Abundant clinical evidences indicate that up-regulation of several cathepsins in many human cancers is correlated with malignant progression and poor patient prognosis. In addition, a decrease in catalase activity or accumulation of hydrogen peroxide correlates with cancer metastasis. Recent studies indicate that cathepsin activation and expression can be modulated via H2O2 treatment. However, the actual relationship between catalase and cathepsins is not yet fully understood. In the present study, we found that catalase expression (or activity) was higher, while intracellular and extracellular Cat S, Cat L, and Cat K activities were lower in the non-invasive CL1-0 cells compared to the highly invasive CL1-5 cells. After CL1-0 cells were transfected with catalase-shRNA, the corresponding ROS (H2O2) level and Cat S, Cat L, or Cat K expression (or activity) was up-regulated, accompanied by an increase in cell migration and invasion. On the other hand, ROS (H2O2) level, cathepsin S, L, and K activities, cell migration and invasion were decreased in catalase-overexpressed CL1-5 cells. It is suggested that catalase may regulate cathepsin activity by controlling the production of ROS (H2O2), leading to variation in migration and invasion ability of lung cancer cells.
Abstract Elevated cathepsin S (Cat S) level is correlated with higher migration ability in tumor cells. This study investigates the inhibitory effect of novel synthetic ?-ketoamide compounds on cathepsin activity and cancer cell migration. The effect of several ?-ketoamide compounds on the activity of recombinant cathepsins (Cat S, Cat L and Cat K) was examined. Two highly metastatic cancer cell lines were incubated with three Cat S-specific compounds (6n, 6w and 6r) to analyze their effect on cellular Cat S activity and cell migration. At a 100?nM concentration, compounds 6n, 6r and 6w effectively inhibited Cat S activity. Cat S activity and cell migration were significantly reduced in CL1-3 cells after treatment with either 6n or 6w at 5??M. Similar results were also obtained when A2058 cells were treated with 6n. These results highlight the therapeutic potential of ?-ketoamide compounds, especially 6n and 6w, to prevent or delay cancer metastasis.
Microarray provides genome-wide transcript profiles, whereas RNA-seq is an alternative approach applied for transcript discovery and genome annotation. Both high-throughput techniques show quantitative measurement of gene expression. To explore differential gene expression rates and understand biological functions, the authors designed a system which utilises annotations from Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways and Gene Ontology (GO) associations for integrating multiple RNA-seq or microarray datasets. The developed system is initiated by either estimating gene expression levels from mapping next generation sequencing short reads onto reference genomes or performing intensity analysis from microarray raw images. Normalisation procedures on expression levels are evaluated and compared through different approaches including Reads Per Kilobase per Million mapped reads (RPKM) and housekeeping gene selection. Such gene expression levels are shown in different colour shades and graphically displayed in designed temporal pathways. To enhance importance of functional relationships of clustered genes, representative GO terms associated with differentially expressed gene cluster are visually illustrated in a tag cloud representation.
Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore, in silico computer modeling and in vitro binding assays suggest critical roles for the following basic amino acids located within heparin binding regions (HBRs) of EDN 34QRRCKN39 (HBR1), 65NKTRKN70 (HBR2), and 113NRDQRRD119 (HBR3) and in particular Arg35, Arg36, and Arg38 within HBR1, and Arg114 and Arg117 within HBR3. Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN.
Heparan sulfate proteoglycans (HSPGs) are glycoproteins consisting of a core protein to which linear heparan sulfate (HS) side chains are covalently attached. These HS side chains mediate a variety of biologic functions involved in inflammation. Radionuclide imaging of HS side chains in tissues with inflammation may be used for the stratification of patients who would most likely benefit from HSPG-targeting therapy. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of HS side chain expression in a mouse model of asthma using the recombinant eosinophil cationic protein (rECP).
Amyotrophic lateral sclerosis (ALS) is a complicate and progressive onset devastating neurodegenerative disease. Its pathogenic mechanisms remain unclear and there is no specific test for diagnosis. For years, researchers have been vigorously searching for biomarkers associated with ALS to assist clinical diagnosis and monitor disease progression. Some specific inflammatory processes in the central nervous system have been reported to participate in the pathogenesis of ALS. As high mobility group box 1 (HMGB1) is elevated in spinal cord tissues of patients with ALS, we hypothesized, therefore, that serum autoantibody against HMGB1 (HMGB1 autoAb) might represent an effective biomarker for ALS. Patients with ALS, Alzheimers disease, Parkinsons disease, and healthy age-matched control subjects were recruited for this study. ALS group consisted of 61 subjects, the other groups each consisted of forty subjects. We generated a polyclonal antibody against HMGB1 and developed an ELISA-based methodology for screening serum samples of these subjects. All samples were coded for masked comparison. For statistic analyses, two-tailed Students t-test, ANOVA, Bonferroni multiple comparison test, Spearman correlation, and receiver operating characteristic curve were applied. We discovered that the level of HMGB1 autoAb significantly increased in patients with ALS as compared with that of patients with Alzheimers disease, Parkinsons disease, and healthy control subjects. The differences between all groups were robust even at the early stages of ALS progression. More importantly, higher HMGB1 autoAb level was found in more severe disease status with significant correlation. Our study demonstrates that serum HMGB1 autoAb may serve as a biomarker for the diagnosis of ALS and can be used to monitor disease progression.
Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP(32-41), derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP(32-41) entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP(32-41) to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP(32-41) internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP(32-41) attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP(32-41) internalization routes. ECP(32-41) was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.
Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, (34)RWRCK(38) (HBR1), (75)RSRFR(79) (HBR2), and (101)RPGRR(105) (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members.
Mechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G2/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12dyn/cm(2) induced death of these four tumor cell lines. In contrast to LSS at 0.5dyn/cm(2), oscillatory shear stress (OSS) at 0.5±4dyn/cm(2) cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V-FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells.
Doxorubicin is an anticancer drug used in a wide range of cancer therapies; however, doxorubicin-induced drug resistance is one of the most serious obstacles of cancer chemotherapy. Recent studies have indicated that reduced oxidative stress levels in cancer cells induce drug resistance. However, the redox-modifications of resistance - associated cellular targets are largely unknown. Thus, the current study employed cysteine-labeling based two-dimensional differential gel electrophoresis (2D-DIGE) combined with MALDI-TOF mass spectrometry (MALDI-TOF MS) to analyze the effect of doxorubicin resistance on redox regulation in uterine cancer and showed 33 spots that were significantly changed in thiol reactivity. These proteins involve cytoskeleton regulation, signal transduction, redox-regulation, glycolysis, and cell-cycle regulation. The current work shows that the redox 2D-DIGE-based proteomic strategy provides a rapid method to study the molecular mechanisms of doxorubicin-induced drug resistance in uterine cancer. The identified targets may be used to further evaluate their roles in drug-resistance formation and for possible diagnostic or therapeutic applications.
Transforming growth factor-?1 (TGF-?1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-?1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-?1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-?1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-? agonists (i.e., WY14,643 and clofibrate), but not a PPAR-?/? agonist (GW501516) or PPAR-? agonist (troglitazone), inhibit TGF-?1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-?1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-?1-induced cell cycle arrest at the G0/G1 phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-?-mediated 10T1/2 cell cycle arrest at the G0/G1 phases is TGF-? receptor independent. These results suggest that PPAR-? mediates cell cycle control and TGF-?1-induced SMC phenotypic changes in 10T1/2 cells.
Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.
A convenient route for the synthesis of heparin oligosaccharides involving regioselective protection of D-glucosamine and a concise preparation of rare L-ido sugars from diacetone ?-D-glucose is described. Stereoselective coupling of a D-glucosamine-derived trichloroacetimidate with a 1,6-anhydro-?-L-idopyranosyl 4-alcohol gave the desired ?-linked disaccharide, which was used as repeating unit for dual chain elongation and termination. Stepwise assembly from the reducing to the non-reducing end with a D-glucosamine-derived monosaccharide as starting unit furnished the oligosaccharide skeletons having different chain lengths. A series of functional group transformations afforded the expected heparin oligosaccharides with 3, 5 and 7 sugar units. Interaction of these oligosaccharides with eosinophil-derived neurotoxin (EDN), a cationic ribonuclease and a mediator produced by human eosinophils, was further investigated. The results revealed that at 5 ?g mL(-1), the heptasaccharide has sufficiently strong interference to block EDN binding to Beas-2B cells. The tri- and pentasaccharides have moderate inhibitory properties at 50 ?g mL(-1) concentration, but no inhibition has been observed at 10 ?g mL(-1). The IC(50) values of the tri-, penta- and heptasaccharides are 69.4, 47.2 and 0.225 ?g mL(-1), respectively.
