Publications by authors named "Giuseppe Ciossani"

17 Publications

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Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint.

EMBO J 2020 Jun 23;39(12):e103180. Epub 2020 Mar 23.

Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK.

Cyclin B:CDK1 is the master kinase regulator of mitosis. We show here that, in addition to its kinase functions, mammalian Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminal region of MAD1, and point mutations in this sequence abolish MAD1 corona localisation and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona, and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness arises because Cyclin B1:MAD1 localisation loses dependence on MPS1 kinase after the corona has been established, ensuring that corona-localised MAD1 can still be phosphorylated when MPS1 activity is low. Therefore, this study explains how corona-MAD1 generates a robust SAC signal, and it reveals a scaffolding role for the key mitotic kinase, Cyclin B1:CDK1, which ultimately helps to inhibit its own degradation.
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http://dx.doi.org/10.15252/embj.2019103180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298293PMC
June 2020

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology.

Elife 2019 07 16;8. Epub 2019 Jul 16.

Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.
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http://dx.doi.org/10.7554/eLife.48287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656429PMC
July 2019

A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex.

Cell Rep 2019 04;27(2):387-399.e7

Department of Biology and Biotechnology "Lazzaro Spallanzani," University of Pavia, via Ferrata 9, 27100 Pavia, Italy. Electronic address:

LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
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http://dx.doi.org/10.1016/j.celrep.2019.03.061DOI Listing
April 2019

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

J Biol Chem 2018 06 10;293(26):10084-10101. Epub 2018 May 10.

From the Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund and

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising ∼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the od/wilch/W10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.
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http://dx.doi.org/10.1074/jbc.RA118.003154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028960PMC
June 2018

Probing the interaction of the p53 C-terminal domain to the histone demethylase LSD1.

Arch Biochem Biophys 2017 10 4;632:202-208. Epub 2017 Aug 4.

Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy. Electronic address:

The p53 transcription factor plays a central role in the regulation of the expression of several genes, and itself is post-translationally regulated through its different domains. Of particular relevance for p53 function is its intrinsically disordered C-terminal domain (CTD), representing a hotspot for post-translational modifications and a docking site for transcriptional regulators. For example, the histone H3 lysine demethylase 1 (LSD1) interacts with p53 via the p53-CTD for mutual regulation. To biochemically and functionally characterize this complex, we evaluated the in vitro interactions of LSD1 with several p53-CTD peptides differing in length and modifications. Binding was demonstrated through thermal shift, enzymatic and fluorescence polarization assays, but no enzymatic activity could be detected on methylated p53-CTD peptides in vitro. These experiments were performed using the wild-type enzyme and LSD1 variants that are mutated on three active-site residues. We found that LSD1 demethylase activity is inhibited by p53-CTD. We also noted that the association between the two proteins is mediated by mostly non-specific electrostatic interactions involving conserved active-site residues of LSD1 and a highly charged segment of the p53-CTD. We conclude that p53-CTD inhibits LSD1 activity and that the direct association between the two proteins can contribute to their functional cross-talk.
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http://dx.doi.org/10.1016/j.abb.2017.07.021DOI Listing
October 2017

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 2: Structure-Based Drug Design and Structure-Activity Relationship.

J Med Chem 2017 03 27;60(5):1693-1715. Epub 2017 Feb 27.

Department of Experimental Oncology, Academic Drug Discovery, European Institute of Oncology , Via Adamello 16, 20139 Milano, Italy.

The balance of methylation levels at histone H3 lysine 4 (H3K4) is regulated by KDM1A (LSD1). KDM1A is overexpressed in several tumor types, thus representing an emerging target for the development of novel cancer therapeutics. We have previously described ( Part 1, DOI 10.1021.acs.jmedchem.6b01018 ) the identification of thieno[3,2-b]pyrrole-5-carboxamides as novel reversible inhibitors of KDM1A, whose preliminary exploration resulted in compound 2 with biochemical IC = 160 nM. We now report the structure-guided optimization of this chemical series based on multiple ligand/KDM1A-CoRest cocrystal structures, which led to several extremely potent inhibitors. In particular, compounds 46, 49, and 50 showed single-digit nanomolar IC values for in vitro inhibition of KDM1A, with high selectivity in secondary assays. In THP-1 cells, these compounds transcriptionally affected the expression of genes regulated by KDM1A such as CD14, CD11b, and CD86. Moreover, 49 and 50 showed a remarkable anticlonogenic cell growth effect on MLL-AF9 human leukemia cells.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01019DOI Listing
March 2017

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 1: High-Throughput Screening and Preliminary Exploration.

