ABSTRACT
The ABO blood group system is the most important factor in clinical transfusion medicine and is implicated in a number of human diseases. ABO antigens are not confined to red blood cells (RBCs) and are widely expressed in a variety of human cells and tissues. To date, many alleles with variant ABO expression have been identified and in many cases traced to one of the >250 reported genetic variations in the respective glycosyltransferase. The role of microRNAs (miRNAs) in the regulation of blood group antigens during erythropoiesis has not been addressed, however. Here, we show that miR-331-3p and miR-1908-5p directly target the mRNA of glycosyltransferases A and B. Expression levels of miR-331-3p and miR-1908-5p inversely correlated with levels of blood group A antigen. In addition, we found that overexpression of these miRNAs in hematopoietic stem cells led to a significantly reduced number of blood group A antigens per RBC. Simultaneous targeting of the transcription factor (TF) SP1 by miR-331-3p further enhanced these effects. The targeting rendered SP1 incapable of binding to the ABO gene promoter, causing further downregulation of blood group A antigen expression by up to 70%. Taken together, expression changes in these miRNAs may account for rare cases of weak A/B phenotypes that genetic variations in the glycosyltransferase coding region cannot explain. These results also suggest an explanation for the disappearance of ABH antigens during carcinogenesis and point to new therapeutic targets in ABO mismatched organ transplantation.
Subject(s)
Blood Group Antigens/genetics , Cell Differentiation/genetics , Gene Expression Regulation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , MicroRNAs/metabolism , Base Sequence , Blood Group Antigens/metabolism , Down-Regulation/genetics , Genotype , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Humans , MicroRNAs/genetics , Models, Biological , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolismABSTRACT
Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis.
Subject(s)
Leukemia, Erythroblastic, Acute , Plasmodium vivax/growth & development , Reticulocytes , Cell Differentiation , Duffy Blood-Group System/biosynthesis , Duffy Blood-Group System/genetics , Gene Expression Regulation, Leukemic , Humans , K562 Cells , Leukemia, Erythroblastic, Acute/genetics , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Erythroblastic, Acute/parasitology , Leukemia, Erythroblastic, Acute/pathology , MicroRNAs/biosynthesis , MicroRNAs/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Reticulocytes/metabolism , Reticulocytes/parasitology , Reticulocytes/pathologyABSTRACT
The treatment or prevention of bleeding in patients with hemophilia A relies on replacement therapy with different factor VIII (FVIII)-containing products or on the use of by-passing agents, i.e., activated prothrombin complex concentrates or recombinant activated factor VII. Emerging approaches include the use of bispecific anti-factor IXa/factor X antibodies, anti-tissue factor pathway inhibitor antibodies, interfering RNA to antithrombin, and activated protein C-specific serpins or gene therapy. The latter strategies are, however, hampered by the short clinical experience and potential adverse effects including the absence of tight temporal and spatial control of coagulation and the risk of uncontrolled insertional mutagenesis. Systemic delivery of mRNA allows endogenous production of the corresponding encoded protein. Thus, injection of erythropoietin-encoding mRNA in a lipid nanoparticle formulation resulted in increased erythropoiesis in mice and macaques. Here, we demonstrate that a single injection of in vitro transcribed B domain-deleted FVIII-encoding mRNA to FVIII-deficient mice enables endogenous production of pro-coagulant FVIII. Circulating FVIII:C levels above 5% of normal levels were maintained for up to 72 h, with an estimated half-life of FVIII production of 17.9 h, and corrected the bleeding phenotype in a tail clipping assay. The endogenously produced FVIII did however exhibit low specific activity and induced a potent neutralizing IgG response upon repeated administration of the mRNA. Our results suggest that the administration of mRNA is a plausible strategy for the endogenous production of proteins characterized by poor translational efficacy. The use of alternative mRNA delivery systems and improved FVIII-encoding mRNA should foster the production of functional molecules and reduce their immunogenicity.
Subject(s)
Antibodies, Bispecific , Hemophilia A , Animals , Factor VIII/genetics , Hemophilia A/drug therapy , Hemophilia A/genetics , Hemorrhage/therapy , Humans , Mice , RNA, Messenger/geneticsABSTRACT
PURPOSE: The aim of this study was to investigate the curve fitting and model selection problem of the torque-velocity relationship of elbow flexors and extensors in untrained females. The second goal was to determine the optimal models in different function classes and the best, among the optimal ones. Lastly, test the best models to predict the torque were tested. METHODS: Using the polynomials (second - fourth degree) and Boltzmann sigmoid functions, and a different presentation of data points (averages, a point cloud, etc.), we determined the optimal models by both error criteria: minimum residual sum of squares and minimum of the maximal absolute residue. To assess the best models, we applied Akaike and Bayesian information criteria, Hausdorff distance and the minimum of the smallest maximal absolute residue and the predictive torque-velocity relationships of the best models with torque values, calculated beyond the experimental velocity interval. RESULTS: The application of different error and model selection criteria showed that the best models in the majority of cases were polynomials of fourth degree, with some exceptions from second and third degree. The criteria values for the optimal Boltzmann sigmoids were very close to those of the best polynomial models. However, the predicted torque-velocity relationships had physiological behavior only in Boltzmann's sigmoid functions, and their parameters had a clear interpretation. CONCLUSION: The results obtained suggest that the Boltzmann sigmoid functions are suitable for modeling and predicting of the torque-velocity relationship of elbow flexors and extensors in untrained females, as compared to polynomials, and their curves are physiologically relevant.