Hypoxia-inducible factors (HIFs) are transcription factors that play a crucial role in response to hypoxic stress in living organisms. The HIF pathway is activated by changes in cellular oxygen levels and has significant impacts on the regulation of gene expression patterns in cancer cells. Identifying functional conservation across species and discovering conserved regulatory motifs can facilitate the selection of reference species for empirical tests. This paper describes a cross-species functional pathway mapping strategy based on evidence of homologous relationships that employs matrix-based searching techniques for identifying transcription factor-binding sites on all retrieved HIF target genes.
Carbohydrate binding modules (CBMs) are found in polysaccharide-targeting enzymes and increase catalytic efficiency. Because only a relatively small number of CBM structures have been solved, computational modeling represents an alternative approach in conjunction with experimental assessment of CBM functionality and ligand-binding properties. An accurate target-template sequence alignment is the crucial step during homology modeling. However, low sequence identities between target/template sequences can be a major bottleneck. We therefore incorporated the predicted hydrophilic aromatic residues (HARs) and secondary structure elements into our feature-incorporated alignment (FIA) algorithm to increase CBM alignment accuracy. An alignment performance comparison for FIA and six others was made, and the greatest average sequence identities and similarities were achieved by FIA. In addition, structure models were built for 817 representative CBMs. Our models possessed the smallest average surface-potential z scores. Besides, a large true positive value for liagnd-binding aromatic residue prediction was obtained by HAR identification. Finally, the pre-simulated CBM structures have been deposited in the Database of Simulated CBM structures (DS-CBMs). The web service is publicly available at http://dscbm.life.nthu.edu.tw/ and http://dscbm.cs.ntou.edu.tw/.
Mesenchymal stem cell (MSC) found in bone marrow (BM-MSCs) and the Whartons jelly matrix of human umbilical cord (WJ-MSCs) are able to transdifferentiate into neuronal lineage cells both in vitro and in vivo and therefore hold the potential to treat neural disorders such as stroke or Parkinsons disease. In bone marrow MSCs, miR-130a and miR-206 have been show to regulate the synthesis of neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cells. However, how neuronal differentiation is controlled in WJ-MSC remains unclear.
Mesenchymal stem cells (MSCs) are an attractive source for providing the cells necessary for regenerating damaged tissues. Fetal MSCs (fMSCs) are known to proliferate fast and have an excellent osteogenic capacity, yet the underlying mechanisms need to be explored. A better understanding of MSCs from different anatomic origins and ages will eventually benefit cell-based therapies, as well as subsequent mechanistic studies in the field of stem cell biology.
The eosinophil cationic protein (ECP) is cytotoxic to bacteria, viruses, parasites and mammalian cells. Cells are damaged via processes of pore formation, permeability alteration and membrane leaking. Some clinical studies indicate that ECP gathers in the bronchial tract of asthma sufferers, damages bronchial and airway epithelial cells, and leads to in breathing tract inflammation; therefore, prevention of the cytotoxicity caused by ECP may serve as an approach to treat airway inflammation. To achieve the purpose, reduction of the ECP-cell interactions is rational. In this work, the Chinese herbal combinative network was generated to predict and identify the functional herbs from the pools of prescriptions. It was useful to select the node herbs and to demonstrate the relative binding ability between ECP and Beas-2B cells with or withour herb treatments.
A series of small molecules bearing an alpha-ketoamide warhead were synthesized and evaluated for their ability to inhibit cathepsin S, a key proteolytic enzyme upregulated in many cancers during tumor progression and metastasis. Most of the synthetic compounds were noncytotoxic, but several robustly inhibited cathepsin S (IC(50) < 10 nM) and potently suppressed cell migration, invasion, and capillary tube formation. These results highlight the potential of alpha-ketoamide therapy for preventing or delaying cancer spread.
