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Protein Expression and Purification Mar 2018l-asparaginase (E.C. 3.5.1.1), an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of l-asparaginase from Pseudomonas...
l-asparaginase (E.C. 3.5.1.1), an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of l-asparaginase from Pseudomonas fluorescens was addressed in the present studies. The ANS gene in Pseudomonas fluorescens MTCC 8127 which produces l-asparaginase was cloned and expressed in E. coli BL21 (DE3). The expressed recombinant protein (PfAns) which was purified, showed l-asparaginase activity. The enzyme was further characterized. The pH and temperature optima were found to be 7.5 and 37 °C. The recombinant enzyme was stable to temperature and pH. The enzyme was homotetramer with the molecular weight of the monomer being 35 kDa and a whole protein molecular weight of 140 kDa. The purified l-asparaginase had a specific activity of 26 U/mg with a K and V of 0.050 M and 4.032 molmin. The enzyme exhibited a high affinity towards l-asparagine and showed a very minimal activity with glutamine as a substrate. The enzyme activity was inhibited by PMSF, suggesting the presence of serine at the active site. The presence of Mg enhanced PfAns activity by 49%, and SDS strongly inhibited the enzyme activity. The in vitro half-life of the recombinant enzyme was ∼40 h. The enzyme demonstrated deglycosylation activity which could exhibit an additional barrier for proliferating cancer cells.
Topics: Asparaginase; Asparagine; Bacterial Proteins; Enzyme Stability; Escherichia coli; Glycosylation; Metals; Pseudomonas fluorescens; Recombinant Proteins; Substrate Specificity
PubMed: 29079538
DOI: 10.1016/j.pep.2017.09.009 -
Amino Acids Aug 2021Malignant cells often demonstrate a proliferative advantage when compared to non-malignant cells. However, the rapid growth and metabolism required for survival can also... (Review)
Review
Malignant cells often demonstrate a proliferative advantage when compared to non-malignant cells. However, the rapid growth and metabolism required for survival can also highlight vulnerabilities specific to these malignant cells. One such vulnerability exhibited by cancer is an increased demand for amino acids (AAs), which often results in a dependency on exogenous sources of AAs or requires upregulation of de novo synthesis. These metabolic alterations can be exploited by therapy, which aims to improve treatment outcome and decrease relapse and reoccurrence. One clinically utilised strategy targeting AA dependency is the use of asparaginase in the treatment of acute lymphoblastic leukaemia (ALL), which results in a depletion of exogenous asparagine and subsequent cancer cell death. Examples of other successful strategies include the exploitation of arginine deiminase and methioninase, nutrient restriction of methionine and the inhibition of glutaminase. In this review, we summarise these treatment strategies into three promising avenues: AA restriction, enzymatic depletion and inhibition of metabolism. This review provides an insight into the complexity of metabolism in cancer, whilst highlighting these three current research avenues that have support in both preclinical and clinical settings.
Topics: Amino Acids; Asparaginase; Dietary Proteins; Electron Transport; Glycolysis; Humans; Oxidative Phosphorylation; Precursor Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 34292410
DOI: 10.1007/s00726-021-03052-1 -
International Journal of Pharmaceutics Feb 2023l-asparaginase is a first-line medicine used for the treatment of acute lymphoblastic leukemia. Differing quality of marketed l-asparaginase biosimilars has been... (Review)
Review
l-asparaginase is a first-line medicine used for the treatment of acute lymphoblastic leukemia. Differing quality of marketed l-asparaginase biosimilars has been reported to adversely influence treatment outcomes. Herein, the quality of l-asparaginase biosimilars intended for clinical use was reviewed in sight of quality assurance parameters using English and Chinese language database searching, which provided information for possible improvements to the manufacture of this medicine. Ten articles met inclusion criteria, and quality attributes that measured potency, specific activity, purity and host cell proteins (HCPs) were identified. Biosimilars manufactured in high-income countries represented good quality in all aspects. Biosimilars manufactured in high-middle/middle-income countries, however, suggested poorer quality control particularly over removal of HCPs. Future work should now focus on establishing pharmacopeia monographs to establish equivalent quality assurance for l-asparaginase biosimilars manufactured between countries. Standardization of the quality profile, analytical methods and the limits of critical quality parameters, are essential to ensure appropriated efficacy and safety of clinical grade l-asparaginase.
Topics: Humans; Asparaginase; Biosimilar Pharmaceuticals; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Treatment Outcome; Antineoplastic Agents
PubMed: 36581108
DOI: 10.1016/j.ijpharm.2022.122523 -
FEMS Yeast Research Oct 2022The study of nitrogen assimilation in yeast is of interest from genetic, evolutionary, and biotechnological perspectives. Over the course of evolution, yeasts have...
The study of nitrogen assimilation in yeast is of interest from genetic, evolutionary, and biotechnological perspectives. Over the course of evolution, yeasts have developed sophisticated control mechanisms to regulate nitrogen metabolism, with domesticated lineages sometimes displaying particular specialisation. The focus of this study was on assimilation of asparagine, which is a significant nutritional source for some alcoholic fermentations. We were particularly interested in ASP3, which encodes a periplasmic asparaginase and that was proposed to have been acquired relatively recently in S. cerevisiae by horizontal gene transfer. We examined 1680 S. cerevisiae genome assemblies to evaluate the distribution and evolutionary trajectory of ASP3. Our findings suggest an alternative hypothesis that ASP3 is an ancient Saccharomyces gene that has generally been lost over the course of evolution but has been retained in certain fermentative environments. As asparagine is the major nitrogen source in apple juice, we explored whether the presence of ASP3 would confer a growth advantage. Interestingly, we found that although ASP3 enhances growth when asparagine is the sole nitrogen source, the same effect is not seen in apple juice. These data indicate that growth in pure culture may not reflect the original selective environment for ASP3+ strains and highlight the role that complex regulation may play in optimising nitrogen assimilation in yeasts.
Topics: Asparaginase; Asparagine; Fermentation; Nitrogen; Saccharomyces cerevisiae
PubMed: 36040324
DOI: 10.1093/femsyr/foac044 -
Medical Oncology (Northwood, London,... Apr 2023L-Asparaginase is an antileukemic drug long approved for clinical use to treat childhood acute lymphoblastic leukemia, the most common cancer in this population... (Review)
Review
L-Asparaginase is an antileukemic drug long approved for clinical use to treat childhood acute lymphoblastic leukemia, the most common cancer in this population worldwide. However, the efficacy and its use as a drug have been subject to debate due to the variety of adverse effects that patients treated with it present, as well as the prompt elimination in plasma, the need for multiple administrations, and high rates of allergic reactions. For this reason, the search for new, less immunogenic variants has long been the subject of study. This review presents the main aspects of the L-asparaginase enzyme from a structural, pharmacological, and clinical point of view, from the perspective of its use in chemotherapy protocols in conjunction with other drugs in the different treatment phases.
Topics: Humans; Child; Asparaginase; Antineoplastic Agents; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Drug Hypersensitivity; Drug-Related Side Effects and Adverse Reactions
PubMed: 37060469
DOI: 10.1007/s12032-023-02014-9 -
Biomeditsinskaia Khimiia 2015For 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... (Review)
Review
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.
Topics: Amino Acid Sequence; Antineoplastic Agents; Asparaginase; Bacterial Proteins; Cell Line, Tumor; Helicobacter pylori; Humans; Leukemia; Molecular Sequence Data; Pectobacterium carotovorum; Protein Engineering; Recombinant Proteins; Rhodospirillum rubrum; Yersinia pseudotuberculosis
PubMed: 26215408
DOI: 10.18097/PBMC20156103312 -
Pharmacogenomics May 2022To investigate the role of gene and rs3809849 in pancreatic cancer (PANC1) and lymphoblastic leukemia (NALM6) cell lines and their response to asparaginase treatment....
To investigate the role of gene and rs3809849 in pancreatic cancer (PANC1) and lymphoblastic leukemia (NALM6) cell lines and their response to asparaginase treatment. The authors applied CRISPR-Cas9 to produce knock-out (KO) and rs3809849 knock-in (KI) cell lines. The authors also interrogated rs3809849's impact on PANC1 cells through allele-specific overexpression. PANC1 KO cells exhibited lower proliferation capacity (p ≤ 0.05), higher asparaginase sensitivity (p = 0.01), reduced colony-forming potential (p = 0.001), cell cycle blockage in S phase, induction of apoptosis and remarkable morphology changes suggestive of an epithelial-mesenchymal transition. Overexpression of the wild-type (but not the mutant) allele of -rs3809849 in PANC1 cells increased asparaginase sensitivity. NALM6 KO displayed resistance to asparaginase (p < 0.0001), whereas no effect for rs3809849 KI was noted. is important for regulating various cellular functions, and it plays, along with its rs3809849 polymorphism, a tissue-specific role in asparaginase treatment response.
Topics: Alleles; Asparaginase; DNA-Binding Proteins; Humans; Pancreatic Neoplasms; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA-Binding Proteins; Transcription Factors
PubMed: 35485735
DOI: 10.2217/pgs-2022-0010 -
Applied Microbiology and Biotechnology Sep 2019Thermostability plays an important role in the application of L-asparaginase in the pharmaceutical and food industries. Understanding the key residues and structures... (Comparative Study)
Comparative Study
Thermostability plays an important role in the application of L-asparaginase in the pharmaceutical and food industries. Understanding the key residues and structures that influence thermostability in L-asparaginase is necessary to obtain suitable L-asparaginase candidates. In this study, special residues and structures that altered thermostability in thermophilic L-asparaginase and non-thermophilic L-asparaginase II were identified. Interchanging these special residues and structures of L-asparaginases from the four strains, that is, Pyrococcus yayanosii CH1 (PYA), Thermococcus gammatolerans (TGA), Bacillus subtilis (BSA II), and Escherichia coli (ECA II), revealed the 51st and 298th residues of PYA (corresponding to 57th, 305th residues of ECA II) as the key residues responsible for thermal stability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II. Moreover, the C terminal tightness, loop rigidity, and low surface charge around activity sites were of great significance to the thermostability of L-asparaginase. This study therefore revealed the crucial amino acid residues and structures responsible for the difference in thermostability of the thermophilic and non-thermophilic L-asparaginase and provides a reference for engineering thermostability in L-asparaginase II.
Topics: Amino Acid Sequence; Archaeal Proteins; Asparaginase; Bacterial Proteins; Catalytic Domain; Computational Biology; Enzyme Stability; Hot Temperature; Models, Molecular; Mutation; Protein Conformation; Structure-Activity Relationship
PubMed: 31273395
DOI: 10.1007/s00253-019-09967-w -
Journal of Cellular Physiology Nov 2019l-Asparaginases hydrolyzing plasma l-asparagine and l-glutamine has attracted tremendous attention in recent years owing to remarkable anticancer properties. This enzyme... (Review)
Review
l-Asparaginases hydrolyzing plasma l-asparagine and l-glutamine has attracted tremendous attention in recent years owing to remarkable anticancer properties. This enzyme is efficiently used for acute lymphoblastic leukemia (ALL) and lymphosarcoma and emerged against ALL in children, neoplasia, and some other malignancies. Cancer cells reduce the expression of l-asparaginase leading to their elimination. The l-asparaginase anticancerous application approach has made incredible breakthrough in the field of modern oncology through depletion of plasma l-asparagine to inhibit the cancer cells growth; particularly among children. High level of l-asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species is highly desirable as bacterial alternative enzyme sources for anticancer therapy. Thermal or harsh conditions stability of those from the two latter bacterial species is considerable. Some enzymes from marine bacteria have conferred stability in adverse conditions being more advantageous in cancer therapy. Several side effects exerted by l-asparaginases such as hypersensitivity should be hindered or decreased through alternative therapies or use of immune-suppressor drugs. The l-asparaginase from Erwinia species has displayed remarkable traits in children with this regard. Noticeably, Erwinia chrysanthemi l-asparaginase exhibited negligible glutaminase activity representing a promising efficiency mitigating related side effects. Application of software such as RSM would optimize conditions for higher levels of enzyme production. Additionally, genetic recombination of the encoding gene would indisputably help improving enzyme traits. Furthermore, the possibility of anticancer combination therapy using two or more l-asparaginases from various sources is plausible in future studies to achieve better therapeutic outcomes with lower side effects.
Topics: Antineoplastic Agents; Asparaginase; Asparagine; Escherichia coli; Glutamine; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Recombination, Genetic
PubMed: 30993718
DOI: 10.1002/jcp.28563 -
Biomolecules Aug 2019With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical... (Review)
Review
With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the 'real structure'.
Topics: Asparaginase; Cryoelectron Microscopy; Crystallography, X-Ray; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Polyethylene Glycols; Scattering, Small Angle; X-Ray Diffraction
PubMed: 31416261
DOI: 10.3390/biom9080370