Publications by authors named "Alessandro Paiardini"

93 Publications

Nuclear localisation of Aurora-A: its regulation and significance for Aurora-A functions in cancer.

Oncogene 2021 Jun 13;40(23):3917-3928. Epub 2021 May 13.

Institute of Molecular Biology and Pathology, National Research Council of Italy, c/o Sapienza University of Rome, Rome, Italy.

The Aurora-A kinase regulates cell division, by controlling centrosome biology and spindle assembly. Cancer cells often display elevated levels of the kinase, due to amplification of the gene locus, increased transcription or post-translational modifications. Several inhibitors of Aurora-A activity have been developed as anti-cancer agents and are under evaluation in clinical trials. Although the well-known mitotic roles of Aurora-A point at chromosomal instability, a hallmark of cancer, as a major link between Aurora-A overexpression and disease, recent evidence highlights the existence of non-mitotic functions of potential relevance. Here we focus on a nuclear-localised fraction of Aurora-A with oncogenic roles. Interestingly, this pool would identify not only non-mitotic, but also kinase-independent functions of the kinase. We review existing data in the literature and databases, examining potential links between Aurora-A stabilisation and localisation, and discuss them in the perspective of a more effective targeting of Aurora-A in cancer therapy.
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http://dx.doi.org/10.1038/s41388-021-01766-wDOI Listing
June 2021

Phosphorylation, Mg-ADP, and Inhibitors Differentially Shape the Conformational Dynamics of the A-Loop of Aurora-A.

Biomolecules 2021 Apr 12;11(4). Epub 2021 Apr 12.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, 00185 Rome, Italy.

The conformational state of the activation loop (A-loop) is pivotal for the activity of most protein kinases. Hence, the characterization of the conformational dynamics of the A-loop is important to increase our understanding of the molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. Here, we carry out a combination of molecular dynamics (MD) and essential dynamics (ED) analyses to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the A-loop of Aurora-A. MD revealed that the stability of the A-loop in an open conformation is enhanced by single phospho-Thr-288, while paradoxically, the presence of a second phosphorylation at Thr-287 decreases such stability and renders the A-loop more fluctuant in time and space. Moreover, we found that this post-translational modification has a significant effect on the direction of the A-loop motions. ED analysis suggests that the presence of the phosphate moiety induces the dynamics of Aurora-A to sample two distinct energy minima, instead of a single large minimum, as in unphosphorylated Aurora-A states. This observation indicates that the conformational distributions of Aurora-A with both single and double phospho-threonine modifications are remarkably different from the unphosphorylated state. In the closed states, binding of CD532 and MLN8054 inhibitors has the effect of increasing the distance of the N- and C-lobes of the kinase domain of Aurora-A, and the angle analysis between those two lobes during MD simulations showed that the N- and C-lobes are kept more open in presence of CD532, compared to MLN8054. As the A-loop is a common feature of Aurora protein kinases, our studies provide a general description of the conformational dynamics of this structure upon phosphorylation and different ligands binding.
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http://dx.doi.org/10.3390/biom11040567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070005PMC
April 2021

The Archaeal Elongation Factor EF-2 Induces the Release of aIF6 From 50S Ribosomal Subunit.

Front Microbiol 2021 24;12:631297. Epub 2021 Mar 24.

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal () IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release.
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http://dx.doi.org/10.3389/fmicb.2021.631297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024482PMC
March 2021

Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts.

Life (Basel) 2021 Jan 6;11(1). Epub 2021 Jan 6.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy.

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.
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http://dx.doi.org/10.3390/life11010031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825114PMC
January 2021

Intronic Determinants Coordinate Charme lncRNA Nuclear Activity through the Interaction with MATR3 and PTBP1.

Cell Rep 2020 12;33(12):108548

Department of Biology and Biotechnology "Charles Darwin," Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. Electronic address:

Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.
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http://dx.doi.org/10.1016/j.celrep.2020.108548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773549PMC
December 2020

A Single Center Retrospective Review of Patients from Central Italy Tested for Melanoma Predisposition Genes.

Int J Mol Sci 2020 Dec 11;21(24). Epub 2020 Dec 11.

Laboratory of Medical Genetics, Department of Molecular Medicine, Sapienza University, San Camillo-Forlanini Hospital, 00100 Rome, Italy.

Background: Cutaneous malignant melanoma (CMM) is one of the most common skin cancers worldwide. CMM pathogenesis involves genetic and environmental factors. Recent studies have led to the identification of new genes involved in CMM susceptibility: beyond CDKN2A and CDK4, BAP1, POT1, and MITF were recently identified as potential high-risk melanoma susceptibility genes.