Carbohydrate-binding modules (CBMs) share similar secondary and tertiary topology, but their primary sequence identity is low. Computational identification of ligand-binding residues allows biologists to better understand the protein-carbohydrate binding mechanism. In general, functional characterization can be alternatively solved by alignment-based manners. As alignment accuracy based on conventional methods is often sensitive to sequence identity, low sequence identity among query sequences makes it difficult to precisely locate small portions of relevant features. Therefore, we propose a feature-incorporated alignment (FIA) to flexibly align conserved signatures in CBMs. Then, an FIA-based target-template prediction model was further implemented to identify functional ligand-binding residues.
Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and alpha-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and alpha-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric alpha-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of alpha-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.
Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.
Somatic CD34+ CD38- stem cells can differentiate into cells of hematopoietic and endothelial lineages and have been clinically used to treat diseases. These stem cells can be obtained from cord blood (CB), bone marrow or granulocyte-macrophage colony-stimulating factor-mobilized peripheral blood. Unmasking genes differentially expressed in hematopoietic stem cells (HSCs) from different anatomic locations can improve our understanding of their basic biological features and help in clinical decision making when applying different HSCs.
Baculovirus has emerged as a novel gene delivery and vaccine vector, and the demand for purified baculovirus is rising due to the increasing in vivo applications. Since the baculoviral envelope protein gp64 is a glycoprotein, we aimed to develop a concanavalin A (Con A) chromatography process, which harnessed the possible affinity interaction between gp64 and Con A, for simple and effective baculovirus purification. Throughout the purification process the virus stability and recovery were assessed by quantifying the virus transducing titers [TT, defined as transducing units (TU) per milliliter] and viral particles (VP). We found that baculovirus stability was sensitive to buffer conditions and diafiltration with a tangential flow filtration system LabScale using 300 K membranes yielded recoveries of approximately 75% in TT and 82% in VP. The diafiltered baculovirus strongly bound to the Con A column as evidenced by the low virus losses to the flow through and wash fractions. The wash steps eliminated >99% of protein impurities and elution with 0.6 M alpha-D-methylmannoside at room temperature led to the recoveries of approximately 16% in VP and approximately 15.3% in TU. The resultant VP/TU ratio was as low as 41.4, attesting the high quality of the purified virus. Further elution with 1 M alpha-D-methylmannoside recovered another 6% virus TU, yielding a cumulative recovery of approximately 21.3% in TU. These data demonstrated for the first time that Con A chromatography is suitable for baculovirus purification, and may be used for the purification of other viruses with surface glycoproteins.
It has been recognized cancer cells acquire characters reminiscent of those of normal stem cells, and the degree of stem cell gene expression correlates with patient prognosis. Lgr5(+) or CD133(+) epithelial stem cells (EpiSCs) have recently been identified and these cells are susceptible to neoplastic transformation. It is unclear, however, whether genes enriched in EpiSCs also contribute in tumor malignancy. Endometrial endometrioid carcinoma (EEC) is a dominant type of the endometrial cancers and is still among the most common female cancers. Clinically endometrial carcinoma is classified into 4 FIGO stages by the degree of tumor invasion and metastasis, and the survival rate is low in patients with higher stages of tumors. Identifying genes shared between advanced tumors and stem cells will not only unmask the mechanisms of tumor malignancy but also provide novel therapeutic targets.
Upon prolonged arrest in mitosis, cells undergo adaptation and exit mitosis without cell division. These tetraploid cells are either eliminated by apoptosis or arrested in the subsequent G(1) phase in a spindle checkpoint- and p53-dependent manner. p53 has long been known to be activated by spindle poisons, such as nocodazole and Taxol, although the underlying mechanism remains elusive. Here we present evidence that stabilization and activation of p53 by spindle disruption requires the spindle checkpoint kinase TTK/hMps1. TTK/hMps1 phoshorylates the N-terminal domain of p53 at Thr18, and this phosphorylation disrupts the interaction with MDM2 and abrogates MDM2-mediated p53 ubiquitination. Phosphorylation at Thr18 enhances p53-dependent activation of not only p21 but also Lats2, two mediators of the postmitotic checkpoint. Furthermore, a phospho-mimicking substitution at Thr18 (T18D) is more competent than the phospho-deficient mutant (T18A) in rescuing the tetraploid checkpoint defect of p53-depleted cells. Our findings therefore provide a mechanism connecting the spindle checkpoint with p53 in the maintenance of genome stability.
Although the differential expression of heat shcok proteins, Hsp90alpha and Hsp90beta was extensively studied in many kinds of cells, the post-transcriptional regulation of Hsp90 isoforms remains unclear. In control and GA-treated rat gliosarcoma cells, it has been reported that the translational efficiency of hsp90alpha is higher than hsp90beta. In this study, we present evidences identifying the roles for leaky scanning and 5-UTR sequence in translational regulation of Hsp90beta. The result of in vitro transcription and translation (IVTT) experiment showed that hsp90alpha exhibited higher translation efficiency than hsp90beta. Sequence analysis revealed that there is an out-of-frame downstream AUG codon in hsp90beta gene. However, elimination of the downstream AUG by site-directly mutagenesis or introducing Kozak context sequence around the initiator AUG of hsp90beta open reading frame increased its translational efficiency, which indicated that leaky scanning might be a possible mechanism regulating hsp90beta. Furthermore, we also constructed a firefly luciferase reporter system to verify the effect of subsequent translation at the downstream out-of-frame AUG codon in 9L and A549 cells. Furthermore, it is believed that 5-untranslated region (5-UTR) also plays a significant role in translational control. We showed hsp90beta 5-UTR gives rise to the reduction of the translation efficiency in IVTT experiment. Additionally, the reductive effect of hsp90beta 5-UTR was further confirmed by luciferase reporter assay using truncated deletion analyses of 5-UTR of hsp90beta. Our results support the hypothesis that ribosome leaky scanning mechanism and 5-UTR sequence acts as negative regulators in hsp90beta mRNA.
The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner.
Human eosinophil-derived neurotoxin (EDN, RNase2) and eosinophil cationic protein (ECP, RNase3) sequences possess as high as 92% identity in their promoter regions. The major difference within this region is a 34-nucleotide (34-nt) segment appeared only in the edn promoter. In addition, six discrete segments existed in the regulatory regions of both edn and ecp. Our previous study indicated that the 34-nt segment is responsive for higher transcription activity of edn in comparison with ecp, via binding to transcription activator Sp1. In this study, the roles of the six discrete segments in transcription regulation were investigated and the -350/-329 region (ednR2) was shown to be involved in the regulation of edn expression. When the ednR2 segment of edn was replaced with that of ecp, a significant decrease in edn promoter activity was detected. Supershift, chromatin immunoprecipitation, and DNA affinity precipitation assays further showed that a transcription factor HNF4 bound to the ednR2 region of edn promoter in vitro. Interestingly, HNF4 overexpression resulted in the reduction of edn promoter activity in HepG2 cells, due to involvement of both ednR2 and the 34-nt regions, and direct interaction between HNF4 and Sp1, which abolishes Sp1 binging to the 34-nt segment. Moreover, when the Sp1 was depleted in the cell, overexpressed HNF4 enhanced edn promoter activity. Our results provide novel mechanisms for HNF4 function as an activator to regulate edn promoter activity, which account for differential transcription regulation of human eosinophil RNases.
Eosinophil cationic protein is a clinical asthma biomarker that would be released into blood, especially gathered in bronchia. The signal peptide of eosinophil cationic protein (ECPsp) plays an important role in translocating ECP to the extracellular space. We previously reported that ECPsp inhibits microbial growth and regulates the expression of mammalian genes encoding tumor growth factor-? (TGF-?) and epidermal growth factor receptor (EGFR).
Drug resistance is a common cause of failure in cancer chemotherapy treatments. In this study, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin-resistant MES-SA/Dx5 as a model system to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes induced by doxorubicin treatment and doxorubicin resistance. A proteomic study revealed that doxorubicin-exposure altered the expression of 87 proteins in MES-SA cells, while no significant response occurred in similarly treated MES-SA/Dx5 cells, associating these proteins with drug specific resistance. By contrast, 37 proteins showed differential expression between MES-SA and MES-SA/Dx5, indicating baseline resistance. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against asparagine synthetase (ASNS) and membrane-associated progesterone receptor component 1 (mPR) proteins, to monitor and evaluate their potency on the formation of doxorubicin resistance. The proteomic approach allowed us to identify numerous proteins, including ASNS and mPR, involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.
The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.