Investigation of molecular details of a bacterial cationic amino acid transporter (GkApcT) during arginine transportation using molecular dynamics simulation and umbrella sampling techniques.

CONTEXT: Cationic amino acid transporters (CATs) facilitate arginine transport across membranes and maintain its levels in various tissues and organs, but their overexpression has been associated with severe cancers. A recent study identified the alternating access mechanism and critical residues involved in arginine transportation in a cationic amino acid transporter from Geobacillus kaustophilus (GkApcT). Here, we used molecular dynamics (MD) simulation methods to investigate the transportation mechanism of arginine (Arg) through GkApcT. The results revealed that arginine strongly interacts with specific binding site residues (Thr43, Asp111, Glu115, Lys191, Phe231, Ile234, and Asp237). Based on the umbrella sampling, the main driving force for arginine transport is the polar interactions of the arginine with channel-lining residues. An in-depth description of the dissociation mechanism and binding energy analysis brings valuable insight into the interactions between arginine and transporter residues, facilitating the design of effective CAT inhibitors in cancer cells. METHODS: The membrane-protein system was constructed by uploading the prokaryotic CAT (PDB ID: 6F34) to the CHARMM-GUI web server. Molecular dynamics simulations were done using the GROMACS package, version 5.1.4, with the CHARMM36 force field and TIP3P water model. The MM-PBSA approach was performed for determining the arginine binding free energy. Furthermore, the hotspot residues were identified through per-residue decomposition analysis. The characteristics of the channel such as bottleneck radius and channel length were analyzed using the CaverWeb 1.1 web server. The proton wire inside the transporter was investigated based on the classic Grotthuss mechanism. We also investigated the atomistic details of arginine transportation using the path-based free energy umbrella sampling technique (US).

Arginine transportation mechanism through cationic amino acid transporter 1: insights from molecular dynamics studies.

Metabolic and signaling mechanisms in mammalian cells are facilitated by the transportation of L-arginine (Arg) across the plasma membrane through cationic amino acid transporter (CAT) proteins. Due to a lack of argininosuccinate synthase (ASS) activity in various tumor cells such as acute myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, these tumor entities are arginine-auxotrophic and therefore depend on the uptake of the amino acid arginine. Cationic amino acid transporter-1 (CAT-1) is the leading arginine importer expressed in the aforementioned tumor entities. Hence, in the present study, to investigate the transportation mechanism of arginine in CAT-1, we performed molecular dynamics (MD) simulation methods on the modeled human CAT-1. The MM-PBSA approach was conducted to determine the critical residues interacting with arginine within the corresponding binding site of CAT-1. In addition, we found out that the water molecules have the leading role in forming the transportation channel within CAT-1. The conductive structure of CAT-1 was formed only when the water molecules were continuously distributed across the channel. Steered molecular dynamics (SMD) simulation approach showed various energy barriers against arginine transportation through CAT-1, especially while crossing the bottlenecks of the related channel. These findings at the molecular level might shed light on identifying the crucial amino acids in the binding of arginine to eukaryotic CATs and also provide fundamental insights into the arginine transportation mechanisms through CAT-1. Understanding the transportation mechanism of arginine is essential to developing CAT-1 blockers, which can be potential medications for some types of cancers.Communicated by Ramaswamy H. Sarma.

Therapeutic effects of L-arginine, L-carnitine, and mesenchymal stem cell-conditioned medium on endometriosis-induced oocyte poor quality in an experimental mouse model.

AIM: The present study aimed to explore the potential ameliorative effects of L-arginine (LA), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on endometriosis (EMS) model in vivo and in vitro. METHODS: The animals were divided into two main groups, normal and EMS-induced mice. Normal and EMS-induced groups were injected with or without LA (250 mg/kg), LC (250 mg/kg), and BMSC-CM (a final volume of 100 µL of CM/mouse). At the end of the study, the level of total antioxidant capacity (TAC), nitric oxide (NO), and total oxidative status (TOS) were measured in plasma. Furthermore, immature oocytes were collected from two groups and cultured in a maturation medium. Subsequently, the rates of in vitro maturation, in vitro fertilization (IVF), and in vitro embryonic development were evaluated. RESULTS: The results revealed that administration of LA, LC, and BMSC-CM ameliorated the oxidative status through maintaining TAC and alleviating TOS and NO levels. More importantly, the maturation and fertilization rates, blastocyst development, and total blastocyst cell numbers significantly increased in LA, LC, and BMSC-CM-administrated groups compared to the control group. In both the normal and EMS groups, the highest IVF, cleavage, and blastocyst percentages were associated with BMSC-CM treatment (p < 0.05). CONCLUSION: Altogether, LA, LC, and BMSC-CM have therapeutic effects on impaired oocyte quality and promote subsequent development in vitro, probably through normalization of nitro-oxidative stress, thus offering potential alternatives to conventional therapies during assisted reproductive technologies for patients with EMS-associated sub/infertility.