Objective: This study is aimed to evaluate the genetic predisposition to CMM in patients from central Italy.

Methods: From 1998 to 2017, genetic testing was performed in 888 cases with multiple primary melanoma and/or familial melanoma. Genetic analyses included the sequencing CDKN2A, CDK4, BAP1, POT1, and MITF in 202 cases, and of only CDKN2A and CDK4 codon 24 in 686 patients. By the evaluation of the personal and familial history, patients were divided in two clinical categories: "low significance" and "high significance" cases.

Results: 128 patients (72% belonging to the "high significance" category, 28% belonging to the "low significance" category) were found to carry a DNA change defined as pathogenic, likely pathogenic, variant of unknown significance (VUS)-favoring pathogenic or VUS.

Conclusions: It is important to verify the genetic predisposition in CMM patients for an early diagnosis of further melanomas and/or other tumors associated with the characterized genotype.
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http://dx.doi.org/10.3390/ijms21249432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763813PMC
December 2020

Cytosolic serine hydroxymethyltransferase controls lung adenocarcinoma cells migratory ability by modulating AMP kinase activity.

Cell Death Dis 2020 11 26;11(11):1012. Epub 2020 Nov 26.

Department of Biochemical Sciences A. Rossi Fanelli, Laboratory Affiliated to Istituto Pasteur Italia, Sapienza University of Rome, Piazzale A. Moro 5, 00185, Rome, Italy.

Nutrient utilization and reshaping of metabolism in cancer cells is a well-known driver of malignant transformation. Less clear is the influence of the local microenvironment on metastasis formation and choice of the final organ to invade. Here we show that the level of the amino acid serine in the cytosol affects the migratory properties of lung adenocarcinoma (LUAD) cells. Inhibition of serine or glycine uptake from the extracellular milieu, as well as knockdown of the cytosolic one-carbon metabolism enzyme serine hydroxymethyltransferase (SHMT1), abolishes migration. Using rescue experiments with a brain extracellular extract, and direct measurements, we demonstrate that cytosolic serine starvation controls cell movement by increasing reactive oxygen species formation and decreasing ATP levels, thereby promoting activation of the AMP sensor kinase (AMPK) by phosphorylation. Activation of AMPK induces remodeling of the cytoskeleton and finally controls cell motility. These results highlight that cytosolic serine metabolism plays a key role in controlling motility, suggesting that cells are able to dynamically exploit the compartmentalization of this metabolism to adapt their metabolic needs to different cell functions (movement vs. proliferation). We propose a model to explain the relevance of serine/glycine metabolism in the preferential colonization of the brain by LUAD cells and suggest that the inhibition of serine/glycine uptake and/or cytosolic SHMT1 might represent a successful strategy to limit the formation of brain metastasis from primary tumors, a major cause of death in these patients.
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http://dx.doi.org/10.1038/s41419-020-03215-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691363PMC
November 2020

PyMod 3: a complete suite for structural bioinformatics in PyMOL.

Bioinformatics 2021 Jun;37(10):1471-1472

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza Università di Roma, 00185 Rome, Italy.

Summary: The PyMod project is designed to act as a fully integrated interface between the popular molecular graphics viewer PyMOL, and some of the most frequently used tools for structural bioinformatics, e.g. BLAST, HMMER, Clustal, MUSCLE, PSIPRED, DOPE and MODELLER. Here we report its latest release, PyMod 3, which has been completely renewed with a graphical interface written in PyQt, to make it compatible with the most recent PyMOL versions, and has been extended with a large set of new functionalities compared to its predecessor, i.e. PyMod 2. Starting from the amino acid sequence of a target protein, users can take advantage of PyMod 3 to carry out all the steps of the homology modeling process (i.e. template searching, target-template sequence alignment, model building and quality assessment). Additionally, the integrated tools in PyMod 3 may also be used alone, in order to extend PyMOL with a wide range of capabilities. Sequence similarity searches, multiple sequence/structure alignment building, phylogenetic trees and evolutionary conservation analyses, domain parsing, single/multiple chains and loop modeling can be performed in the PyMod 3/PyMOL environment.

Availability And Implementation: A cross-platform PyMod 3 installer package for Windows, Linux and Mac OS X and a complete user guide with tutorials, are available at https://github.com/pymodproject/pymod.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa849DOI Listing
June 2021

A novel CDKN2A in-frame deletion associated with pancreatic cancer-melanoma syndrome.

