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Anthony E Pegg - One of the best experts on this subject based on the ideXlab platform.

  • functions of polyamines in mammals
    Journal of Biological Chemistry, 2016
    Co-Authors: Anthony E Pegg
    Abstract:

    The content of spermidine and Spermine in mammalian cells has important roles in protein and nucleic acid synthesis and structure, protection from oxidative damage, activity of ion channels, cell proliferation, differentiation, and apoptosis. Spermidine is essential for viability and acts as the precursor of hypusine, a post-translational addition to eIF5A allowing the translation of mRNAs encoding proteins containing polyproline tracts. Studies with Gy mice and human patients with the very rare X-linked genetic condition Snyder-Robinson syndrome that both lack Spermine Synthase show clearly that the correct Spermine:spermidine ratio is critical for normal growth and development.

  • enhancing human Spermine Synthase activity by site directed mutations
    Biophysical Journal, 2013
    Co-Authors: Yoshihiko Ikeguchi, Anthony E Pegg, Zhe Zhang, Emil Alexov
    Abstract:

    Spermine Synthase (SMS) is an enzyme converting spermidine into Spermine, both of which are polyamines controlling normal cell growth and development. Several missense mutations in human SMS (HsSMS) are known to cause Snyder-Robinson Syndrome (SRS) by either destabilizing the monomer/dimer conformation or directly affecting the hydrogen bond network in the active sites. Recently a comparison of protein sequence and crystal structure between the HsSMS and its homologous protein Thermotoga maritima (Tm) spermidine Synthase (TmSRM) was performed. Tm is the only bacterium known to grow at a high temperature as well as 90°C, and the half-life of TmSRM is longer than 25h under this temperature. In contrast, HsSMS is much less stable than TmSRM under the same temperature. Sequence alignment between HsSMS and TmSRM suggests that some key residues may be essential players for the elevate stability of TmSRM. Such key residues were identified based on various biophysical and sequence criteria and four mutations (S165D, L175E, T178H and C206R) were selected for HsSMS. Both in silico and in vitro experiments indicated that these four mutations strongly stabilize the monomer structure and dramatically improve the efficiency of SPM synthesis. The enhanced reaction rate in the mutant HsSMS is attributed to the increase of the strength of negative electrostatic potential, calculated with DelPhi, in the dimer cleft between HsSMS units, which presumably facilitates the substrate delivery to the active site.The work was supported by a grant from the Institute of General Medical Sciences, National Institutes of Health, and the grant number is 1R01GM093937.

  • brain infarction correlates more closely with acrolein than with reactive oxygen species
    Biochemical and Biophysical Research Communications, 2011
    Co-Authors: Ryotaro Saiki, Kazuhiro Nishimura, Toshihiko Toida, Hyerim Park, Itsuko Ishii, Madoka Yoshida, Hideki Tatsukawa, Soichi Kojima, Yoshihiko Ikeguchi, Anthony E Pegg
    Abstract:

    Although it is thought that the major factor responsible for cell damage is reactive oxygen species (ROS), our recent studies have shown that acrolein is more toxic than ROS. Thus, the relative importance of acrolein and ROS in cell damage during brain infarction was compared using photochemically induced thrombosis model mice. The levels of acrolein-conjugated albumin, and of 4-hydroxynonenal (HNE)-conjugated albumin and 8-OHdG were evaluated as indicators of damage produced by acrolein and ROS, respectively. The increase in acrolein-conjugated albumin was much greater than the increase in HNE-conjugated albumin or 8-OHdG, suggesting that acrolein is more strongly involved in cell damage than ROS during brain infarction. It was also shown that infarction led more readily to RNA damage than to DNA or phospholipid damage. As a consequence, polyamines were released from RNA, and acrolein was produced from polyamines, especially from Spermine by Spermine oxidase. Production of acrolein from Spermine by Spermine oxidase was clarified using Spermine Synthase-deficient Gy mice and transglutaminase 2-knockout mice, in which Spermine content is negligible or spermidine/Spermine N(1)-acetyltransferase activity is elevated.

