RESUMEN
Asparaginase is one of the most important chemotherapeutic agents against acute lymphoblastic leukemia, the most common form of blood cancer. To date, both asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi), used in hematology, induce chemoresistance in cancer cells and side effects in the form of hypersensitivity of immune reactions. Leukemic cells may be resistant to asparaginase due to the increased activity of asparagine synthetase and other mechanisms associated with resistance to asparaginase. Therefore, the search for new sources of L-asparaginases with improved pharmacological properties remains a promising and prospective study. This article discusses the mechanisms of development of resistance and drug resistance to L-asparaginase, as well as possible ways to overcome them.
Asunto(s)
Asparaginasa , Leucemia-Linfoma Linfoblástico de Células Precursoras , Asparaginasa/efectos adversos , Resistencia a Medicamentos , Escherichia coli , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/inducido químicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Estudios ProspectivosRESUMEN
L-asparaginase is extensively used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is also of prospective use in food industry to reduce the formation of acrylamide, which is classified as probably neurotoxic and carcinogenic to human, and in biosensors for determination of L-asparagine level in medicine and food chemistry. The importance of L-asparaginases in different fields, disadvantages of commercial ferments, and the fact that they are widespread in nature stimuli the search for biobetter L-asparaginases from new producing microorganisms. In this regard, extremofile microorganisms exhibit unique physiological properties such as thermal stability, adaptability to extreme cold conditions, salt and pH tolerance and so provide one of the most valuable sources for novel L-asparaginases. The present review summarizes the recent results on studying the structural, functional, physicochemical and kinetic properties, stability of extremophilic L-asparaginases in comparison with their mesophilic homologues.
Asunto(s)
Asparaginasa/química , Extremófilos/enzimología , AsparaginaRESUMEN
In the process of optimization of heterologous expression of thermostable chemotaxis proteins CheW and CheY as industrially useful polypeptides, their direct influence on the cell growth kinetics and morphology of Escherichia coli was observed. CheW and CheY of bacteria of the genus Thermotoga, being expressed in recombinant form in E. coli cells, are involved in the corresponding signal pathways of the mesophilic microorganisms. The effects of such involvement in the metabolism of "host" cells are extremely diverse: from rapid aging of the culture to elongation of the stationary growth phase. We also discuss the mechanisms of the influence of the heterologous chemotaxis proteins on cells, their positive and negative effects, as well as potential applications in industry and biomedicine.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/biosíntesis , Escherichia coli/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/biosíntesis , Proteínas Bacterianas/genética , Reactores Biológicos/microbiología , Quimiotaxis/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Expresión Génica/genética , Proteínas Quimiotácticas Aceptoras de Metilo/genética , ThermotogaRESUMEN
Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.
Asunto(s)
Proteínas Bacterianas/genética , Quimiotaxis , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/genética , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Escherichia coli , Proteínas Mutantes/genética , Proteínas RecombinantesRESUMEN
Mutant homologues of small chemotactic and DNA-binding proteins from thermophilic bacteria Thermotoga petrophila RKU-1 and Thermotoga naphthophila were obtained. These proteins can be expressed in the recombinant form in E. coli cells. A wide range of properties and parameters that are important for isolation of these proteins were revealed: stability in a wide temperature and pH range, high level of expression, solubility, and the possibility of using simple purification schemes with low number of successive steps. The positive effect of proteins on in vitro fibroblasts growth was demonstrated. The described properties of the target proteins indicate the possibility of their use in different biotechnology industries as an inexpensive source of L-amino acids.
Asunto(s)
Aminoácidos/química , Aminoácidos/metabolismo , Medios de Cultivo/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fibroblastos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
The effect of apoptotic endonuclease EndoG on alternative splicing of mRNA of human telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) and telomerase activity in normal human lymphocytes were studied. Human CD4+, CD8+, B, and NK cells were transfected with a plasmid pEndoG-GFP containing EndoG gene or control plasmid pGFP. The levels of mRNA of EndoG or hTERT splicing variants were analyzed by real-time PCR. Protein content was assessed by Western blotting. Telomerase activity was measured by the telomere repeats amplification protocol. EndoG overexpression reduced the expression of active full-length hTERT and increased the expression of inactive splice variant. Shifted balance of hTERT splice variants in cells led to a significant decrease in telomerase activity within 72 h after transfection.
Asunto(s)
Empalme Alternativo , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Endonucleasas/genética , Células Asesinas Naturales/inmunología , Telomerasa/genética , Apoptosis/genética , Apoptosis/inmunología , Linfocitos B/citología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Endonucleasas/inmunología , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunofenotipificación , Células Asesinas Naturales/citología , Transducción de Señal , Telomerasa/inmunologíaRESUMEN
In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.
Asunto(s)
Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales , Proteínas Bacterianas , Escherichia coli , Proteínas de Escherichia coli , Proteínas RecombinantesRESUMEN
Activity of telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At present time exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ T-lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ T-cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.