Dermatol Online J 2020 Aug 15;26(8). Epub 2020 Aug 15.

Division/Laboratory of Medical Genetics, Department of Molecular Medicine, Sapienza University, San Camillo-Forlanini Hospital, Rome.

Pancreatic cancer-melanoma syndrome (PCMS) is an inherited condition in which mutation carriers have an increased risk of malignant melanoma and/or pancreatic cancer. About 30% of PCMS cases carry mutations in CDKN2A. This gene encodes several protein isoforms, one of which, known as p16, regulates the cell-cycle by interacting with CDK4/CDK6 kinases and with several non-CDK proteins. Herein, we report on a novel CDKN2A germline in-frame deletion (c.52_57delACGGCC) found in an Italian family with PCMS. By segregation analysis, the c.52_57delACGGCC was proven to segregate in kindred with cutaneous melanoma (CM), in kindred with CM and pancreatic cancer, and in a single case presenting only with pancreatic cancer. In the literature, duplication mapping in the same genic region has been already reported at the germline level in several unrelated CM cases as a variant of unknown clinical significance. A computational approach for studying the effect of mutational changes over p16 protein structure showed that both the deletion and the duplication of the c.52_57 nucleotides result in protein misfolding and loss of interactors' binding. In conclusion, the present results argue that the quantitative alteration of nucleotides c.52_57 has a pathogenic role in p16 function and that the c.52_57delACGGCC is associated with PCMS.
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August 2020

Bacterial Oxidases of the Cytochrome Family: Redox Enzymes of Unique Structure, Function, and Utility As Drug Targets.

Antioxid Redox Signal 2021 Jun 9;34(16):1280-1318. Epub 2020 Nov 9.

Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, United Kingdom.

Cytochrome is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome is found in many bacterial pathogens and, surprisingly, in bacteria formally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target. We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed. Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands. It is clear that cytochrome is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase. Since cytochrome is a uniquely bacterial enzyme, future research should focus on harnessing fundamental knowledge of its structure and function to the development of novel and effective antibacterial agents.
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http://dx.doi.org/10.1089/ars.2020.8039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112716PMC
June 2021

Artemisinin Derivatives with Antimelanoma Activity Show Inhibitory Effect against Human DNA Topoisomerase 1.

ACS Med Chem Lett 2020 May 10;11(5):1035-1040. Epub 2020 Apr 10.

Dipartimento di Biologia, Università di Padova Distaccato presso il "Centro Linceo Beniamino Segre" Accademia Nazionale dei Lincei, Palazzo Corsini, Via della Lungara 10, 00165 Rome, Italy.

Artesunic acid and artemisinin are natural substances with promiscuous anticancer activity against different types of cancer cell lines. The mechanism of action of these compounds is associated with the formation of reactive radical species by cleavage of the sesquiterpene pharmacophore endoperoxide bridge. Here we suggested topoisomerase 1 as a possible molecular target for the improvement of the anticancer activity of these compounds. In this context, we report that novel hybrid and dimer derivatives of artesunic acid and artemisinin, bearing camptothecin and SN38 as side-chain biological effectors, can inhibit growth of yeast cells overexpressing human topoisomerase 1 and its enzymatic activity . These derivatives showed also anticancer activity in melanoma cell lines higher than camptothecin and paclitaxel. molecular docking calculations highlighted a common binding mode for the novel derivatives, with the sesquiterpene lactone scaffold being located near the traditional recognition site for camptothecin, while the bioactive side-chain effector laid in the camptothecin cleft.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236541PMC
May 2020

From PARP1 to TNKS2 Inhibition: A Structure-Based Approach.

ACS Med Chem Lett 2020 May 3;11(5):862-868. Epub 2020 Feb 3.

Department of Chemistry and Technology of Drugs, ″Sapienza" University of Rome, 00185 Rome, Italy.

Tankyrases (TNKSs) have recently gained great consideration as potential targets in Wnt/β-catenin pathway-dependent solid tumors. Previously, we reported the 2-mercaptoquinazolin-4-one MC2050 as a micromolar PARP1 inhibitor. Here we show how the resolution of the X-ray structure of PARP1 in complex with MC2050, combined with the computational investigation of the structural differences between TNKSs and PARP1/2 active sites, provided the rationale for a structure-based drug design campaign that with a limited synthetic effort led to the discovery of the bis-quinazolinone as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236224PMC
May 2020

SMaRT lncRNA controls translation of a G-quadruplex-containing mRNA antagonizing the DHX36 helicase.