  • Use of (Gyro) Gy and Spermine Synthase Transgenic Mice to Study Functions of Spermine
    Methods in molecular biology (Clifton N.J.), 2011
    Co-Authors: Xiaojing Wang, Anthony E Pegg
    Abstract:

    The polyamines putrescine, spermidine, and Spermine are essential for mammalian cell growth, -differentiation, and cell death and have important physiological roles in all tissues. Many of the properties of polyamines that can be demonstrated in vitro are common to all three molecules with differences only in potency. Loss of any of the enzymes needed to make either putrescine or spermidine (which also -prevent the production of Spermine) is lethal, but male mice lacking Spermine Synthase (SpmS) due to a deletion of part of the X chromosome are viable on the B6C3H background. These mice are termed Gyro (Gy) due to their circling behavior. They have a variety of abnormalities including deafness, neurological problems, small size, and a tendency to early death. They can therefore be used to evaluate the physiological function(s) uniquely provided by Spermine. They also provide a potential animal model for Snyder-Robinson syndrome (SRS), a rare human inherited disease due to a loss of SpmS activity. An essential control in experiments using Gy mice is to demonstrate that the abnormal phenotypes exhibited by these mice are abolished by providing replacement Spermine and this can be accomplished by breeding with CAG-SMS mice that express SpmS from a ubiquitous promoter. Techniques for identifying, characterizing, and using these mouse strains and limitations of this approach are described in this chapter.

  • Spermine Synthase activity affects the content of decarboxylated s adenosylmethionine
    Biochemical Journal, 2011
    Co-Authors: Anthony E Pegg, Xiaojing Wang, Charles E Schwartz, Diane E Mccloskey
    Abstract:

    dcAdoMet (decarboxylated S-adenosylmethionine) is an essential intermediate in the synthesis of polyamines. Its content is normally very low, amounting to less than 5% of that of S-adenosylmethionine itself. It was found that in mice lacking Spermine Synthase there was a large increase in dcAdoMet and that overexpression of Spermine Synthase reduced the amount of this nucleoside. There was also an increase in dcAdoMet in cells derived from patients with Snyder-Robinson syndrome, a rare X-linked recessive human disease caused by SMS gene mutations that greatly reduce the content of Spermine Synthase. These results suggest that there is an inverse relationship between the amount of Spermine Synthase protein and the content of dcAdoMet and raise the possibility that some of the abnormalities seen in mammals deficient in Spermine Synthase might be due to changes in dcAdoMet pools.

Charles E Schwartz - One of the best experts on this subject based on the ideXlab platform.

  • Spermine Synthase and myc cooperate to maintain colorectal cancer cell survival by repressing bim expression
    Nature Communications, 2020
    Co-Authors: Charles E Schwartz, Yubin Guo, Pan Deng, Yanan Cao, Zhaohe Zhou, Chi Wang, Yekaterina Y. Zaytseva, Eun Y. Lee, Mark B Evers
    Abstract:

    Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC), but the underlying mechanism is incompletely characterized. Here, we report that Spermine Synthase (SMS), a polyamine biosynthetic enzyme, is overexpressed in CRC. Targeted disruption of SMS in CRC cells results in spermidine accumulation, which inhibits FOXO3a acetylation and allows subsequent translocation to the nucleus to transcriptionally induce expression of the proapoptotic protein Bim. However, this induction is blunted by MYC-driven expression of miR-19a and miR-19b that repress Bim production. Pharmacological or genetic inhibition of MYC activity in SMS-depleted CRC cells dramatically induces Bim expression and apoptosis and causes tumor regression, but these effects are profoundly attenuated by silencing Bim. These findings uncover a key survival signal in CRC through convergent repression of Bim expression by distinct SMS- and MYC-mediated signaling pathways. Thus, combined inhibition of SMS and MYC signaling may be an effective therapy for CRC. Polyamine metabolism is frequently dysregulated in cancers. Here, the authors show that a polyamine biosynthetic enzyme, Spermine Synthase, is overexpressed in colorectal cancers and cooperates with MYC to prevent cancer cell apoptosis by repression of proapoptotic protein, Bim.

