreased accumulation of active peroxisome proliferator-activated receptor γ coactivator 1α in the nucleus and altered the translocation of mitochondrial transcription factor A to the mitochondria. Genetic or pharmacologic approaches that block mitochondrial biogenesis or glycolysis resulted in decreased contraction and reduced expression of TGF-β1-induced α-smooth muscle actin and collagen α-2(I) but not of fibronectin or collagen α-1(I). These data indicate a critical role for TGF-β1-induced metabolic reprogramming in regulating myofibroblast-specific contractile signaling and support the concept of integrating bioenergetics with cellular differentiation.

Nrf2: new insight in cell apoptosis.

Nrf2: new insight in cell apoptosis.

Detailed Biological Profiling of a Photoactivated and Apoptosis Inducing pdppz Ruthenium(II) Polypyridyl Complex in Cancer Cells

Ruthenium polypyridyl complexes show great promise as new photodynamic therapy (PDT) agents. However, a lack of detailed understanding of their mode of action in cells poses a challenge to their development. We have designed a new Ru(II) PDT candidate that efficiently enters cells by incorporation of the lipophilic aromatic pdppz ([2,3-h]dipyrido[3,2-a:2',3'-c]phenazine) ligand and exhibits photoactivity through incorporation of 1,4,5,8-tetraazaphenanthrene ancillary ligands. Its photoreactivity toward biomolecules was studied in vitro, where light activation caused DNA cleavage. Cellular internalization occurred via an energy dependent mechanism. Confocal and transmission electron microscopy revealed that the complex localizes in various organelles, including the mitochondria. The complex is nontoxic in the dark, with cellular clearance within 96 h; however, upon visible light activation it induces caspase-dependent and reactive-oxygen-species-dependent apoptosis, with low micromolar IC50 values. This investigation greatly increases our understanding of such systems in cellulo, aiding development and realization of their application in cancer therapy.

Identification and characterization of nuclear and nucleolar localization signals in 58-kDa microspherule protein (MSP58).

BackgroundMSP58 is a nucleolar protein associated with rRNA transcription and cell proliferation. Its mechanism of translocation into the nucleus or the nucleolus, however, is not entirely known. In order to address this lack, the present study aims to determine a crucial part of this mechanism: the nuclear localization signal (NLS) and the nucleolar localization signal (NoLS) associated with the MSP58 protein.ResultsWe have identified and characterized two NLSs in MSP58. The first is located between residues 32 and 56 (NLS1) and constitutes three clusters of basic amino acids (KRASSQALGTIPKRRSSSRFIKRKK); the second is situated between residues 113 and 123 (NLS2) and harbors a monopartite signal (PGLTKRVKKSK). Both NLS1 and NLS2 are highly conserved among different vertebrate species. Notably, one bipartite motif within the NLS1 (residues 44–56) appears to be absolutely necessary for MSP58 nucleolar localization. By yeast two-hybrid, pull-down, and coimmunoprecipitation analysis, we show that MSP58 binds to importin α1 and α6, suggesting that nuclear targeting of MSP58 utilizes a receptor-mediated and energy-dependent import mechanism. Functionally, our data show that both nuclear and nucleolar localization of MSP58 are crucial for transcriptional regulation on p21 and ribosomal RNA genes, and context-dependent effects on cell proliferation.ConclusionsResults suggest that MSP58 subnuclear localization is regulated by two nuclear import signals, and that proper subcellular localization of MSP58 is critical for its role in transcriptional regulation. Our study reveals a molecular mechanism that controls nuclear and nucleolar localization of MSP58, a finding that might help future researchers understand the MSP58 biological signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0136-0) contains supplementary material, which is available to authorized users.

Structure based investigation on the binding interaction of transport proteins in leishmaniasis: insights from molecular simulation.

Leishmania major is the causative agent of cutaneous leishmaniasis which affects over 1 million people in 88 different countries. The incidence of this disease is on the rise due to the current problems associated with the present chemotherapeutics. In addition, Leishmania confronts resistance to the traditional drugs like sodium stibogluconate and newer repurposed drugs like miltefosine. ABC transporters are involved in the development of drug resistance. Miltefosine, the drug used for the treatment of leishmaniasis, is effluxed by P4 ATPase and ABC transporter, which is the prime focus of our study in this paper. P4 ATPase (MDR1) along with an unnamed protein (cdc50) translocates miltefosine from the outer to the inner leaflet by the process of flipping which is ATP driven. In contrast, miltefosine also escapes from the cells by an energy dependent mechanism that involves the ABC transporter protein (ABC). It is known that certain genes in the parasite amplify the portions of a gene which encodes ABC transporter and P4 ATPase involved in translocating phospholipids and hence resistance to miltefosine. We observed the ABC and P4 ATPase genes, 39 T-box elements were observed in the ABC transporter protein and three elements were observed in the P4 ATPase gene suggesting its role in transcription regulation. To the best of our knowledge, there are no structural and regulatory reports on these two proteins in L. major. Computational structural biology tools may aid in understanding the interaction of miltefosine with the P4-ATPase-cdc50 complex and the ABC transporter. This can be achieved by modeling the target protein structures, studying the dynamics associated with the different domains of the protein and later using activators and inhibitors to alter the functioning of the protein. Molecular dynamics simulation with a lipid bilayer is performed to investigate the conformational changes and structure-activity relationship. As transporters are difficult to model, the relevant structural motifs and domains may help to understand the allosteric relation with the substrate and the cofactors. The dynamics of a protein molecule ultimately defines the functional mechanism involving excursions of multiple conformational states. To understand these functional mechanisms of transporter proteins, computational modeling and simulations will be carried out with the goal of elucidating the atomistic details of allosteric conformational transitions and propagations during the transport processes. In particular these studies are designed to investigate the critical structural and dynamic elements that determine individual and combined ligand-binding specificities, the interactions among transporters, their coupled-proteins and the associations of transporters within the lipid bilayer. The nature of results from such studies also makes it possible to rationally optimize existing ligands for these proteins and develop some new compounds that can shift the conformational equilibrium of transporters which may aid in functional studies leading to drug discovery.

Characterization of the intestinal absorption of inorganic mercury in Caco-2 cells

The main form of mercury exposure in the general population is through food. Intestinal absorption is therefore a key step in the penetration of mercury into the systemic circulation, and should be considered when evaluating exposure risk. Many studies have investigated the transport of mercury species in different cell lines, though the mechanisms underlying their intestinal absorption are not clear. This study evaluates the accumulation and transport of Hg(II), one of the mercury species ingested in food, using Caco-2 cells as intestinal epithelium model with the purpose of clarifying the mechanisms involved in its absorption. Hg(II) shows moderate absorption, and its transport fundamentally takes place via a carrier-mediated transcellular mechanism. The experiments indicate the participation of an energy-dependent transport mechanism. In addition, H+- and Na+-dependent transport is also observed. These data, together with those obtained from inhibition studies using specific substrates or inhibitors of different transporter families, suggest the participation of divalent cation and amino acid transporters, and even some organic anion transporters, in Hg(II) intestinal transport. An important cellular accumulation of up to 51% is observed – a situation which in view of the toxic nature of this species could affect intestinal mucosal function. This study contributes new information on the mechanisms of transport of Hg(II) at intestinal level, and which may be responsible for penetration of this mercurial form into the systemic circulation.