EMBO Rep 2020 06 26;21(6):e49942. Epub 2020 Apr 26.

Department of Biology and Biotechnology, Charles Darwin, Sapienza University of Rome, Rome, Italy.

Guanine-quadruplexes (G4) included in RNA molecules exert several functions in controlling gene expression at post-transcriptional level; however, the molecular mechanisms of G4-mediated regulation are still poorly understood. Here, we describe a regulatory circuitry operating in the early phases of murine muscle differentiation in which a long non-coding RNA (SMaRT) base pairs with a G4-containing mRNA (Mlx-γ) and represses its translation by counteracting the activity of the DHX36 RNA helicase. The time-restricted, specific effect of lnc-SMaRT on the translation of Mlx-γ isoform modulates the general subcellular localization of total MLX proteins, impacting on their transcriptional output and promoting proper myogenesis and mature myotube formation. Therefore, the circuitry made of lnc-SMaRT, Mlx-γ, and DHX36 not only plays an important role in the control of myogenesis but also unravels a molecular mechanism where G4 structures and G4 unwinding activities are regulated in living cells.
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http://dx.doi.org/10.15252/embr.201949942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271651PMC
June 2020

Insights into PPARγ Phosphorylation and Its Inhibition Mechanism.

J Med Chem 2020 05 15;63(9):4811-4823. Epub 2020 Apr 15.

Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Via Salaria km. 29.300, 00015 Monterotondo Stazione, Rome, Italy.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00048DOI Listing
May 2020

Revisiting the "satisfaction of spatial restraints" approach of MODELLER for protein homology modeling.

PLoS Comput Biol 2019 12 17;15(12):e1007219. Epub 2019 Dec 17.

Department of Biochemical Sciences "A. Rossi Fanelli", "Sapienza" University of Rome, Roma, Italy.

The most frequently used approach for protein structure prediction is currently homology modeling. The 3D model building phase of this methodology is critical for obtaining an accurate and biologically useful prediction. The most widely employed tool to perform this task is MODELLER. This program implements the "modeling by satisfaction of spatial restraints" strategy and its core algorithm has not been altered significantly since the early 1990s. In this work, we have explored the idea of modifying MODELLER with two effective, yet computationally light strategies to improve its 3D modeling performance. Firstly, we have investigated how the level of accuracy in the estimation of structural variability between a target protein and its templates in the form of σ values profoundly influences 3D modeling. We show that the σ values produced by MODELLER are on average weakly correlated to the true level of structural divergence between target-template pairs and that increasing this correlation greatly improves the program's predictions, especially in multiple-template modeling. Secondly, we have inquired into how the incorporation of statistical potential terms (such as the DOPE potential) in the MODELLER's objective function impacts positively 3D modeling quality by providing a small but consistent improvement in metrics such as GDT-HA and lDDT and a large increase in stereochemical quality. Python modules to harness this second strategy are freely available at https://github.com/pymodproject/altmod. In summary, we show that there is a large room for improving MODELLER in terms of 3D modeling quality and we propose strategies that could be pursued in order to further increase its performance.
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http://dx.doi.org/10.1371/journal.pcbi.1007219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938380PMC
December 2019

Prenatal whole exome sequencing detects a new homozygous fukutin (FKTN) mutation in a fetus with an ultrasound suspicion of familial Dandy-Walker malformation.

Mol Genet Genomic Med 2020 01 22;8(1):e1054. Epub 2019 Nov 22.

Fondazione IRCCS Casa Sollievo della Sofferenza, Laboratory of Clinical Genomics, San Giovanni Rotondo (FG), Italy.

Background: Posterior fossa malformations are among the most diagnosed central nervous system (CNS) anomalies detected by ultrasound (US) in prenatal age. We identified the pathogenic gene mutation in a male fetus of 17 weeks of gestation with US suspicion of familial Dandy-Walker spectrum malformation, using Next Generation Sequencing approach in prenatal diagnosis.

Methods: Whole exome sequencing (WES) approach has been performed on fetal genomic DNA. After reads preprocessing, mapping, variant calling, and annotation, a filtering strategy based on allelic frequency, recessive inheritance, and phenotypic ontologies has been applied. A fetal magnetic resonance imaging (MRI) at 18 weeks of gestation has been performed. An in silico analysis of a potential causative missense variant in the fukutin protein has been carried out through a structural modeling approach.

Results: We identified a new homozygous missense mutation in fukutin gene (FKTN, NM_006731.2: c.898G>A; NP_006722.2: p.Gly300Arg). Fetal MRI supported molecular findings. Structural modeling analyses indicated a potential pathogenetic mechanism of the variant, through a reduced activation of the sugar moieties, which in turn impairs transfer to dystroglycan and thus its glycosylation. These findings pointed to a redefinition of the US suspicion of recurrence of Dandy-Walker malformation (DWM) to a muscular dystrophy-dystroglycanopathy type A4.

Conclusions: The present case confirmed WES as a reliable tool for the prenatal identification of the molecular bases of early-detected CNS malformations.
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http://dx.doi.org/10.1002/mgg3.1054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978243PMC
January 2020

A novel compound heterozygous genotype associated with aromatic amino acid decarboxylase deficiency: Clinical aspects and biochemical studies.

Mol Genet Metab 2019 06 10;127(2):132-137. Epub 2019 May 10.

Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy. Electronic address:

Aromatic amino acid decarboxylase (AADC) deficiency is a rare autosomal neurometabolic disorder caused by a deficit of AADC, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which catalyzes the synthesis of dopamine and serotonin. While many studies have highlighted the molecular defects of the homozygous pathogenic variants, so far only a study investigated heterozygous variants at protein level. Here, we report a clinical case of one AADC deficiency compound heterozygous patient bearing the A91V mutation and the novel C410G mutation. To elucidate its enzymatic phenotype, the A91V and C410G homodimers were first expressed in Escherichia coli, purified and characterized. Although both apo variants display an unaltered overall tertiary structure, they show a ̴ 20-fold decreased PLP binding affinity. The C410G mutation only causes a ̴ 4-fold decrease of the catalytic efficiency, while the A91V mutation causes a 1300-fold decrease of the k/K, and changes in the holoAADC consisting in a marked alteration of the tertiary structure and the coenzyme microenvironment. Structural analyses of these mutations are in agreement with these data. Unfortunately, the C410G/A91V heterodimer was constructed, expressed and purified in rather modest amount. Anyway, measurements of decarboxylase activity indicate that its putative k value is lower than that predicted by averaging the k values of the two parental enzymes. This indicates a negative interallelic complementation between the C410G and A91V monomers. Overall, this study allowed to relate the clinical to the enzymatic phenotype of the patient and to extend knowledge in the clinical and molecular pathogenesis of AADC deficiency.
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http://dx.doi.org/10.1016/j.ymgme.2019.05.004DOI Listing
June 2019

N-Methyladenosine (mA): A Promising New Molecular Target in Acute Myeloid Leukemia.

Front Oncol 2019 9;9:251. Epub 2019 Apr 9.

Department of Biology and Biotechnology "Charles Darwin," Sapienza University of Rome, Rome, Italy.

Recent studies have uncovered an important role for RNA modifications in gene expression regulation, which led to the birth of the epitranscriptomics field. It is now acknowledged that RNA modifiers play a crucial role in the control of differentiation of stem and progenitor cells and that changes in their levels are a relevant feature of different types of cancer. To date, among more than 160 different RNA chemical modifications, the more relevant in cancer biology is the reversible and dynamic N-methylation of adenosine, yielding N-methyladenosine (mA). mA is the more abundant internal modification in mRNA, regulating the expression of the latter at different levels, from maturation to translation. Here, we will describe the emerging role of mA modification in acute myeloid leukemia (AML), which, among first, has demonstrated how mis-regulation of the m6A modifying system can contribute to the development and progression of cancer. Moreover, we will discuss how AML is paving the way to the development of new therapeutic options based on the inhibition of mA deposition.
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http://dx.doi.org/10.3389/fonc.2019.00251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465620PMC
April 2019

R180T variant of δ-ornithine aminotransferase associated with gyrate atrophy: biochemical, computational, X-ray and NMR studies provide insight into its catalytic features.

FEBS J 2019 07 15;286(14):2787-2798. Epub 2019 Apr 15.

Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Italy.

Among the over 50 gyrate atrophy-causing mutations of ornithine δ-aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of OAT complexed with 5-fluoromethylornithine engages a salt bridge with the α-carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO-K cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild-type, its crystal structure solved at 1.8 Ǻ reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild-type OAT, shows (a) an increased K value for l-ornithine (l-Orn), (b) an altered pyridoxal 5'-phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ-transamination but also, albeit to a lesser extent, the α-transamination of l-Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l-Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency. ENZYMES: Ornithine aminotransferase EC 2.6.1.13. DATABASES: 6HX7.pdb.
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http://dx.doi.org/10.1111/febs.14843DOI Listing
July 2019

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism.