  • Spermine Synthase and MYC cooperate to maintain colorectal cancer cell survival by repressing Bim expression
    'Springer Science and Business Media LLC', 2020
    Co-Authors: Yubin Guo, Charles E Schwartz, Pan Deng, Yanan Cao, Zhaohe Zhou, Chi Wang, Yekaterina Y. Zaytseva, Eun Y. Lee
    Abstract:

    Polyamine metabolism is frequently dysregulated in cancers. Here, the authors show that a polyamine biosynthetic enzyme, Spermine Synthase, is overexpressed in colorectal cancers and cooperates with MYC to prevent cancer cell apoptosis by repression of proapoptotic protein, Bim

  • Polyamine Homeostasis in Snyder-Robinson Syndrome
    MDPI AG, 2018
    Co-Authors: Tracy Murray-stewart, Charles E Schwartz, Matthew Dunworth, Jackson R. Foley, Robert A. Casero
    Abstract:

    Loss-of-function mutations of the Spermine Synthase gene (SMS) result in Snyder-Robinson Syndrome (SRS), a recessive X-linked syndrome characterized by intellectual disability, osteoporosis, hypotonia, speech abnormalities, kyphoscoliosis, and seizures. As SMS catalyzes the biosynthesis of the polyamine Spermine from its precursor spermidine, SMS deficiency causes a lack of Spermine with an accumulation of spermidine. As polyamines, Spermine, and spermidine play essential cellular roles that require tight homeostatic control to ensure normal cell growth, differentiation, and survival. Using patient-derived lymphoblast cell lines, we sought to comprehensively investigate the effects of SMS deficiency on polyamine homeostatic mechanisms including polyamine biosynthetic and catabolic enzymes, derivatives of the natural polyamines, and polyamine transport activity. In addition to decreased Spermine and increased spermidine in SRS cells, ornithine decarboxylase activity and its product putrescine were significantly decreased. Treatment of SRS cells with exogenous Spermine revealed that polyamine transport was active, as the cells accumulated Spermine, decreased their spermidine level, and established a spermidine-to-Spermine ratio within the range of wildtype cells. SRS cells also demonstrated elevated levels of tissue transglutaminase, a change associated with certain neurodegenerative diseases. These studies form a basis for further investigations into the leading biochemical changes and properties of SMS-mutant cells that potentially represent therapeutic targets for the treatment of Snyder-Robinson Syndrome

  • RESEARCH Open Access Protein sector analysis for the clustering of disease-associated mutations
    2016
    Co-Authors: Jose Guevara-coto, Charles E Schwartz, Liangjiang Wang
    Abstract:

    Background: The importance of mutations in disease phenotype has been studied, with information available in databases such as OMIM. However, it remains a research challenge for the possibility of clustering amino acid residues based on an underlying interaction, such as co-evolution, to understand how mutations in these related sites can lead to different disease phenotypes. Results: This paper presents an integrative approach to identify groups of co-evolving residues, known as protein sectors. By studying a protein family using multiple sequence alignments and statistical coupling analysis, we attempted to determine if it is possible that these groups of residues could be related to disease phenotypes. After the protein sectors were identified, disease-associated residues within these groups of amino acids were mapped to a structure representing the protein family. In this study, we used the proposed pipeline to analyze two test cases of Spermine Synthase and Rab GDP dissociation inhibitor. Conclusions: The results suggest that there is a possible link between certain groups of co-evolving residues and different disease phenotypes. The pipeline described in this work could also be used to study other protein families associated with human diseases. Backgroun

  • revealing the effects of missense mutations causing snyder robinson syndrome on the stability and dimerization of Spermine Synthase
    International Journal of Molecular Sciences, 2016
    Co-Authors: Yunhui Peng, Charles E Schwartz, Joy Norris, Emil Alexov
    Abstract:

    Missense mutations in Spermine Synthase (SpmSyn) protein have been shown to cause the Snyder-Robinson syndrome (SRS). Depending on the location within the structure of SpmSyn and type of amino acid substitution, different mechanisms resulting in SRS were proposed. Here we focus on naturally occurring amino acid substitutions causing SRS, which are situated away from the active center of SpmSyn and thus are not directly involved in the catalysis. Two of the mutations, M35R and P112L, are reported for the first time in this study. It is demonstrated, both experimentally and computationally, that for such mutations the major effect resulting in dysfunctional SpmSyn is the destabilization of the protein. In vitro experiments indicated either no presence or very little amount of the mutant SpmSyn in patient cells. In silico modeling predicted that all studied mutations in this work destabilize SpmSyn and some of them abolish homo-dimer formation. Since dimerization and structural stability are equally important for the wild type function of SpmSyn, it is proposed that the SRS caused by mutations occurring in the N-domain of SpmSyn is a result of dysfunctional mutant proteins being partially unfolded and degraded by the proteomic machinery of the cell or being unable to form a homo-dimer.

Zhe Zhang - One of the best experts on this subject based on the ideXlab platform.

  • Enhancing Human Spermine Synthase Activity by Engineered Mutations
    2016
    Co-Authors: Zhe Zhang, Yoshihiko Ikeguchi, Yueli Zheng, Margo Petukh, Anthony Pegg, Emil Alexov
    Abstract:

    Spermine Synthase (SMS) is an enzyme which function is to convert spermidine into Spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine R Spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculate

  • Alexov E: In silico and in vitro investigations of the mutability of disease-causing missense mutation sites in Spermine Synthase. PLoS One 2011
    2016
    Co-Authors: Zhe Zhang, Charles Schwartz, Joy Norris, Emil Alexov
    Abstract:

    Background: Spermine Synthase (SMS) is a key enzyme controlling the concentration of spermidine and Spermine in the cell. The importance of SMS is manifested by the fact that single missense mutations were found to cause Snyder-Robinson Syndrome (SRS). At the same time, currently there are no non-synonymous single nucleoside polymorphisms, nsSNPs (harmless mutations), found in SMS, which may imply that the SMS does not tolerate amino acid substitutions, i.e. is not mutable. Methodology/Principal Findings: To investigate the mutability of the SMS, we carried out in silico analysis and in vitro experiments of the effects of amino acid substitutions at the missense mutation sites (G56, V132 and I150) that have been shown to cause SRS. Our investigation showed that the mutation sites have different degree of mutability depending on their structural micro-environment and involvement in the function and structural integrity of the SMS. It was found that the I150 site does not tolerate any mutation, while V132, despite its key position at the interface of SMS dimer, is quite mutable. The G56 site is in the middle of the spectra, but still quite sensitive to charge residue replacement. Conclusions/Significance: The performed analysis showed that mutability depends on the detail of the structural and functional factors and cannot be predicted based on conservation of wild type properties alone. Also, harmless nsSNPs ca

  • rational design of small molecule stabilizers of Spermine Synthase dimer by virtual screening and free energy based approach
    PLOS ONE, 2014
    Co-Authors: Yoshihiko Ikeguchi, Zhe Zhang, Emil Alexov, David Lagorce, Virginie Y Martiny, Maria A. Miteva
    Abstract:

    Snyder-Robinson Syndrome (SRS) is a rare mental retardation disorder which is caused by the malfunctioning of an enzyme, the Spermine Synthase (SMS), which functions as a homo-dimer. The malfunctioning of SMS in SRS patients is associated with several identified missense mutations that occur away from the active site. This investigation deals with a particular SRS-causing mutation, the G56S mutation, which was shown computationally and experimentally to destabilize the SMS homo-dimer and thus to abolish SMS enzymatic activity. As a proof-of-concept, we explore the possibility to restore the enzymatic activity of the malfunctioning SMS mutant G56S by stabilizing the dimer through small molecule binding at the mutant homo-dimer interface. For this purpose, we designed an in silico protocol that couples virtual screening and a free binding energy-based approach to identify potential small-molecule binders on the destabilized G56S dimer, with the goal to stabilize it and thus to increase SMS G56S mutant activity. The protocol resulted in extensive list of plausible stabilizers, among which we selected and tested 51 compounds experimentally for their capability to increase SMS G56S mutant enzymatic activity. In silico analysis of the experimentally identified stabilizers suggested five distinctive chemical scaffolds. This investigation suggests that druggable pockets exist in the vicinity of the mutation sites at protein-protein interfaces which can be used to alter the disease-causing effects by small molecule binding. The identified chemical scaffolds are drug-like and can serve as original starting points for development of lead molecules to further rescue the disease-causing effects of the Snyder-Robinson syndrome for which no efficient treatment exists up to now.

  • Rational design of small-molecule stabilizers of Spermine Synthase dimer by virtual screening and free energy-based approach
    2014
    Co-Authors: Zhe Zhang, Yoshihiko Ikeguchi, Emil Alexov, Virginie Martiny, David Lagorce, Maria A. Miteva
    Abstract:

    Snyder-Robinson Syndrome (SRS) is a rare mental retardation disorder which is caused by the malfunctioning of an enzyme, the Spermine Synthase (SMS), which functions as a homo-dimer. The malfunctioning of SMS in SRS patients is associated with several identified missense mutations that occur away from the active site. This investigation deals with a particular SRS-causing mutation, the G56S mutation, which was shown computationally and experimentally to destabilize the SMS homo-dimer and thus to abolish SMS enzymatic activity. As a proof-of-concept, we explore the possibility to restore the enzymatic activity of the malfunctioning SMS mutant G56S by stabilizing the dimer through small molecule binding at the mutant homo-dimer interface. For this purpose, we designed an in silico protocol that couples virtual screening and a free binding energy-based approach to identify potential small-molecule binders on the destabilized G56S dimer, with the goal to stabilize it and thus to increase SMS G56S mutant activity. The protocol resulted in extensive list of plausible stabilizers, among which we selected and tested 51 compounds experimentally for their capability to increase SMS G56S mutant enzymatic activity. In silico analysis of the experimentally identified stabilizers suggested five distinctive chemical scaffolds. This investigation suggests that druggable pockets exist in the vicinity of the mutation sites at protein-protein interfaces which can be used to alter the disease-causing effects by small molecule binding. The identified chemical scaffolds are drug-like and can serve as original starting points for development of lead molecules to further rescue the disease-causing effect

  • a y328c missense mutation in Spermine Synthase causes a mild form of snyder robinson syndrome
    Human Molecular Genetics, 2013
    Co-Authors: Zhe Zhang, Charles E Schwartz, Emil Alexov, Joy Norris, Tim Wood, Vera M Kalscheuer, Lin Wang, Hilde Van Esch
    Abstract:

    Snyder-Robinson syndrome (SRS, OMIM: 309583) is an X-linked intellectual disability (XLID) syndrome, characterized by a collection of clinical features including facial asymmetry, marfanoid habitus, hypertonia, osteoporosis and unsteady gait. It is caused by a significant decrease or loss of Spermine Synthase (SMS) activity. Here, we report a new missense mutation, p.Y328C (c.1084A>G), in SMS in a family with XLID. The affected males available for evaluation had mild ID, speech and global delay, an asthenic build, short stature with long fingers and mild kyphosis. The Spermine/spermidine ratio in lymphoblasts was 0.53, significantly reduced compared with normal (1.87 average). Activity analysis of SMS in the index patient failed to detect any activity above background. In silico modeling demonstrated that the Y328C mutation has a significant effect on SMS stability, resulting in decreased folding free energy and larger structural fluctuations compared with those of wild-type SMS. The loss of activity was attributed to the increase in conformational dynamics in the mutant which affects the active site geometry, rather than preventing dimer formation. Taken together, the biochemical and in silico studies confirm the p.Y328C mutation in SMS is responsible for the patients having a mild form of SRS and reveal yet another molecular mechanism resulting in a non-functional SMS causing SRS.