Asunto(s)
Empalme Alternativo , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD8-positivos/enzimología , Daño del ADN , Endodesoxirribonucleasas/metabolismo , Telomerasa/genética , Células Cultivadas , Cisplatino , HumanosRESUMEN
The activity of telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) can be regulated by alternative splicing of its mRNA. The mechanism of hTERT splicing is not understood in detail. Apoptotic endonuclease EndoG is known to participate in this process. In the present work, the intracellular colocalization and mRNA levels of EndoG and hTERT splice-variants in normal and apoptotic cancer cells were studied. We found that the development of apoptosis increased the expression of EndoG and changed the ratio of hTERT splice-variants, which decreased the telomerase activity in the cells. The development of apoptosis was accompanied by changes in the amount of mRNA and in the localization of EndoG and hTERT splice-variants in the cytoplasm, nuclei, and mitochondria of the cells. The suppression of EndoG expression using RNA interference prevented induction of the α+ß- splice-variant of hTERT and inhibition of the telomerase activity. A high degree of the intracellular colocalization of EndoG and hTERT was shown. The changes in the expression and localization of EndoG corresponded with changes in the amount and localization of hTERT splice-variants. These data confirm the participation of EndoG in the alternative splicing of mRNA of the telomerase catalytic subunit and in regulation of the telomerase activity.
Asunto(s)
Endonucleasas/metabolismo , Telomerasa/metabolismo , Empalme Alternativo , Apoptosis , Células CACO-2 , Dominio Catalítico , Endonucleasas/antagonistas & inhibidores , Endonucleasas/genética , Humanos , Transporte de Proteínas , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Telomerasa/química , Telomerasa/genéticaRESUMEN
The construction of proteins and peptides with desired properties, including resistance to high temperatures, as well as optimization of their amino acid composition, is an important and complex task, which attracts much attention in various branches of the basic sciences, and also in biomedicine and biotechnology. This raises the question: what method is more relevant for the at the pilot stage of research in order to estimate the influence of the planned amino acid substitutions on the thermostability of the resultant protein construct? In this brief review we have classified existing basic practical and theoretical approaches used in studies and predicting the thermal stability of native and recombinant polypeptides. Particular attention has been paid to the predictive potential of statistical methods for studying the thermodynamic parameters of the primary protein structure and prospects of their use.
Asunto(s)
Algoritmos , Aminoácidos/química , Ingeniería de Proteínas , Proteínas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Archaea/química , Bacterias/química , Calor , Humanos , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/ultraestructura , TermodinámicaRESUMEN
Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.
Asunto(s)
Antineoplásicos/toxicidad , Linfocitos T CD4-Positivos/efectos de los fármacos , Transformación Celular Neoplásica/genética , Cisplatino/toxicidad , Endodesoxirribonucleasas/genética , Telomerasa/genética , Empalme Alternativo/efectos de los fármacos , Animales , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/patología , Endodesoxirribonucleasas/metabolismo , Humanos , Linfoma/genética , Linfoma/inmunología , Linfoma/mortalidad , Linfoma/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Cultivo Primario de Células , Análisis de Supervivencia , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismo , Telómero/química , Telómero/efectos de los fármacos , Acortamiento del Telómero/efectos de los fármacosRESUMEN
Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active full-length hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.
Asunto(s)
Empalme Alternativo , Linfocitos T CD4-Positivos/enzimología , Transformación Celular Neoplásica/metabolismo , Endodesoxirribonucleasas/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Telomerasa/biosíntesis , Homeostasis del Telómero , Animales , Linfocitos T CD4-Positivos/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Endodesoxirribonucleasas/genética , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Telomerasa/genéticaRESUMEN
The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrÐE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrÐ+N17, D60K, F61L and RrÐ+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.
Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/farmacología , Proteínas Bacterianas/farmacología , Mutación Puntual , Rhodospirillum rubrum/enzimología , Telomerasa/antagonistas & inhibidores , Telómero/efectos de los fármacos , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Asparaginasa/química , Asparaginasa/genética , Asparaginasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Células HL-60 , Humanos , Células Jurkat , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimología , Pectobacterium carotovorum/genética , Plásmidos/química , Plásmidos/metabolismo , Estructura Secundaria de Proteína , Rhodospirillum rubrum/química , Rhodospirillum rubrum/genética , Especificidad de la Especie , Relación Estructura-Actividad , Telomerasa/genética , Telomerasa/metabolismo , Telómero/química , Wolinella/química , Wolinella/enzimología , Wolinella/genéticaRESUMEN
Proteins and polypeptides play a key role in the life of a human body. Scientific and practical interest to the natural proteinaceous substances could be explained by the diversity of their functions in metabolic processes. Biologically active substances of the protein origin have a rich history of applications in different sectors of the economy. In this case, the close relationship is observed between food industry, biomedicine and fodder production, because efficient conversion of feed protein in productive agricultural animals provides, as a result, the required level of metabolism in a human, as the main consumer of final products derived from these animals. Obviously, for normal growth, development and resistance to infectious agents, both people and farm animals need a constant consumption of L-amino acids in certain proportions and in available for absorption form. This review considers the bioactive polypeptides used in nutrition and food industry, main trends and practical approaches to generating protein products with the desired characteristics.