Nucleic Acids Res 2019 05;47(8):4240-4254

Department of Biochemical Sciences, Sapienza University of Rome - P. le Aldo Moro 5, 00185 Rome, Italy.

Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5'untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1-UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.
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http://dx.doi.org/10.1093/nar/gkz129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486632PMC
May 2019

Cysteine 180 Is a Redox Sensor Modulating the Activity of Human Pyridoxal 5'-Phosphate Histidine Decarboxylase.

Biochemistry 2018 11 29;57(44):6336-6348. Epub 2018 Oct 29.

Department of Neuroscience, Biomedicine and Movement , University of Verona , Strada Le Grazie, 8 , 37134 Verona , Italy.

Histidine decarboxylase is a pyridoxal 5'-phosphate enzyme catalyzing the conversion of histidine to histamine, a bioactive molecule exerting its role in many modulatory processes. The human enzyme is involved in many physiological functions, such as neurotransmission, gastrointestinal track function, cell growth, and differentiation. Here, we studied the functional properties of the human enzyme and, in particular, the effects exerted at the protein level by two cysteine residues: Cys-180 and Cys-418. Surprisingly, the enzyme exists in an equilibrium between a reduced and an oxidized form whose extent depends on the redox state of Cys-180. Moreover, we determined that (i) the two enzymatic redox species exhibit modest structural changes in the coenzyme microenvironment and (ii) the oxidized form is slightly more active and stable than the reduced one. These data are consistent with the model proposed by bioinformatics analyses and molecular dynamics simulations in which the Cys-180 redox state could be responsible for a structural transition affecting the C-terminal domain reorientation leading to active site alterations. Furthermore, the biochemical properties of the purified C180S and C418S variants reveal that C180S behaves like the reduced form of the wild-type enzyme, while C418S is sensitive to reductants like the wild-type enzyme, thus allowing the identification of Cys-180 as the redox sensitive switch. On the other hand, Cys-418 appears to be a residue involved in aggregation propensity. A possible role for Cys-180 as a regulatory switch in response to different cellular redox conditions could be suggested.
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http://dx.doi.org/10.1021/acs.biochem.8b00625DOI Listing
November 2018

Insights into the GTP-dependent allosteric control of c-di-GMP hydrolysis from the crystal structure of PA0575 protein from Pseudomonas aeruginosa.

FEBS J 2018 10 7;285(20):3815-3834. Epub 2018 Sep 7.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Italy.

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.
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http://dx.doi.org/10.1111/febs.14634DOI Listing
October 2018

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from .

Int J Mol Sci 2018 Jul 20;19(7). Epub 2018 Jul 20.

Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy.

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite includes the genes for cystathionine β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of . However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from . Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of l-cystathionine to form l-cysteine and displays marginal reactivity toward l-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,β- versus α,γ-elimination of l-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards l-cystathionine. Our results suggest that CGL is an important functional enzyme in , likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes.
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http://dx.doi.org/10.3390/ijms19072111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073527PMC
July 2018

The catalytic activity of serine hydroxymethyltransferase is essential for de novo nuclear dTMP synthesis in lung cancer cells.

FEBS J 2018 09 7;285(17):3238-3253. Epub 2018 Aug 7.

Department of Biochemical Sciences, Sapienza University of Rome, Italy.

Cancer cells reprogramme one-carbon metabolism (OCM) to sustain growth and proliferation. Depending on cell demands, serine hydroxymethyltransferase (SHMT) dynamically changes the fluxes of OCM by reversibly converting serine and tetrahydrofolate (THF) into 5,10-methylene-THF and glycine. SHMT is a tetrameric enzyme that mainly exists in three isoforms; two localize in the cytosol (SHMT1/SHMT2α) and one (SHMT2) in the mitochondria. Both the cytosolic isoforms can also translocate to the nucleus to sustain de novo thymidylate synthesis and support cell proliferation. Finally, the expression levels of the different isoforms are regulated to a certain extent by a yet unknown crosstalk mechanism. We have designed and fully characterized a set of three SHMT1 mutants, which uncouple the oligomeric state of the enzyme from its catalytic activity. We have then investigated the effects of the mutations on SHMT1 nuclear localization, cell viability and crosstalk in lung cancer cells (A549; H1299). Our data reveal that in these cell lines de novo thymidylate synthesis requires SHMT1 to be active, regardless of its oligomeric state. We have also confirmed that the crosstalk between the cytosolic and mitochondrial SHMT actually takes place and regulates the expression of the two isoforms. Apparently, the crosstalk mechanism is independent from the oligomeric state and the catalytic activity of SHMT1.

Database: Structural data are available in the PDB under the accession number 6FL5.
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http://dx.doi.org/10.1111/febs.14610DOI Listing
September 2018

Familial hypomagnesaemia, Hypercalciuria and Nephrocalcinosis associated with a novel mutation of the highly conserved leucine residue 116 of Claudin 16 in a Chinese patient with a delayed diagnosis: a case report.

BMC Nephrol 2018 07 13;19(1):181. Epub 2018 Jul 13.

Department of Nephrology, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao, 266003, China.

Background: Sixty mutations of claudin 16 coding gene have been reported in familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) patients. Recent investigations revealed that a highly conserved glycine-leucine-tryptophan (G-L-W) motif in the first extracellular segment (ESC1) of claudin 16 might be essential for stabilization of the appropriately folded ECS1 structure and conservation of normal claudin 16 function. However, neither missense nor nonsense mutation has ever been described in this motif. Our study aimed at identifying mutations in a Chinese patient with FHHNC and exploring the association between genotype and phenotype.

Case Presentation: A 33-year-old female presented with 4 years history of recurrent acute pyelonephritis without other notable past medical history. Her healthy parents, who aged 56 and 53 respectively, were second cousins, and her only sibling died from renal failure without definite cause at age 25. Renal ultrasound imaging demonstrated atrophic kidneys and bilateral nephrocalcinosis. The laboratory workup revealed impaired renal function (Stage CKD IV), hypocalcemia and mild hypomagnesemia, accompanied with marked renal loss of magnesium and hypercalciuria. During the follow-up, treatment with calcitriol and calcium but not with magnesium was difficult to achieve normal serum calcium levels, whereas her serum magnesium concentration fluctuated within normal ranges. In the end, the patient unavoidably reached ESRD at 36 years old. The clinical features and family history suggested the diagnosis of FHHNC. To make a definite diagnosis, we use whole-exome sequencing to identify the disease-causing mutations and Sanger sequencing to confirm the mutation co-segregation in the family. As a result, a novel homozygous mutation (c.346C > G, p.Leu116Val) in G-L-W motif of claudin 16 was identified. Her parents, grandmother and one of her cousins carried heterozygous p.Leu116Val, whereas 200 unrelated controls did not carry this mutation.

Conclusions: We described a delayed diagnosis patient with FHHNC in the Chinese population and identified a novel missense mutation in the highly conserved G-L-W motif of claudin 16 for the first time. According to the reported data and the information deduced from 3D modeling, we speculate that this mutation probably reserve partial residual function which might be related to the slight phenotype of the patient.
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http://dx.doi.org/10.1186/s12882-018-0979-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045852PMC
July 2018

Differential inhibitory effect of a pyrazolopyran compound on human serine hydroxymethyltransferase-amino acid complexes.

Arch Biochem Biophys 2018 09 4;653:71-79. Epub 2018 Jul 4.

Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185, Roma, Italy. Electronic address:

Serine hydroxymethyltransferase (SHMT) is a pivotal enzyme in one-carbon metabolism that catalyses the reversible conversion of serine and tetrahydrofolate into glycine and methylenetetrahydrofolate. It exists in cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms. Research on one-carbon metabolism in cancer cell lines has shown that SHMT1 preferentially catalyses serine synthesis, whereas in mitochondria SHMT2 is involved in serine breakdown. Recent research has focused on the identification of inhibitors that bind at the folate pocket. We have previously found that a representative derivative of the pyrazolopyran scaffold, namely 2.12, inhibits both SHMT isoforms, with a preference for SHMT1, causing apoptosis in lung cancer cell lines. Here we show that the affinity of 2.12 for SHMT depends on the identity of the amino acid substrate bound to the enzyme. The dissociation constant of 2.12 is 50-fold lower when it binds to SHMT1 enzyme-serine complex, as compared to the enzyme-glycine complex. Evidence is presented for a similar behaviour of compound 2.12 in the cellular environment. These findings suggest that the presence and identity of the amino acid substrate should be considered when designing SHMT inhibitors. Moreover, our data provide the proof-of-concept that SHMT inhibitors selectively targeting the directionality of one-carbon metabolism flux could be designed.
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http://dx.doi.org/10.1016/j.abb.2018.07.001DOI Listing
September 2018

Biochemical data from the characterization of a new pathogenic mutation of human pyridoxine-5'-phosphate oxidase (PNPO).