Emil Alexov - One of the best experts on this subject based on the ideXlab platform.

  • dfmd fast and effective delphiforce steered molecular dynamics approach to model ligand approach toward a receptor application to Spermine Synthase enzyme
    Frontiers in Molecular Biosciences, 2019
    Co-Authors: Yunhui Peng, Ye Yang, Zhe Jia, Weiguo Cao, Emil Alexov
    Abstract:

    Here we report a novel approach, the DelPhiForce Molecular Dynamics (DFMD) method, for steered molecular dynamics simulations to model receptor-ligand association involving charged species. The main purpose of developing DFMD is to simulate ligand's trajectory toward the receptor and thus to predict the "entrance" of the binding pocket and conformational changes associated with the binding. We demonstrate that the DFMD is superior compared with molecular dynamics simulations applying standard cut-offs, provides correct binding forces, allows for modeling the ligand approach at long distances and thus guides the ligand toward the correct binding spot, and it is very fast (frequently the binding is completed in <1 ns). The DFMD is applied to model the binding of two ligands to a receptor (Spermine Synthase) and it is demonstrated that it guides the ligands toward the corresponding pockets despite of the initial ligand's position with respect to the receptor. Predicted conformational changes and the order of ligand binding are experimentally verified.

  • On the electrostatic properties of homodimeric proteins
    2016
    Co-Authors: On Campbell, Marharyta Petukh, Emil Alexov
    Abstract:

    A large fraction of proteins function as homodimers, but it is not always clear why the dimerization is important for functionality since frequently each monomer possesses a distinctive active site. Recent work (PLoS Computational Biology, 9(2), e1002924) indicates that homodimerization may be important for forming an electrostatic funnel in the Spermine Synthase homodimer which guides changed substrates toward the active centers. This prompted us to investigate the electrostatic properties of a large set of homodimeric proteins and resulted in an observation that in a vast majority of the cases the dimerization indeed results in specific electrostatic features, although not necessarily in an electrostatic funnel. It is demonstrated that the electrostatic dipole moment of the dimer is predominantly perpendicular to the axis connecting the centers of the mass of the monomers. In addition, the surface points with highest potential are located in the proximity of the interfacial plane of the homodimeric complexes. These findings indicate that frequently homodimerization provides specific electrostatic features needed for the function of proteins. Keywords electrostatics; Poisson-Boltzmann equation; homodimers; electrostatic field; electrostatic funnelin

  • Enhancing Human Spermine Synthase Activity by Engineered Mutations
    2016
    Co-Authors: Zhe Zhang, Yoshihiko Ikeguchi, Yueli Zheng, Margo Petukh, Anthony Pegg, Emil Alexov
    Abstract:

    Spermine Synthase (SMS) is an enzyme which function is to convert spermidine into Spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine R Spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculate

  • Alexov E: In silico and in vitro investigations of the mutability of disease-causing missense mutation sites in Spermine Synthase. PLoS One 2011
    2016
    Co-Authors: Zhe Zhang, Charles Schwartz, Joy Norris, Emil Alexov
    Abstract:

    Background: Spermine Synthase (SMS) is a key enzyme controlling the concentration of spermidine and Spermine in the cell. The importance of SMS is manifested by the fact that single missense mutations were found to cause Snyder-Robinson Syndrome (SRS). At the same time, currently there are no non-synonymous single nucleoside polymorphisms, nsSNPs (harmless mutations), found in SMS, which may imply that the SMS does not tolerate amino acid substitutions, i.e. is not mutable. Methodology/Principal Findings: To investigate the mutability of the SMS, we carried out in silico analysis and in vitro experiments of the effects of amino acid substitutions at the missense mutation sites (G56, V132 and I150) that have been shown to cause SRS. Our investigation showed that the mutation sites have different degree of mutability depending on their structural micro-environment and involvement in the function and structural integrity of the SMS. It was found that the I150 site does not tolerate any mutation, while V132, despite its key position at the interface of SMS dimer, is quite mutable. The G56 site is in the middle of the spectra, but still quite sensitive to charge residue replacement. Conclusions/Significance: The performed analysis showed that mutability depends on the detail of the structural and functional factors and cannot be predicted based on conservation of wild type properties alone. Also, harmless nsSNPs ca