LRH-1 (liver receptor homolog-1) derived affinity peptide ligand to inhibit interactions between ß-catenin and LRH-1 in pancreatic cancer cells: from computational design to experimental validation.

Poor prognosis, rapid progression and the lack of an effective treatment make pancreatic cancer one of the most lethal malignancies. Recent studies point to a role for liver receptor homolog-1 (LRH-1) in pathogenesis of pancreatic cancer and suggest prevention of the ß-catenin/LRH-1 complex formation as a potential strategy for inhibition of the pancreas cancer cells progression. In the current investigation, we have followed a biomimetic strategy and designed an affinity peptide with sequence DEMEEPQQTE to inhibit formation of the ß-catenin/LRH-1 complex. Quantitative real-time PCR experiments on the AsPC-1 pancreatic metastatic cells showed that the peptide has an inhibitory effect on the Wnt signaling proliferation line by reducing the expression levels of the CCND1, CCNE1, and MYC genes. Furthermore, the increased expression level of BAX gene showed that AsPC-1 cells were directed to the apoptosis pathway. At last, POU5F1, KLF4, and CD44 gene expression levels suggested that the peptide has an inhibitory effect on the stemness feature of the AsPC-1 cells. Here, we introduced a novel peptide inhibitor targeting an important protein-protein interaction, the ß-catenin/LRH-1 complex, which may provide highly promising starting points for subsequent drug design. Communicated by Ramaswamy H. Sarma.

Comparative experimental/theoretical studies on the EGFR dimerization under the effect of EGF/EGF analogues binding: Highlighting the importance of EGF/EGFR interactions at site III interface.

Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play significant roles in cell proliferation and signaling. All the members of the EGFR/ErbB family are primary goals for cancer therapy, particularly for tumors of breast, cervix, ovaries, kidney, esophagus, prostate and non-small-cell lung carcinoma and head and neck tumors. However, the therapeutic ability of accessible anti-ErbB agents is limited. Therefore, recognizing EGF analogues or small organic molecules with high affinity for the extracellular domain of the EGFR is a critical target on cancer research. An effective EGF analogue should have a comparable binding affinity for EGFR in order to create an effective ligand competitive inhibition against circulating wild EGF while fails to transduce appropriate downstream signaling into the cancer cell. In our earlier study we have developed a mutant form of human EGF (mEGF, lacking the four critical amino acid residues; Gln43, Tyr44, Arg45 and Asp46 at the C-terminal of the protein) and its binding properties and mitogenic activity were assessed. The mEGF showed high affinity for EGFR binding domains but caused poor EGFR dimerization and phosphorylation and especially, mEGF induced EGFR internalization. However, underlying mechanism of action of EGF analogues is still unclear and thus considered to be worthwhile for further study. With regard to different effects of the EGF analogue on EGFR activating process, computational analysis of wild EGF/EGFR and mEGF/EGFR complexes (along with EGFt/EGFR complex) were done. Results of the protein dissection identified several interactions within "ligand/EGFR" that are common among EGF and EGFt/mEGF. These results disclose that while several interactions are conserved within EGF/EGFR interfaces, EGF/EGFR interactions on site III interface controls the affinity, EGFR dimerization and subsequent downstream signaling through a heterogeneous set of non-covalent interactions. These findings not only represent the EGFR dynamics complexity but also smooth the path for structure-based design of therapeutics targeting C-terminal region of EGF (and the related domain within the receptor) or EGFR-based imaging probes.

Comprehensive Dissection of Transcriptome Data and Regulatory Factors in Pancreatic Cancer Cells.

Features of pancreatic cancers include high mortality rates caused by rapid tumor progression and a lack of effective therapy. Underpinning the molecular mechanisms involved in the alteration of the gene expression program in the pancreatic cancer remains to be understood. In the current study, we performed a comprehensive analysis using 282 pancreatic tumor and normal samples from seven independent expression data sets to provide a better view on the interactions between different transcription factors (TFs) and the most affected biological pathways in pancreatic cancer. We highlighted common differentially expressed genes (DEGs) and common affected processes within pancreatic cancer samples. We revealed 16 main DE-TFs that regulated gene expression alterations as well as the most significant processes in pancreatic cancer compared to normal cells. For example, we found the upregulated FOXM1 to be a top regulator of pancreatic cellular transformation based on results from different analyses, including from its regulation of gene regulatory networks, its presence in protein complex, its significant regulation of genes related to cancer pathways, and its regulation of most of the identified DE-TFs. Furthermore, we provided a model and assessed the role of different DE-TFs in the regulation of the most affected pancreatic- and cancer-specific processes. In conclusion, our bioinformatics meta-analysis of high throughput expression data sets, besides clarifying common affected genes and pathways, also showed the mechanisms involved in regulating these common profiles. Our results, especially for DE-TFs, could potentially be useful for screening for pancreatic cancer, and for confirming or determining novel pharmacological targets. J. Cell. Biochem. 118: 3976-3985, 2017. © 2017 Wiley Periodicals, Inc.

Appraisal of role of the polyanionic inducer length on amyloid formation by 412-residue 1N4R Tau protein: A comparative study.

In many neurodegenerative diseases, formation of protein fibrillar aggregates has been observed as a major pathological change. Neurofibrillary tangles, mainly composed of fibrils formed by the microtubule-associated protein; Tau, are a hallmark of a group of neurodegenerative diseases such as Alzheimer's disease. Tau belongs to the class of natively unfolded proteins and partially folds into an ordered ß-structure during aggregation. Polyanionic cofactors such as heparin are commonly used as inducer of Tau aggregation in vitro. The role of heparin in nucleation and elongation steps during Tau fibril formation is not fully understood. In the current study, aggregation kinetics as well as structure of Tau amyloid fibrils, by using the 1N4R isoform, have been reproducibly determined in the presence of heparin and the shorter molecule; enoxaparin. The kinetic studies demonstrated that heparin (not enoxaparin) efficiently accelerates Tau amyloid formation and revealed, mechanistically, that the molecular weight of the inducer is important in accelerating amyloidogenesis. The kinetic parameter values of Tau amyloid aggregation, especially, the amyloid aggregation extent, were relatively different in the presence of heparin and enoxaparin, at various stoichiometries of the inducers binding. Also, based on the results, obtained from CD, FTIR, AFM and XRD studies, it may be suggested that the inducer length plays a critical role mainly in the nucleation process, so that it determines that oligomers lie on or off the pathway of Tau fibrillization. The biochemical results herein suggest that the chemical environment of the extracellular matrix as well as localization of distinct glycosaminoglycans may influence deposition behavior of Tau amyloidosis.

Role of CHK2 inhibitors in the cellular responses to ionizing radiation.

Ionizing radiation is the more effective therapy to reduce tumor growth through damaging the DNA of cells. In response to DNA damage, cells activate the checkpoint kinases such as CHK2, which signal to initiate repair processes and cell-cycle arrest, until the damaged DNA is repaired. At present, the development of CHK2 inhibitors has provided an interesting strategy for the treatment of cancer by introducing new radiation modifier agents. CHK2 inhibitors can contribute for the improvement of cancer therapy through sensitizing cancerous cells and radioprotection of normal cells against ionizing radiation. This review describes and discusses the most recent inhibitors of CHK2 and presents an evaluation of chemical structures and biological activities. As well as their role in cell growth during exposure to ionizing radiation.

Anti-tumor Activity of Ferulago angulata Boiss. Extract in Gastric Cancer Cell Line via Induction of Apoptosis.

Ferulago angulata Boiss. known in Iran as Chavir, has some bioactive compounds having antioxidant activity. Because of its antioxidant activities, it sounded Chavir extract can be a good candidate for finding chemopreventive agents having inductive apoptosis properties on cancer cells. In this study, the cytotoxic effects and proapoptotic activities of Chavir's leaf and flower extracts were investigated on human adenocarcinoma gastric cell line (AGS). The ferric reducing antioxidant power (FRAP) assay was used to determine antioxidant activity of the extract. Cytotoxic effects of the extract were performed by trypan blue and neutral red assays. For apoptosis detection, we used Annexin V staining, flow cytometry and DNA fragmentation assays. The FRAP assay results showed that antioxidant activity of leaf extract was higher than flower extract. Cytotoxicity and apoptosis-inducing activity of flower and leaf extracts changed coordinately, indicating the cytotoxicity of chavir extracts is due probably to induce apoptosis. Our results revealed that the cytotoxic effects of F. angulate Boiss. extracts on AGS cell line is close to some other plant extracts such as Rhus verniciflua Stokes (RVS) and Scutellaria litwinowii. This is the first study on cytotoxic and apoptosis-inducing effects of chavir leaf and flower extracts against AGS cell line. The Further investigation can be identification of the agent(s) by which these effects is observed.