J Med Chem 2017 03 27;60(5):1673-1692. Epub 2017 Feb 27.

Department of Experimental Oncology, Academic Drug Discovery, European Institute of Oncology , Via Adamello 16, 20139 Milano, Italy.

Lysine specific demethylase 1 KDM1A (LSD1) regulates histone methylation and it is increasingly recognized as a potential therapeutic target in oncology. We report on a high-throughput screening campaign performed on KDM1A/CoREST, using a time-resolved fluorescence resonance energy transfer (TR-FRET) technology, to identify reversible inhibitors. The screening led to 115 hits for which we determined biochemical IC, thus identifying four chemical series. After data analysis, we have prioritized the chemical series of N-phenyl-4H-thieno[3, 2-b]pyrrole-5-carboxamide for which we obtained X-ray structures of the most potent hit (compound 19, IC = 2.9 μM) in complex with the enzyme. Initial expansion of this chemical class, both modifying core structure and decorating benzamide moiety, was directed toward the definition of the moieties responsible for the interaction with the enzyme. Preliminary optimization led to compound 90, which inhibited the enzyme with a submicromolar IC (0.162 μM), capable of inhibiting the target in cells.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01018DOI Listing
March 2017

Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features.

Sci Adv 2016 09 9;2(9):e1601017. Epub 2016 Sep 9.

Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy.

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors.
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http://dx.doi.org/10.1126/sciadv.1601017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017823PMC
September 2016

Pure enantiomers of benzoylamino-tranylcypromine: LSD1 inhibition, gene modulation in human leukemia cells and effects on clonogenic potential of murine promyelocytic blasts.

Eur J Med Chem 2015 Apr 3;94:163-74. Epub 2015 Mar 3.

Department of Drug Chemistry and Technologies, Sapienza University of Roma, P.le A. Moro 5, 00185 Roma, Italy; Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Roma, P.le A. Moro 5, 00185 Roma, Italy. Electronic address:

The pure enantiomers of the N-(2-, 3-, and 4-(2-aminocyclopropyl)phenyl)benzamides hydrochlorides 11a-j were prepared and tested against LSD1 and MAO enzymes. The evaluation of the regioisomers 11a-j highlighted a net increase of the anti-LSD1 potency by shifting the benzamide moiety from ortho to meta and mainly to para position of tranylcypromine phenyl ring, independently from their trans or cis stereochemistry. In particular, the para-substituted 11a,b (trans) and 11g,h (cis) compounds displayed LSD1 and MAO-A inhibition at low nanomolar levels, while were less potent against MAO-B. The meta analogs 11c,d (trans) and 11i,j (cis) were in general less potent, but more efficient against MAO-A than against LSD1. In cellular assays, all the para and meta enantiomers were able to inhibit LSD1 by inducing Gfi-1b and ITGAM gene expression, with 11b,c and 11g-i giving the highest effects. Moreover, 11b and 11g,h strongly inhibited the clonogenic potential of murine promyelocytic blasts.
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http://dx.doi.org/10.1016/j.ejmech.2015.02.060DOI Listing
April 2015

Pure Diastereomers of a Tranylcypromine-Based LSD1 Inhibitor: Enzyme Selectivity and In-Cell Studies.

ACS Med Chem Lett 2015 Feb 8;6(2):173-7. Epub 2014 Dec 8.

Department of Drug Chemistry and Technologies, Sapienza University of Roma , P.le A. Moro 5, 00185 Roma, Italy ; Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Roma , P.le A. Moro 5, 00185 Roma, Italy.

The pure four diastereomers (11a-d) of trans-benzyl (1-((4-(2-aminocyclopropyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate hydrochloride 11, previously described by us as LSD1 inhibitor, were obtained by enantiospecific synthesis/chiral HPLC separation method. Tested in LSD1 and MAO assays, 11b (S,1S,2R) and 11d (R,1S,2R) were the most potent isomers against LSD1 and were less active against MAO-A and practically inactive against MAO-B. In cells, all the four diastereomers induced Gfi-1b and ITGAM gene expression in NB4 cells, accordingly with their LSD1 inhibition, and 11b and 11d inhibited the colony forming potential in murine promyelocytic blasts.
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http://dx.doi.org/10.1021/ml500424zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329595PMC
February 2015

Synthesis, biological activity and mechanistic insights of 1-substituted cyclopropylamine derivatives: a novel class of irreversible inhibitors of histone demethylase KDM1A.