  • revealing the effects of missense mutations causing snyder robinson syndrome on the stability and dimerization of Spermine Synthase
    International Journal of Molecular Sciences, 2016
    Co-Authors: Yunhui Peng, Charles E Schwartz, Joy Norris, Emil Alexov
    Abstract:

    Missense mutations in Spermine Synthase (SpmSyn) protein have been shown to cause the Snyder-Robinson syndrome (SRS). Depending on the location within the structure of SpmSyn and type of amino acid substitution, different mechanisms resulting in SRS were proposed. Here we focus on naturally occurring amino acid substitutions causing SRS, which are situated away from the active center of SpmSyn and thus are not directly involved in the catalysis. Two of the mutations, M35R and P112L, are reported for the first time in this study. It is demonstrated, both experimentally and computationally, that for such mutations the major effect resulting in dysfunctional SpmSyn is the destabilization of the protein. In vitro experiments indicated either no presence or very little amount of the mutant SpmSyn in patient cells. In silico modeling predicted that all studied mutations in this work destabilize SpmSyn and some of them abolish homo-dimer formation. Since dimerization and structural stability are equally important for the wild type function of SpmSyn, it is proposed that the SRS caused by mutations occurring in the N-domain of SpmSyn is a result of dysfunctional mutant proteins being partially unfolded and degraded by the proteomic machinery of the cell or being unable to form a homo-dimer.

Yoshihiko Ikeguchi - One of the best experts on this subject based on the ideXlab platform.

  • Enhancing Human Spermine Synthase Activity by Engineered Mutations
    2016
    Co-Authors: Zhe Zhang, Yoshihiko Ikeguchi, Yueli Zheng, Margo Petukh, Anthony Pegg, Emil Alexov
    Abstract:

    Spermine Synthase (SMS) is an enzyme which function is to convert spermidine into Spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine R Spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculate

  • rational design of small molecule stabilizers of Spermine Synthase dimer by virtual screening and free energy based approach
    PLOS ONE, 2014
    Co-Authors: Yoshihiko Ikeguchi, Zhe Zhang, Emil Alexov, David Lagorce, Virginie Y Martiny, Maria A. Miteva
    Abstract:

    Snyder-Robinson Syndrome (SRS) is a rare mental retardation disorder which is caused by the malfunctioning of an enzyme, the Spermine Synthase (SMS), which functions as a homo-dimer. The malfunctioning of SMS in SRS patients is associated with several identified missense mutations that occur away from the active site. This investigation deals with a particular SRS-causing mutation, the G56S mutation, which was shown computationally and experimentally to destabilize the SMS homo-dimer and thus to abolish SMS enzymatic activity. As a proof-of-concept, we explore the possibility to restore the enzymatic activity of the malfunctioning SMS mutant G56S by stabilizing the dimer through small molecule binding at the mutant homo-dimer interface. For this purpose, we designed an in silico protocol that couples virtual screening and a free binding energy-based approach to identify potential small-molecule binders on the destabilized G56S dimer, with the goal to stabilize it and thus to increase SMS G56S mutant activity. The protocol resulted in extensive list of plausible stabilizers, among which we selected and tested 51 compounds experimentally for their capability to increase SMS G56S mutant enzymatic activity. In silico analysis of the experimentally identified stabilizers suggested five distinctive chemical scaffolds. This investigation suggests that druggable pockets exist in the vicinity of the mutation sites at protein-protein interfaces which can be used to alter the disease-causing effects by small molecule binding. The identified chemical scaffolds are drug-like and can serve as original starting points for development of lead molecules to further rescue the disease-causing effects of the Snyder-Robinson syndrome for which no efficient treatment exists up to now.