RESUMEN
N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.
Asunto(s)
Antineoplásicos Fitogénicos/química , Asparaginasa/química , Proteínas Bacterianas/química , Pectobacterium carotovorum/química , Polietilenglicoles/química , Antineoplásicos Fitogénicos/biosíntesis , Antineoplásicos Fitogénicos/farmacología , Asparaginasa/biosíntesis , Asparaginasa/genética , Asparaginasa/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Supervivencia Celular/efectos de los fármacos , Cromatografía en Gel , Clonación Molecular , Reactivos de Enlaces Cruzados/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HL-60 , Humanos , Células Jurkat , Células K562 , Pectobacterium carotovorum/enzimología , Polietilenglicoles/farmacología , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Succinimidas/químicaRESUMEN
Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of b-deletion splice variant was determined during EndoG over-expression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.
Asunto(s)
Empalme Alternativo/fisiología , Endodesoxirribonucleasas/metabolismo , Exones , ARN no Traducido/biosíntesis , Telomerasa/biosíntesis , Células CACO-2 , Endodesoxirribonucleasas/genética , Humanos , ARN no Traducido/genética , Telomerasa/genéticaRESUMEN
Telomerase activity is known to be regulated by alternative splicing of its catalytic subunit hTERT (human Telomerase Reverse Transcriptase) mRNA. Induction of non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. Strong correlation was found between expression of EndoG and hTERT splice-variants in 12 colon cancer cell lines. Overexpression of EndoG in СаСо-2 cells downregulated the expression of active full-length hTERT variant and upregulated non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, dramatically shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. These data indicated the participation of EndoG in alternative splicing of mRNA of telomerase catalytic subunit, regulation of telomerase activity and cell fate.
Asunto(s)
Empalme Alternativo , Apoptosis , Endodesoxirribonucleasas/metabolismo , Telomerasa/genética , Células CACO-2 , Endodesoxirribonucleasas/genética , Células HCT116 , Células HT29 , Humanos , Telomerasa/metabolismoRESUMEN
A new approach to the regulation of catalytic properties of medically relevant enzymes has been proposed using the novel recombinant preparation of L-asparaginase from Erwinia carotovora (EwA), a promising antitumor agent. New branched co-polymers of different composition based on chitosan modified with polyethylene glycol (PEG) molecules, designated as PEG-chitosan, have been synthesized. PEG-chitosan copolymers were further conjugated with EwA. In order to optimize the catalytic properties of asparaginase two types of conjugates differing in their architecture have been synthesized: (1) crown-type conjugates were synthesized by reductive amination reaction between the reducing end of the PEG-chitosan copolymer and enzyme amino groups; (2) multipoint-conjugates were synthesized using the reaction of multipoint amide bond formation between PEG-chitosan amino groups and carboxyl groups of the enzyme in the presence of the Woodward's reagent. The structure and composition of these conjugates were determined by IR spectroscopy. The content of the copolymers in the conjugates was controlled by the characteristic absorption band of C-O-C bonds in the PEG structure at the frequency of 1089 cm-1. The study of catalytic characteristics of EwA preparations by conductometry showed that at physiological pH values the enzyme conjugates with PEG-chitosan with optimized structure and the optimal composition demonstrated 5-8-fold higher catalytic efficiency (kcat/Km) than the native enzyme. To certain extent, this can be attributed to favorable shift of pH-optima in result of positively charged amino-groups introduction in the vicinity of the active site. The proposed approach, chito-pegylation, is effective for regulating the catalytic and pharmacokinetic properties of asparaginase, and is promising for the development of prolonged action dosage forms for other enzyme therapeutics.
Asunto(s)
Antineoplásicos/química , Asparaginasa/química , Proteínas Bacterianas/química , Quitosano/química , Polietilenglicoles/química , Antineoplásicos/metabolismo , Asparaginasa/genética , Asparaginasa/metabolismo , Asparagina/química , Asparagina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Concentración de Iones de Hidrógeno , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimología , Polimerizacion , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría Infrarroja , Relación Estructura-Actividad , Succinimidas/químicaRESUMEN
For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.
Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/química , Asparaginasa/farmacología , Proteínas Bacterianas/farmacología , Secuencia de Aminoácidos , Antineoplásicos/química , Asparaginasa/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular Tumoral/efectos de los fármacos , Helicobacter pylori/enzimología , Humanos , Leucemia/tratamiento farmacológico , Leucemia/patología , Datos de Secuencia Molecular , Pectobacterium carotovorum/enzimología , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Rhodospirillum rubrum/enzimología , Yersinia pseudotuberculosis/enzimologíaRESUMEN
Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrÐI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrÐD), and R118H, G120R(RrÐJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrÐB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrÐE), considerably destabilized recombinant enzyme.