Eur J Med Chem 2014 Oct 27;86:352-63. Epub 2014 Aug 27.

Drug Discovery Unit, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy.

Histone demethylase KDM1A (also known as LSD1) has become an attractive therapeutic target for the treatment of cancer as well as other disorders such as viral infections. We report on the synthesis of compounds derived from the expansion of tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. These compounds, which are substituted on the cyclopropyl core moiety, were evaluated for their ability to inhibit KDM1A in vitro as well as to function in cells by modulating the expression of Gfi-1b, a well recognized KDM1A target gene. The molecules were all found to covalently inhibit KDM1A and to become increasingly selective against human monoamine oxidases MAO A and MAO B through the introduction of bulkier substituents on the cyclopropylamine ring. Structural and biochemical analysis of selected trans isomers showed that the two stereoisomers are endowed with similar inhibitory activities against KDM1A, but form different covalent adducts with the FAD co-enzyme.
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http://dx.doi.org/10.1016/j.ejmech.2014.08.068DOI Listing
October 2014

Differential properties of transcriptional complexes formed by the CoREST family.

Mol Cell Biol 2014 Jul;34(14):2760-70

Mammalian genomes harbor three CoREST genes. rcor1 encodes CoREST (CoREST1), and the paralogues rcor2 and rcor3 encode CoREST2 and CoREST3, respectively. Here, we describe specific properties of transcriptional complexes formed by CoREST proteins with the histone demethylase LSD1/KDM1A and histone deacetylases 1 and 2 (HDAC1/2) and the finding that all three CoRESTs are expressed in the adult rat brain. CoRESTs interact equally strongly with LSD1/KDM1A. Structural analysis shows that the overall conformation of CoREST3 is similar to that of CoREST1 complexed with LSD1/KDM1A. Nonetheless, transcriptional repressive capacity of CoREST3 is lower than that of CoREST1, which correlates with the observation that CoREST3 leads to a reduced LSD1/KDM1A catalytic efficiency. Also, CoREST2 shows a lower transcriptional repression than CoREST1, which is resistant to HDAC inhibitors. CoREST2 displays lower interaction with HDAC1/2, which is barely present in LSD1/KDM1A-CoREST2 complexes. A nonconserved leucine in the first SANT domain of CoREST2 severely weakens its association with HDAC1/2. Furthermore, CoREST2 mutants with increased HDAC1/2 interaction and those without HDAC1/2 interaction exhibit equivalent transcriptional repression capacities, indicating that CoREST2 represses in an HDAC-independent manner. In conclusion, differences among CoREST proteins are instrumental in the modulation of protein-protein interactions and catalytic activities of LSD1/KDM1A-CoREST-HDAC complexes, fine-tuning gene expression regulation.
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http://dx.doi.org/10.1128/MCB.00083-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097654PMC
July 2014

Pan-histone demethylase inhibitors simultaneously targeting Jumonji C and lysine-specific demethylases display high anticancer activities.

J Med Chem 2014 Jan 19;57(1):42-55. Epub 2013 Dec 19.

Department of Drug Chemistry and Technologies, Sapienza University of Rome , P. le A. Moro 5, 00185 Rome, Italy.

In prostate cancer, two different types of histone lysine demethylases (KDM), LSD1/KDM1 and JMJD2/KDM4, are coexpressed and colocalize with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC or "pan-KDM" inhibitors 1-6 by coupling the skeleton of tranylcypromine 7, a known LSD1 inhibitor, with 4-carboxy-4'-carbomethoxy-2,2'-bipyridine 8 or 5-carboxy-8-hydroxyquinoline 9, two 2-oxoglutarate competitive templates developed for JmjC inhibition. Hybrid compounds 1-6 are able to simultaneously target both KDM families and have been validated as potential antitumor agents in cells. Among them, 2 and 3 increase H3K4 and H3K9 methylation levels in cells and cause growth arrest and substantial apoptosis in LNCaP prostate and HCT116 colon cancer cells. When tested in noncancer mesenchymal progenitor (MePR) cells, 2 and 3 induced little and no apoptosis, respectively, thus showing cancer-selective inhibiting action.
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http://dx.doi.org/10.1021/jm4012802DOI Listing
January 2014

Expanding the druggable space of the LSD1/CoREST epigenetic target: new potential binding regions for drug-like molecules, peptides, protein partners, and chromatin.

