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Elena Uribe – One of the best experts on this subject based on the ideXlab platform.

  • Cloning of two LIMCH1 isoforms: characterization of their distribution in rat brain and their Agmatinase activity
    Histochemistry and Cell Biology, 2016
    Co-Authors: David García, Nelson Carvajal, José Benítez, Arlette González, Vasthi López, Patricio Ordenes, María A. García-robles, Elena Uribe

    Abstract:

    Agmatine, a precursor for polyamine biosynthesis, is also associated with neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. This molecule results from the decarboxylation of l -arginine by arginine decarboxylase, and it is hydrolyzed to urea and putrescine by Agmatinase. Recently, we have described a new protein that also hydrolyzes agmatine, Agmatinase-like protein (ALP), which was identified through immunohistochemical analysis in the hypothalamus and hippocampus of rats. However, its sequence differs greatly from all known Agmatinases and does not contain the typical Mn^2+ ligands associated with the urea hydrolase family of proteins. ALP has a LIM-like domain close to its carboxyl terminus, and the removal of which results in a truncated variant with a tenfold increased k _cat value and a threefold decreased K _ m value for agmatine. Analysis of the gene database revealed several transcripts, denominated LIMCH1 isoforms, with extreme 3′ sequences identical to ALP. Limch1 gene products have been described as members of a multi-domain family of proteins with the biggest isoform containing a calponin homology (CH) domain at its N-terminus. Here, we cloned two LIMCH1 transcripts, one of 3177 bp and the other of 2709 bp (ALP contains 1569 bp) and analyzed LIMCH1 expression and distribution in rat brain using RT-PCR, Western blot and immunohistochemical analyses. LIMCH1 was detected mainly in the hypothalamic and hippocampal regions, which is similar to the distribution of ALP and agmatine in brain. In addition, we cloned and expressed both isoforms in E. coli and confirmed that they were catalytically active on agmatine with kinetic parameters similar to ALP. LIM domain-truncated variants of both isoforms moderately increased the k _cat and catalytic efficiency. Thus, we propose that LIMCH1 is useful to regulate the intracellular concentrations of the neurotransmitter/neuromodulator, agmatine.

  • further insight into the inhibitory action of a lim double zinc finger motif of an Agmatinase like protein
    Journal of Inorganic Biochemistry, 2014
    Co-Authors: Jaime Cofré, Nelson Carvajal, José Benítez, David García, Alejandro Vallejos, Paola Montes, Jose Martinezoyanedel, Elena Uribe

    Abstract:

    Abstract Agmatine is a precursor for polyamine biosynthesis also associated to neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. It results from decarboxylation of l -arginine by arginine decarboxylase and it is hydrolyzed to urea and putrescine by Agmatinase. Recently, we have described a new protein which also hydrolyzes agmatine although its sequence greatly differs from all known Agmatinases. This Agmatinase-like protein (ALP) contains a LIM-like double Zn-finger domain close to its carboxyl terminus, whose removal results in a truncated variant with a 10-fold increased k cat , and a 3-fold decreased K m value for agmatine. Our proposal was that the LIM-domain functions as an autoinhibitory, regulatory entity for ALP. Results in this report provide additional support for the postulated inhibitory effect. The purified isolated LIM domain was shown to be competitively inhibitory to a truncated variant ALP (lacking the LIM-domain), but not to the wild-type species. The C453A variant was shown to be a Zn 2 + -free enzyme with kinetic parameters similar to those of the truncated-ALP. A molecular dynamic simulation of a modeled LIM-domain 3D structure showed that, as a consequence of C453A mutation, the coordination of the zinc ion is broken and the structure of the zinc finger is melted. The inhibitory action of the LIM/double Zinc-finger motif was associated to a significant conformational change, as detected by tryptophan fluorescence studies, but was not related to changes in the association of the enzyme with the catalytically essential Mn 2 + .

  • Further insight into the inhibitory action of a LIM/double zinc-finger motif of an Agmatinase-like protein.
    Journal of inorganic biochemistry, 2013
    Co-Authors: Jaime Cofré, Nelson Carvajal, José Benítez, David García, Alejandro Vallejos, Paola Montes, José Martínez-oyanedel, Elena Uribe

    Abstract:

    Abstract Agmatine is a precursor for polyamine biosynthesis also associated to neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. It results from decarboxylation of l -arginine by arginine decarboxylase and it is hydrolyzed to urea and putrescine by Agmatinase. Recently, we have described a new protein which also hydrolyzes agmatine although its sequence greatly differs from all known Agmatinases. This Agmatinase-like protein (ALP) contains a LIM-like double Zn-finger domain close to its carboxyl terminus, whose removal results in a truncated variant with a 10-fold increased k cat , and a 3-fold decreased K m value for agmatine. Our proposal was that the LIM-domain functions as an autoinhibitory, regulatory entity for ALP. Results in this report provide additional support for the postulated inhibitory effect. The purified isolated LIM domain was shown to be competitively inhibitory to a truncated variant ALP (lacking the LIM-domain), but not to the wild-type species. The C453A variant was shown to be a Zn 2 + -free enzyme with kinetic parameters similar to those of the truncated-ALP. A molecular dynamic simulation of a modeled LIM-domain 3D structure showed that, as a consequence of C453A mutation, the coordination of the zinc ion is broken and the structure of the zinc finger is melted. The inhibitory action of the LIM/double Zinc-finger motif was associated to a significant conformational change, as detected by tryptophan fluorescence studies, but was not related to changes in the association of the enzyme with the catalytically essential Mn 2 + .

Nelson Carvajal – One of the best experts on this subject based on the ideXlab platform.

  • Mammalian Agmatinases constitute unusual members in the family of Mn2+-dependent ureahydrolases.
    Journal of inorganic biochemistry, 2016
    Co-Authors: Nicol Romero, Gerhard Schenk, José Benítez, David García, Arlette González, Vasthi López, María De Los Ángeles García-robles, Leonardo Bennun, Liam A. Wilson, Nelson Carvajal

    Abstract:

    Agmatine (1-amino-4-guanidinobutane) plays an important role in a range of metabolic functions, in particular in the brain. Agmatinases (AGMs) are enzymes capable of converting agmatine to the polyamine putrescine and urea. AGMs belong to the family of Mn-dependent ureahydrolases. However, no AGM from a mammalian source has yet been extracted in catalytically active form. While in human AGM the six amino acid ligands that coordinate the two Mn ions in the active site are conserved, four mutations are observed in the murine enzyme. Here, we demonstrate that similar to its human counterpart murine AGM does not appear to have in vitro catalytic activity, independent of the presence of Mn. However, in presence of agmatine both enzymes are very efficient in promoting cell growth of a yeast strain that is deficient in polyamine biosynthesis (Saccharomyces cerevisiae strain TRY104Δspe1). Furthermore, mutations among the putative Mn binding residues had no effect on the ability of murine AGM to promote growth of the yeast culture. It thus appears that mammalian AGMs form a distinct group within the family of ureahydrolases that (i) either fold in a manner distinct from other members in this family, or (ii) require accessory proteins to bind Mn in a mechanism related to that observed for the Ni-dependent urease.

  • Cloning of two LIMCH1 isoforms: characterization of their distribution in rat brain and their Agmatinase activity
    Histochemistry and Cell Biology, 2016
    Co-Authors: David García, Nelson Carvajal, José Benítez, Arlette González, Vasthi López, Patricio Ordenes, María A. García-robles, Elena Uribe

    Abstract:

    Agmatine, a precursor for polyamine biosynthesis, is also associated with neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. This molecule results from the decarboxylation of l -arginine by arginine decarboxylase, and it is hydrolyzed to urea and putrescine by Agmatinase. Recently, we have described a new protein that also hydrolyzes agmatine, Agmatinase-like protein (ALP), which was identified through immunohistochemical analysis in the hypothalamus and hippocampus of rats. However, its sequence differs greatly from all known Agmatinases and does not contain the typical Mn^2+ ligands associated with the urea hydrolase family of proteins. ALP has a LIM-like domain close to its carboxyl terminus, and the removal of which results in a truncated variant with a tenfold increased k _cat value and a threefold decreased K _ m value for agmatine. Analysis of the gene database revealed several transcripts, denominated LIMCH1 isoforms, with extreme 3′ sequences identical to ALP. Limch1 gene products have been described as members of a multi-domain family of proteins with the biggest isoform containing a calponin homology (CH) domain at its N-terminus. Here, we cloned two LIMCH1 transcripts, one of 3177 bp and the other of 2709 bp (ALP contains 1569 bp) and analyzed LIMCH1 expression and distribution in rat brain using RT-PCR, Western blot and immunohistochemical analyses. LIMCH1 was detected mainly in the hypothalamic and hippocampal regions, which is similar to the distribution of ALP and agmatine in brain. In addition, we cloned and expressed both isoforms in E. coli and confirmed that they were catalytically active on agmatine with kinetic parameters similar to ALP. LIM domain-truncated variants of both isoforms moderately increased the k _cat and catalytic efficiency. Thus, we propose that LIMCH1 is useful to regulate the intracellular concentrations of the neurotransmitter/neuromodulator, agmatine.