Subject(s)
Algorithms , Torque , Bayes Theorem , Biomechanical Phenomena , Elbow/physiology , Female , Humans , Models, BiologicalABSTRACT
The tertiary structure of the proteins determines their functions. Therefore, the predicting of protein's tertiary structure, based on the primary amino acid sequence from long time, is the most important and challenging subject in biochemistry, molecular biology, and biophysics. One of the most popular protein structure prediction methods, called Hydrophobic-Polar (HP) model, is based on the observation that in polar environment hydrophobic amino acids are in the core of the molecule-in contact between them and more polar amino acids are in contact with the polar environment. In this study, we present a new mixed integer programming formulation, exact algorithm, and two heuristic algorithms to solve the protein folding problem stated as a combinatorial optimization problem in a simple cubic lattice. The results from computational runs on a set of benchmarks are favorably compared to known algorithms for solving the 3D lattice HP model as genetic algorithms, ant colony optimization algorithm, and Monte Carlo algorithm.
Subject(s)
Amino Acids/chemistry , Computational Biology/methods , Proteins/chemistry , Algorithms , Amino Acid Sequence , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Monte Carlo Method , Protein Conformation , Protein Folding , Protein Structure, TertiaryABSTRACT
Arginine residues are very important for the structure of proteins and their action. Arginine is essential for many natural processes because it has unique ionizable group under physiological conditions. Numerous mimetics of arginine were synthesized and their biological effects were evaluated, but the mechanisms of actions are still unknown. The aim of this study is to see if oxy- and sulfoanalogues of arginine can be recognized by human arginyl-tRNA synthetase (HArgS)-an enzyme responsible for coupling of L-arginine with its cognate tRNA in a two-step catalytic reaction. We make use of modeling and docking studies of adenylate kinase (ADK) to reveal the effects produced by the incorporation of the arginine mimetics on the structure of ADK and its action. Three analogues of arginine, L-canavanine (Cav), L-norcanavanine (NCav), and L-sulfoarginine (sArg), can be recognized as substrates of HArgS when incorporated in different peptide and protein sequences instead of L-arginine. Mutation in the enzyme active center by arginine mimetics leads to conformational changes, which produce a decrease the rate of the enzyme catalyzed reaction and even a loss of enzymatic action. All these observations could explain the long-lasting nature of the effects of the arginine analogues.
ABSTRACT
Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
Subject(s)
Factor VIII/metabolism , Hemophilia A/metabolism , Molecular Chaperones/metabolism , Analysis of Variance , Animals , Betaine/metabolism , Betaine/pharmacology , Blotting, Western , CHO Cells , Cricetinae , Cricetulus , Factor VIII/genetics , Flow Cytometry , Genetic Vectors , Hemophilia A/drug therapy , Humans , Lentivirus , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Protein Folding , Protein Transport/drug effects , Protein Transport/physiology , Recombinant Proteins/biosynthesisABSTRACT
The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.
Subject(s)
Factor IX/genetics , Factor VIII/physiology , Genetic Engineering , Genetic Therapy , Genetic Variation/genetics , Hemophilia A/therapy , Hemorrhage/therapy , Animals , Disease Models, Animal , Factor IX/immunology , Hemophilia A/complications , Hemorrhage/etiology , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mutagenesis, Site-Directed , Phenotype , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , VaccinationABSTRACT
Advances in delivery techniques and in expression construct design have renewed interest in nonviral gene transfer. Here, we test plasmid or bacterial backbone free minicircle vectors for factor IX (FIX) expression by hydrodynamic liver-directed delivery. Both constructs are driven by a hepatic control region, the human α(1)-antitrypsin promoter, which results in long-term expression in FIX knockout mice. However, levels of expression were higher and expression loss over time was reduced when using minicircles. Even at the highest expression levels (>700% of normal) FIX was fully functional. Transgene loss was the main determinant for expression loss over time for both vector types. A significant effect of gene silencing was observed only for the plasmid, not for the minicircle vector. To determine the influence of promoter methylation, we performed bisulfite-mediated conversion and sequencing of vector DNA on days 14 and 100 after gene transfer. We determined a higher frequency of methyl-protected cytosines in CpGs and a lower degree of demethylation at bacterial Dcm methylation sequences near transcription factor-binding sites in the α(1)-antitrypsin promoter in plasmid compared with minicircle mice on day 100. Therefore, the methylation status might reflect differences in the levels and durability of expression. Judging from the high levels of functional FIX obtained, small fractions of liver or single liver segments should be sufficient to reach therapeutic efficacy in translating hydrodynamic delivery to humans. However, transgene loss remains to be addressed to further guarantee sustained expression over time.
Subject(s)
Factor IX/metabolism , Genetic Vectors , Plasmids/genetics , Promoter Regions, Genetic , Transgenes , Animals , Factor IX/genetics , Gene Expression , Gene Silencing , Gene Transfer Techniques , Genetic Therapy , Hydrodynamics , Liver/metabolism , Methylation , Mice , Mice, Inbred C57BL , Mice, Knockout , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolismABSTRACT
Considering the difficulty in detecting factor (F)VIII in vivo, fluorescently labelled FVIII protein provides a tool to analyse the intracellular localisation, bio distribution, and pharmacokinetics of the protein in living organisms. Here, we report the use of FVIII full length and B-domain deleted proteins, fused to enhanced green fluorescent protein (eGFP) at the C-terminus of the coagulation protein via a nine amino acid spanning linker. Comparison of the FVIII-eGFP fusion proteins to their unlabelled counterparts showed no impairment with respect to recombinant expression levels, intracellular processing, specific coagulant activity and decay at physiological temperature. Confocal live cell imaging demonstrated ER-Golgi-transport of B-domain deleted FVIII-eGFP in vesicular tubular carriers. Using temperature blocks and release experiments, imaging of FVIII-eGFP fusion proteins enabled for the first time the visualisation of the early secretory pathway of B-domain deleted FVIII in living cells and in particular highlighted the apparent deficit of active transport carriers, an observation consistent with the low rates of FVIII secretion seen in recombinant expression systems.
Subject(s)
Factor VIII/pharmacology , Green Fluorescent Proteins/genetics , Animals , Blood Coagulation/drug effects , CHO Cells , COS Cells , Cell Line , Chlorocebus aethiops , Cricetinae , Cricetulus , Endoplasmic Reticulum/metabolism , Factor VIII/chemistry , Factor VIII/genetics , Factor VIII/metabolism , Factor VIIIa/analysis , Golgi Apparatus/metabolism , Humans , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Conformation , Protein Structure, Tertiary , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacologyABSTRACT
To elucidate the structural features determining delta-opioid receptor properties of enkephalin analogues containing Cys(O2NH2) in position 2, a series of Cys2-containing derivatives were synthesized and tested for their effectiveness in depressing electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective delta-opioid receptors) and the guinea-pig ileum (mu- and kappa-opioid receptors). The peptidase resistance of the compounds was also tested. The ratio IC50 in the guinea-pig ileum/IC50 in the mouse vas deferens, indicating selectivity for delta-opioid receptors, was high for Cys(O2NH2)2-containing analogues and especially for [Cys(O2NH2)2, Leu5]enkephalin, which was about seven times more selective than delta-opioid receptor selective ligand cyclic [D-Pen2, D-Pen5]enkephalin (DPDPE). The dissociation constant (KA) and relative efficacy (e(rel)) of the compounds in the mouse-isolated vas deferens were determined using explicit formulae derived by fitting of the data points with two-parametric hyperbolic function. The obtained values for KA and e(rel) suggest that: (i) incorporation of Cys(O2NH2)2 in the molecule of [Leu5]enkephalin highly increases the efficacy and does not change significantly the affinity of the respective analogues to delta-opioid receptors; [Cys(O2NH2)2, Leu5]enkephalin has higher affinity than DPDPE, but is less resistant to enzyme degradation; the effect of this modification on the efficacy is decreased when methionine is in position 5; (ii) D-configuration of Cys(O2NH2)2-containing analogues increases their peptidase resistance, but reduces efficacy and affinity of the peptides towards delta-opioid receptors; (iii) the substitution of Cys(O2NH2) with Hcy(O2NH2) reduces the efficacy, affinity and potency of the respective analogues and maintains their sensitivity to endogenous peptidases; (iv) the substitution of the sulfonamide group with benzyl group in the molecule of Cys in position 2 decreases their efficacy and affinity toward delta-opioid receptors, but attaches resistance to enzyme degradation. The results obtained in this study allow: (i) to involve the receptor affinity and agonist efficacy as drug-design consideration for delta-opioid receptor properties of newly synthesized compounds and (ii) to characterize some of the structural features, which set the pattern for their opioid profiles.