ABSTRACT
G-quadruplex structures within the nuclear genome (nG4) is an important regulatory factor, while the function of G4 in the mitochondrial genome (mtG4) still needs to be explored, especially in human sperms. To gain a better understanding of the relationship between mtG4 and mitochondrial function, it is crucial to develop excellent probes that can selectively visualize and track mtG4 in both somatic cells and sperms. Herein, based on our previous research on purine frameworks, we attempted for the first time to extend the conjugated structure from the C-8 site of purine skeleton and discovered that the purine derivative modified by the C-8 aldehyde group is an ideal platform for constructing near-infrared probes with extremely large Stokes shift (>220 nm). Compared with the compound substituted with methylpyridine (PAP), the molecule substituted with methylthiazole orange (PATO) showed better G4 recognition ability, including longer emission (â¼720 nm), more significant fluorescent enhancement (â¼67-fold), lower background, and excellent photostability. PATO exhibited a sensitive response to mtG4 variation in both somatic cells and human sperms. Most importantly, PATO helped us to discover that mtG4 was significantly increased in cells with mitochondrial respiratory chain damage caused by complex I inhibitors (6-OHDA and rotenone), as well as in human sperms that suffer from oxidative stress. Altogether, our study not only provides a novel ideal molecular platform for constructing high-performance probes but also develops an effective tool for studying the relationship between mtG4 and mitochondrial function in both somatic cells and human sperms.
Subject(s)
Fluorescent Dyes , Purines , Humans , Purines/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Mitochondrial Diseases/metabolism , Up-Regulation , Genome, Mitochondrial , G-Quadruplexes , Mitochondria/metabolism , Infrared Rays , HeLa CellsABSTRACT
Parkinson's disease (PD) is one of the neurodegenerative diseases that is characterized by obvious motor and some nonmotor symptoms. Various therapeutics failed in the effective treatment of PD because of impaired neurological function in the brain and various complications. Periplaneta Americana oligosaccharides (OPA), the main active ingredients extracted from the medicine residues of Periplaneta Americana (P. Americana), have been reported to exert anti-inflammatory effects. The purpose of this study was to evaluate the possible mechanisms of OPA against 1-methyl-4-phenylpyridinium (MPP+)-induced apotosis in SH-SY5Y cells and its potential neuroprotective effects in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD subacute model mice. The data demonstrated that OPA significantly reversed the MPP+-induced decrease in SH-SY5Y cell viability, reduced the proportion of apoptotic cells, and protected SH-SY5Y cells from apoptosis in a dose-dependent manner by regulating the expression of apoptosis-related genes. Furthermore, OPA also alleviated the motor dysfunction of PD model mice, prevented the loss of tyrosine hydroxylase positive cells, suppressed the apoptosis of substantia nigra cells, and improved the dysbiosis of gut microbiota in vivo, suggesting that OPA demonstrated a significantly neuroprotective effect on PD model mice. These results indicated that OPA might be the possibility of PD therapeutics with economic utility and high safety.
ABSTRACT
INTRODUCTION: In many diseased states, especially fibrosis and cancer, TGF-ß family members are overexpressed and the outcome of signaling is diverted toward disease progression. As the result of activin receptor-like kinase 1 (ALK1) plays a key role in TGF-ß signaling, discovering inhibitors of ALK1 to block TGF-ß signaling for a therapeutic benefit has become an effective strategy. METHODS: In this work, ZINC15894217 and ZINC12404282 were identified as potential ALK1 inhibitors using molecular docking, molecular dynamics simulation and MM/PBSA calculations studies. The analysis of energy decomposition found that Val208, Val216, Lys229, Gly283, Arg334 and Leu337 acted as crucial residues for ligand binding and system stabilizing. RESULTS: In addition, these compounds displayed excellent pharmacological and structural properties, which can be further evaluated through in vitro and in vivo experiments for the inhibition of ALK1 to be developed as drugs against fibrosis and tumor. CONCLUSION: Overall, our study illustrated a time- and cost-effective computer aided drug design procedure to identify potential ALK1 inhibitors. It would provide useful information for further development of ALK1 inhibitors to improve disease related to TGF-ß signal pathway.
Subject(s)
Neoplasms , Transforming Growth Factor beta , Humans , Molecular Docking Simulation , Neoplasms/drug therapy , Signal TransductionABSTRACT
The Ras/Raf/MEK/ERK (MAPK) signaling pathway has gained much attention from scientific community for therapeutic intervention in the past decades, specifically in oncology. Notably, a most prevalent B-Raf(v600e) mutant in Raf kinase family exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, virtual screening is applied to identify its potential inhibitors. Following the 25 ns molecular dynamic (MD) simulations, ZINC38541768, ZINC38541767, and ZINC12496469 are identified as B-Raf(v600e) potential inhibitors in a DFG-in conformation. Furthermore, according to the molecular mechanics/generalized born surface area (MM/GBSA) method, these three small molecules exhibit similar and good binding affinity toward B-Raf(v600e) (-38.76 kcal mol(-1), -42.60 kcal mol(-1), and -39.04 kcal mol(-1)). At the same time, several critical residues, such as I463, V471 in the P-loop, and DFG motif residue D594 within the A-loop, are also well clarified. All these results may not only indicate some future applications of inhibitors targeting B-Raf(v600e), but also benefit B-Raf(v600e) harboring cancer patients.