  • insight on the interaction of an Agmatinase like protein with mn2 activator ions
    Journal of Inorganic Biochemistry, 2015
    Co-Authors: Matías Quiñones, Nelson Carvajal, Jaime Cofré, José Benítez, David García, Nicol Romero, Arlette González, María De Los Angeles García, Vasthi López, Gerhard Schenk

    Abstract:

    Agmatinase is an enzyme that catalyzes the hydrolysis of agmatine, a compound that is associated with numerous functions in the brain of mammalian organisms such as neurotransmitter, anticonvulsant, antinociceptive, anxiolytic and antidepressant-like actions. To date the only characterized Agmatinases with significant enzymatic activity were extracted from bacterial organisms. These Agmatinases are closely related to another ureahydrolase, arginase; both have binuclear Mn2+ centers in their active sites. An Agmatinase-like protein (ALP) from rat brain was identified that bears no sequence homology to known Agmatinases (E. Uribe, M. Salas, S. Enriquez, M.S. Orellana, N. Carvajal, Arch. Biochem. Biophys. 461(2007) 146-150). Since all known ureahydrolases contain histidines in their binuclear Mn2+ site each of the five histidine residues in ALP was individually replaced by alanines to identify those that may be involved in metal ion binding. Reactivation assays and thermal stability measurements indicated that His206 is likely to interact with a Mn2+ bound to a high affinity site. In contrast, His65 and possibly His435 are important for binding of a second Mn2+ to a lower affinity site. Metal ion binding to that site is not only leading to an increase in reactivity but also enzyme stability. Thus, similar to bacterial Agmatinases and some of the antibiotic-degrading, Zn2+-dependent metallo-beta-lactamases ALP appears to be active in the mono and binuclear form, with binding of the second metal ion increasing both reactivity and stability. (C) 2015 Elsevier Inc. All rights reserved.

Fuller W Bazer – One of the best experts on this subject based on the ideXlab platform.

  • Effects of catecholamines on secretion of interferon tau and expression of genes for synthesis of polyamines and apoptosis by ovine trophectoderm
    Biology of reproduction, 2018
    Co-Authors: Mohammed A. Elmetwally, Yasser Y. Lenis, Wanjin Tang, Fuller W Bazer

    Abstract:

    Ovine trophectoderm (oTr1) cells were used to investigate effects of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on their proliferation, migration and adhesion, secretion of interferon tau (IFNT), and expression of genes for synthesis of polyamines and apoptosis. Expression of mRNAs for Agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1), and solute carrier family 7 (SLC7A1) (cationic amino acid transporter, Y + system), member 1 increased (P 

  • Effects of Bisphenol-A on proliferation and expression of genes related to synthesis of polyamines, interferon tau and insulin-like growth factor 2 by ovine trophectoderm cells.
    Reproductive toxicology (Elmsford N.Y.), 2018
    Co-Authors: Mohammed A. Elmetwally, Yasser Y. Lenis, Wanjin Tang, Amal A. Halawa, Fuller W Bazer

    Abstract:

    Abstract This study evaluated the effects of bisphenol A (BPA) on proliferation of ovine trophectoderm (oTr1) cells, as well as expression of genes for transport of arginine and synthesis of polyamines. BPA reduced proliferation of oTr1 cells at concentrations of 1 × 10−6, 1 × 10−5, 1 × 10−4 M compared to concentrations of 0, 1 × 10−9, and 1 × 10−8 M at 24 and 96 h of culture. Lower concentrations of BPA significantly increased expression of mRNAs for Agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1) and solute carrier family 7 member 1 (SLC7A1). Similarly, synthesis of polyamines by oTr1 cells was greatest at lower concentrations of BPA and decreased as the dose of BPA increased. Expression of mRNAs for interferon tau (IFNT) and insulin-like growth factor 2 (IGF2) by oTr1 cells was greater than for controls at 1 × 10−9 M BPA. Overall, the effects of BPA on proliferation and gene expression by oTr1 cells were highly dose-dependent.

  • Functional roles of Agmatinase during the peri-implantation period of pregnancy in sheep
    Amino Acids, 2018
    Co-Authors: Yasser Y. Lenis, Mohammed A. Elmetwally, Wanjin Tang, Carey Satterfield, Kathrin Dunlap, Guoyao Wu, Fuller W Bazer

    Abstract:

    This study investigated the effect of agmatine (Agm) in proliferation of ovine trophecdoderm cells (oTr1) as well as the importance of the arginine decarboxylase (ADC) and Agmatinase (AGMAT) alternative pathway for synthesis of polyamines in ovine conceptuses during the peri-implantation period of pregnancy. Morpholino antisense oligonucleotides (MAOs) were used to inhibit translation of mRNAs for ODC1 alone, AGMAT alone, and their combination. Rambouillet ewes (N = 50) were assigned randomly to the following treatments on Day 8 of pregnancy: MAO control (n = 10); MAO-ODC1 (n = 8); MAO-ADC (n = 6); MAO-ODC1:MAO-ADC (n = 9); or MAO-ODC1:MAO-AGMAT (n = 9). Ewes were ovario-hysterectomized on Day 16 of pregnancy to obtain uterine flushings, uterine endometrium, and conceptus tissues. Inhibition of translation of both ODC1 and AGMAT resulted in 22% of ewes having morphologically and functionally normal (elongated and healthy) conceptuses designated MAO-ODC1:MAO-AGMAT (A). But, 78% of the MAO-ODC1:MAO-AGMAT ewes had morphologically and functionally abnormal (not elongated and fragmented) conceptuses designated MAO-ODC1:MAO-AGMAT (B). The pregnancy rate was less (22%; P