Data Brief 2017 Dec 28;15:868-875. Epub 2017 Oct 28.

Dipartimento di Pediatria e Neuropsichiatria Infantile, Sapienza Università di Roma, Via dei Sabelli 108, 00141 Roma, Italy.

PNPO deficiency is responsible of severe neonatal encephalopathy, responsive to pyridoxal-5'-phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this condition and detected new genetic variants on PNPO gene, whose pathogenetic role and clinical expression remain to be established. One of these mutations, Arg116Gln, is of particular interest because of its later onset of symptoms (beyond the first months of life) and its peculiar epileptic manifestations in patients. This protein variant was expressed as recombinant protein in , purified to homogeneity, and characterized with respect to structural and kinetic properties, stability, binding constants of cofactor flavin mononucleotide (FMN) and product (PLP) in order to define the molecular and structural bases of its pathogenicity. For interpretation and discussion of reported data, together with the description of clinical studies, refer to the article [1] (doi: 10.1016/j.ymgme.2017.08.003).
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http://dx.doi.org/10.1016/j.dib.2017.10.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5779537PMC
December 2017

Heterozygosis in aromatic amino acid decarboxylase deficiency: Evidence for a positive interallelic complementation between R347Q and R358H mutations.

IUBMB Life 2018 03 22;70(3):215-223. Epub 2018 Jan 22.

Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy.

Aromatic amino acid or Dopa decarboxylase (AADC or DDC) is a homodimeric pyridoxal 5'-phosphate (PLP) enzyme responsible for the generation of the neurotransmitters dopamine and serotonin. AADC deficiency is a rare inborn disease caused by mutations of the AADC gene leading to a defect of AADC enzyme and resulting in impaired dopamine and serotonin synthesis. Until now, only the molecular effects of homozygous mutations were analyzed. However, although heterozygous carriers of AADC deficiency were identified, the molecular aspects of their enzymatic phenotypes are not yet investigated. Here, we focus our attention on the R347Q/R358H and R347Q/R160W heterozygous mutations, and report for the first time the isolation and characterization, in the purified recombinant form, of the R347Q/R358H heterodimer and of the R358H homodimer. The results, integrated with those already known of the R347Q homodimeric variant, provide evidence that (i) the R358H mutation strongly reduces the PLP-binding affinity and the catalytic activity, and (ii) a positive interallelic complementation exists between the R347Q and the R358H mutations. Bioinformatics analyses provide the structural basis for these data. Unfortunately, the R347Q/R160W heterodimer was not obtained in a sufficient amount to allow its purification and characterization. Nevertheless, the biochemical features of the R160W homodimer give a contribution to the enzymatic phenotype of the heterozygous R347Q/R160W and suggest the possible relevance of Arg160 in the proper folding of human DDC. © 2018 IUBMB Life, 70(3):215-223, 2018.
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http://dx.doi.org/10.1002/iub.1718DOI Listing
March 2018

Radiation damage at the active site of human alanine:glyoxylate aminotransferase reveals that the cofactor position is finely tuned during catalysis.

Sci Rep 2017 09 15;7(1):11704. Epub 2017 Sep 15.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy.

The alanine:glyoxylate aminotransferase (AGT), a hepatocyte-specific pyridoxal-5'-phosphate (PLP) dependent enzyme, transaminates L-alanine and glyoxylate to glycine and pyruvate, thus detoxifying glyoxylate and preventing pathological oxalate precipitation in tissues. In the widely accepted catalytic mechanism of the aminotransferase family, the lysine binding to PLP acts as a catalyst in the stepwise 1,3-proton transfer, interconverting the external aldimine to ketimine. This step requires protonation by a conserved aspartate of the pyridine nitrogen of PLP to enhance its ability to stabilize the carbanionic intermediate. The aspartate residue is also responsible for a significant geometrical distortion of the internal aldimine, crucial for catalysis. We present the structure of human AGT in which complete X-ray photoreduction of the Schiff base has occurred. This result, together with two crystal structures of the conserved aspartate pathogenic variant (D183N) and the molecular modeling of the transaldimination step, led us to propose that an interplay of opposite forces, which we named spring mechanism, finely tunes PLP geometry during catalysis and is essential to move the external aldimine in the correct position in order for the 1,3-proton transfer to occur.
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http://dx.doi.org/10.1038/s41598-017-11948-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601474PMC
September 2017