Subject(s)
Mitogen-Activated Protein Kinase Kinases/chemistry , Neoplasms/drug therapy , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/chemistry , Biological Products/pharmacology , Cell Proliferation/drug effects , Humans , Mitogen-Activated Protein Kinase Kinases/genetics , Mutation , Neoplasms/genetics , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Signal Transduction/drug effectsABSTRACT
Development of agricultural biotechnology requires rapid and convenient methods for crop plant genotyping. Real-time PCR is sensitive and reliable, and has been a routine technique in plant research. However, its application is limited by the cumbersome DNA template preparation procedures. We tested three PCR master mixes for direct amplification of crude seed DNA extracts without extensive purification. One mix had higher resistance to plant-derived PCR inhibitors and was shown to be applicable to various important crop plants. Furthermore, this method is capable of detecting single-copy genes from 2 mg pieces of seeds repetitively. Meanwhile, melting curve analysis could detect amplicons directly without electrophoresis manipulations. Taken together, this direct real-time PCR method provides a rapid and convenient tool for seed genotypic screening in crop plants.
Subject(s)
Crops, Agricultural/genetics , DNA, Plant/genetics , Genotype , Real-Time Polymerase Chain Reaction/methods , Seeds/genetics , Seeds/chemistryABSTRACT
Autophagy, which degrades redundant or damaged cellular constituents, is intricately relevant to a variety of human diseases, most notably cancer. Autophagy exerts distinct effects on cancer initiation and progression, due to the intrinsic overlapping of autophagic and cancer signalling pathways. However, due to the complexity of cancer as a systemic disease, the fate of cancer cells is not decided by any one signalling pathway. Numerous autophagic inter-connectivity and cross-talk pathways need to be further clarified at a systems level. In this review, we propose a systems biology perspective for the comprehensive analysis of the autophagy-cancer network, focusing on systems biology analysis in autophagy and cancer therapy. Together, these analyses may not only improve our understanding on autophagy-cancer relationships, but also facilitate cancer drug discovery.
Subject(s)
Antineoplastic Agents/therapeutic use , Autophagy/drug effects , Neoplasms/drug therapy , Systems Biology , Animals , Drug Discovery , Humans , Molecular Targeted Therapy , Neoplasms/metabolism , Neoplasms/pathology , Signal Transduction/drug effectsABSTRACT
Several lines of evidence support the notion that increased RNA-binding ability of polypyrimidine tract-binding (PTB) protein-associated splicing factor (PSF) and aberrant expression of long non-coding RNAs (lncRNAs) are associated with mouse and human tumors. To identify the PSF-binding lncRNA involved in human oncogenesis, we screened a nuclear RNA repertoire of human melanoma cell line, YUSAC, through RNA-SELEX affinity chromatography. A previously unreported lncRNA, termed as Llme23, was found to bind immobilized PSF resin. The specific binding of Llme23 to both recombinant and native PSF protein was confirmed in vitro and in vivo. The expression of PSF-binding Llme23 is exclusively detected in human melanoma lines. Knocking down Llme23 remarkably suppressed the malignant property of YUSAC cells, accompanied by the repressed expression of proto-oncogene Rab23. These results may indicate that Llme23 can function as an oncogenic RNA and directly associate the PSF-binding lncRNA with human melanoma.
Subject(s)
Carcinogenesis/genetics , Melanoma/genetics , RNA, Long Noncoding/genetics , RNA-Binding Proteins/genetics , Blotting, Western , Carcinogenesis/metabolism , Cell Line , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , HCT116 Cells , HEK293 Cells , Hep G2 Cells , Humans , MCF-7 Cells , Melanoma/metabolism , Melanoma/pathology , PTB-Associated Splicing Factor , Protein Binding , Proto-Oncogene Mas , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
A 48 kDa, chitin-binding lectin with antifungal, antiviral and apoptosis-inducing activities was isolated from the rhizomes of Setcreasea purpurea Boom, a member of family Commelinaceae. Setcreasea purpurea lectin (designated as SPL) is a homotetrameric protein consisting of 12031.9 Da subunits linked by non-covalent bonds as determined by SDS-PAGE, gel filtration and MS. The N-terminal 25 amino-acid sequence of SPL, NVLGRDAYCGSQNPGATCPGLCCSK was determined and homology analysis suggested that SPL belongs to the family of chitin-binding plant lectins composed of hevein domains. The lectin exhibited strong hemagglutinating activity towards rabbit erythrocytes at 0.95 µg/ml and the activity could be reversed exclusively by chitin hydrolysate (oligomers of GlcNAc). Its hemagglutinating activity was stable in pH range of 2.0-9.0 and it showed excellent thermal tolerance. SPL showed antifungal activity against Rhizoctonia solani, Sclerotinia sclerotiorum, Penicillium italicum and Helminthosporiun maydis. It also exhibited inhibitory effect on HIV-1 (IIIB) and HIV-2 (ROD), with an EC50 of 13.8 ± 1.3 and 57.1 ± 15 µg/ml, respectively. Subsequently, MTT method, cell morphological analysis and LDH activity-based cytotoxicity assays demonstrated that SPL was highly cytotoxic to CNE-1 cells and induced apoptosis in a dose-dependent manner. Moreover, due to the caspase inhibitors analyses, caspase was also found to play an important role in the potential apoptotic mechanism of SPL.
ABSTRACT
The anti-human immunodeficiency virus (HIV) I/II activity of a mannose and sialic acid binding lectin isolated from rhizomes of Polygonatum cyrtonema Hua was elucidated by comparing its HIV infection inhibitory activity in MT-4 and CEM cells with that of other mannose-binding lectins (MBLs). The anti-HIV activity of Polygonatum cyrtonema Hua lectin (PCL) was 10- to 100-fold more potent than other tested MBLs, but without significant cytotoxicity towards MT-4 or CEM cells. To amplify cDNA of PCL by 3'/5'-rapid amplification of cDNA ends (RACE), the 30 amino acids of N-terminal were determined by sequencing and the degenerate oligonucleotide primers were designed. The full-length cDNA of PCL contained 693 bp with an open reading frame encoding a precursor protein of 160 amino acid residues, consisting of a 28-residue signal peptide, a 22-residue C-terminal cleavage peptide and a 110-residue mature polypeptide which contained three tandemly arranged subdomains with an obvious sequence homology to the monocot MBL. However, only one active mannose-binding site (QDNVY) was found in subdomain I of PCL, that of subdomain II and III changed to HNNVY and PDNVY, respectively. There was no intron in PCL, which was in good agreement with other monocot MBLs. Molecular modeling of PCL indicated that its three-dimensional structure resembles that of the snowdrop agglutinin. By docking, an active sialic acid-binding site was found in PCL. The instabilization of translation initiation region (TIR) in mRNA of PCL benefits its high expression in rhizomes.
Subject(s)
Antiviral Agents/pharmacology , HIV-1/drug effects , HIV-2/drug effects , Lectins/pharmacology , Mannose-Binding Lectin/pharmacology , Rhizome/chemistry , Amino Acid Sequence , Base Sequence , Cell Line , Cloning, Molecular , DNA, Complementary/analysis , HIV-1/metabolism , HIV-2/metabolism , Humans , Lectins/genetics , Lectins/isolation & purification , Mannose-Binding Lectin/genetics , Mannose-Binding Lectin/isolation & purification , Mannose-Binding Lectins/chemistry , Models, Molecular , Molecular Sequence Data , Plant Lectins/chemistry , Sialic Acid Binding Immunoglobulin-like LectinsABSTRACT
The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in lambda(max). The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively. However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I(-) was 0.15 M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.
Subject(s)
Millettia/metabolism , Oligosaccharides/chemistry , Plant Lectins/chemistry , Acrylamide/chemistry , Amino Acids/chemistry , Arginine/chemistry , Binding Sites , Carbohydrates/chemistry , Circular Dichroism , Hemagglutination , Mannose/chemistry , Oxygen/metabolism , Protein Conformation , Spectrometry, Fluorescence , Spectrophotometry/methods , Tryptophan/chemistry , Ultraviolet RaysABSTRACT
A sialic acid-binding lectin (Hericium erinaceum lectin, HEL), isolated from fresh fruiting bodies of Hericium erinaceum, was treated with various temperature and pH to investigate its fluorescence spectra and hemagglutinating activity. It was found that the hemagglutinating activity of HEL was relatively steady below 60 degrees and at pH from 6 to 11, and the change of hemagglutinating activity was relative to the change of hydrophobic areas where tryptophan residues located. In fluorescence quenching study of HEL by acrylamide and KI, it was indicated that nearly all the tryptophan residues of HEL located on the surface of the molecule, and most of them were in hydrophobic areas or negatively charged areas. Chemical modification of HEL proved that there were about twelve tryptophan residues in a HEL molecule and all of them were located on the surface or in the shallow groove of the molecule, and eight of them were essential for hemagglutinating activity; aspartic acid or glutamic acid residues were involved in maintaining the crucial conformation of activity center and made great contribution to the hemagglutinating activity of HEL, but they could not touch the sialic acid molecule directly; tyrosine residues also played a role in the hemagglutinating activity of HEL; while arginine, serine, threonine, histidine residues had no effect on the hemagglutinating activity of HEL.