  • Rational design of small-molecule stabilizers of Spermine Synthase dimer by virtual screening and free energy-based approach
    2014
    Co-Authors: Zhe Zhang, Yoshihiko Ikeguchi, Emil Alexov, Virginie Martiny, David Lagorce, Maria A. Miteva
    Abstract:

    Snyder-Robinson Syndrome (SRS) is a rare mental retardation disorder which is caused by the malfunctioning of an enzyme, the Spermine Synthase (SMS), which functions as a homo-dimer. The malfunctioning of SMS in SRS patients is associated with several identified missense mutations that occur away from the active site. This investigation deals with a particular SRS-causing mutation, the G56S mutation, which was shown computationally and experimentally to destabilize the SMS homo-dimer and thus to abolish SMS enzymatic activity. As a proof-of-concept, we explore the possibility to restore the enzymatic activity of the malfunctioning SMS mutant G56S by stabilizing the dimer through small molecule binding at the mutant homo-dimer interface. For this purpose, we designed an in silico protocol that couples virtual screening and a free binding energy-based approach to identify potential small-molecule binders on the destabilized G56S dimer, with the goal to stabilize it and thus to increase SMS G56S mutant activity. The protocol resulted in extensive list of plausible stabilizers, among which we selected and tested 51 compounds experimentally for their capability to increase SMS G56S mutant enzymatic activity. In silico analysis of the experimentally identified stabilizers suggested five distinctive chemical scaffolds. This investigation suggests that druggable pockets exist in the vicinity of the mutation sites at protein-protein interfaces which can be used to alter the disease-causing effects by small molecule binding. The identified chemical scaffolds are drug-like and can serve as original starting points for development of lead molecules to further rescue the disease-causing effect

  • enhancing human Spermine Synthase activity by site directed mutations
    Biophysical Journal, 2013
    Co-Authors: Yoshihiko Ikeguchi, Anthony E Pegg, Zhe Zhang, Emil Alexov
    Abstract:

    Spermine Synthase (SMS) is an enzyme converting spermidine into Spermine, both of which are polyamines controlling normal cell growth and development. Several missense mutations in human SMS (HsSMS) are known to cause Snyder-Robinson Syndrome (SRS) by either destabilizing the monomer/dimer conformation or directly affecting the hydrogen bond network in the active sites. Recently a comparison of protein sequence and crystal structure between the HsSMS and its homologous protein Thermotoga maritima (Tm) spermidine Synthase (TmSRM) was performed. Tm is the only bacterium known to grow at a high temperature as well as 90°C, and the half-life of TmSRM is longer than 25h under this temperature. In contrast, HsSMS is much less stable than TmSRM under the same temperature. Sequence alignment between HsSMS and TmSRM suggests that some key residues may be essential players for the elevate stability of TmSRM. Such key residues were identified based on various biophysical and sequence criteria and four mutations (S165D, L175E, T178H and C206R) were selected for HsSMS. Both in silico and in vitro experiments indicated that these four mutations strongly stabilize the monomer structure and dramatically improve the efficiency of SPM synthesis. The enhanced reaction rate in the mutant HsSMS is attributed to the increase of the strength of negative electrostatic potential, calculated with DelPhi, in the dimer cleft between HsSMS units, which presumably facilitates the substrate delivery to the active site.The work was supported by a grant from the Institute of General Medical Sciences, National Institutes of Health, and the grant number is 1R01GM093937.

  • Enhancing Human Spermine Synthase Activity by Engineered Mutations
    2013
    Co-Authors: Zhe Zhang, Yoshihiko Ikeguchi, Yueli Zheng, Margo Petukh, Anthony Pegg, Emil Alexov
    Abstract:

    Spermine Synthase (SMS) is an enzyme which function is to convert spermidine into Spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → Spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing.