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Asparaginase 1 from Saccharomyces cerevisiae: investigation of the functional and structural effects of the N-terminal domain

Grant number: 17/25272-4
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): July 01, 2018
Effective date (End): June 30, 2019
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Marcos Antonio de Oliveira
Grantee:Gabriella Costa Fernandes
Host Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
Associated research grant:13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical, AP.TEM


The treatment of acute lymphoblastic leukemia (ALL) is commonly done using bacterial asparaginases (ASNase), as ASNase of Escherichia coli (EcA) native or conjugated with polyethylene glycol (PEG-EcA) or Erwinia chrysanthemi (ErA). These enzymes are homotetrameric and present high molecular weight (~140kDa). The antitumoral action of ASNase is based on the inability of the leukemic cells to produce the 1-asparagine amino acid (Asn) in proper levels, using the Asn of the bloodstream. The ASNase catalyzes efficiently the hydrolysis of L-Asn into aspartic acid (Asp) and ammonia (NH3), depleting completely the serum source of Asn to lymphoblastic tumor cells, inducing apoptosis. Additionally, some ASNases present the secondary glutaminase activity (GLNase), which catalyzes the hydrolysis of glutamine (Gln) into glutamic acid (Glu) and NH3, an activity most related to a series of side effects, including neurological. In some cases, the EcA can cause immunologic effects, which can be healed by the introduction of a PEG molecule in the drug. However, such exchange may lead to a cross-reaction by the anti-ASNase antibodies action, being the replacement for ErA a better alternative. Even though, a portion of the patients still show side effects. Then, the search for new ASNases show significant importance in obtaining alternative enzymes with less adverse effects. It is worth mentioning that in Brazil, only the EcA and PEG-EcA enzymes present the authorization by ANVISA, which impairs the treatment of ALL. Recently, our research group characterized a new ASNase derived of Saccharomyces cerevisiae (Asp1), with high affinity for Asn, free of GLNase activity and presenting cytotoxicity for tumor ALL cells. Nevertheless, when expressed in oxidant conditions, size exclusion chromatography experiments revealed a monomeric enzyme, with about a ¼ of the molecular weight when compared to bacterial enzymes, this characteristic must be relevant since the reduced size tends to decrease the immunological effects. The Asp1 amino acid sequence analysis revealed a N-terminal domain of about 50 amino acid residues absent in the EcA and ErA enzymes, and seen in only a small yeast group (< 10 species). In fact, this protein portion can responsible to your monomeric activity and determinate to which cellular compartment the enzyme should migrate or even exportation in S. cerevisiae, as seen in isoform Asp3. In this context, the recombinant expression in bacteria with oxidant conditions, which results in the formation of a monomer with elevated asparaginase activity, may also produce an enzyme with characteristic similar to the native protein. However, to date no work has attempted to investigate the function of this protein portion of Asp1.This project aims to evaluate the existence of a signal function in this portion and evaluate the structure and activity of the Asp1 enzyme with deletion of the N-terminal domain, using constructions with the truncated protein (Asp1DNT). Therefore, methodologies involving molecular biology will be used (constructions Asp1DNT and Asp1wt), biochemical (ASNase and GLNase activity tests) and biophysical (circular dichroism - secundary structure; size exclusion chromatography - tertiary/ quaternary structure; thermic displacement - stability).We believe the results obtained in this project will assist significantly in Asp1 enzyme characterization, aiming its alternative biopharmaceutical drugs potential to ALL.

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DE MOURA, WERNER ALFINITO FEIO; SCHULTZ, LEONARDO; BREYER, CARLOS ALEXANDRE; DE OLIVEIRA, ANA LAURA PIRES; TAIRUM, CARLOS ABRUNHOSA; FERNANDES, GABRIELLA COSTA; TOYAMA, MARCOS HIKARI; PESSOA-JR, ADALBERTO; MONTEIRO, GISELE; DE OLIVEIRA, MARCOS ANTONIO. Functional and structural evaluation of the antileukaemic enzymel-asparaginase II expressed at low temperature by differentEscherichia colistrains. Biotechnology Letters, . (13/08617-7, 17/19942-7, 17/25272-4, 19/04054-4, 14/22039-9, 16/19245-1, 17/20291-0, 11/13500-6)

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