PLoS Comput Biol 2013 18;9(7):e1003158. Epub 2013 Jul 18.

Department of Medicinal Chemistry, College of Pharmacy, The University of Utah, Salt Lake City, Utah, USA.

Lysine specific demethylase-1 (LSD1/KDM1A) in complex with its corepressor protein CoREST is a promising target for epigenetic drugs. No therapeutic that targets LSD1/CoREST, however, has been reported to date. Recently, extended molecular dynamics (MD) simulations indicated that LSD1/CoREST nanoscale clamp dynamics is regulated by substrate binding and highlighted key hinge points of this large-scale motion as well as the relevance of local residue dynamics. Prompted by the urgent need for new molecular probes and inhibitors to understand LSD1/CoREST interactions with small-molecules, peptides, protein partners, and chromatin, we undertake here a configurational ensemble approach to expand LSD1/CoREST druggability. The independent algorithms FTMap and SiteMap and our newly developed Druggable Site Visualizer (DSV) software tool were used to predict and inspect favorable binding sites. We find that the hinge points revealed by MD simulations at the SANT2/Tower interface, at the SWIRM/AOD interface, and at the AOD/Tower interface are new targets for the discovery of molecular probes to block association of LSD1/CoREST with chromatin or protein partners. A fourth region was also predicted from simulated configurational ensembles and was experimentally validated to have strong binding propensity. The observation that this prediction would be prevented when using only the X-ray structures available (including the X-ray structure bound to the same peptide) underscores the relevance of protein dynamics in protein interactions. A fifth region was highlighted corresponding to a small pocket on the AOD domain. This study sets the basis for future virtual screening campaigns targeting the five novel regions reported herein and for the design of LSD1/CoREST mutants to probe LSD1/CoREST binding with chromatin and various protein partners.
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http://dx.doi.org/10.1371/journal.pcbi.1003158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715402PMC
February 2014

Protein recognition by short peptide reversible inhibitors of the chromatin-modifying LSD1/CoREST lysine demethylase.

ACS Chem Biol 2013 Aug 11;8(8):1677-82. Epub 2013 Jun 11.

Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, 27100 Pavia, Italy.

The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1.
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http://dx.doi.org/10.1021/cb4001926DOI Listing
August 2013

Biochemical, structural, and biological evaluation of tranylcypromine derivatives as inhibitors of histone demethylases LSD1 and LSD2.

J Am Chem Soc 2010 May;132(19):6827-33

Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy.

LSD1 and LSD2 histone demethylases are implicated in a number of physiological and pathological processes, ranging from tumorigenesis to herpes virus infection. A comprehensive structural, biochemical, and cellular study is presented here to probe the potential of these enzymes for epigenetic therapies. This approach employs tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. This drug is a clinically validated antidepressant known to target monoamine oxidases A and B. These two flavoenzymes are structurally related to LSD1 and LSD2. Mechanistic and crystallographic studies of tranylcypromine inhibition reveal a lack of selectivity and differing covalent modifications of the FAD cofactor depending on the enantiomeric form. These findings are pharmacologically relevant, since tranylcypromine is currently administered as a racemic mixture. A large set of tranylcypromine analogues were synthesized and screened for inhibitory activities. We found that the common evolutionary origin of LSD and MAO enzymes, despite their unrelated functions and substrate specificities, is reflected in related ligand-binding properties. A few compounds with partial enzyme selectivity were identified. The biological activity of one of these new inhibitors was evaluated with a cellular model of acute promyelocytic leukemia chosen since its pathogenesis includes aberrant activities of several chromatin modifiers. Marked effects on cell differentiation and an unprecedented synergistic activity with antileukemia drugs were observed. These data demonstrate that these LSD1/2 inhibitors are of potential relevance for the treatment of promyelocytic leukemia and, more generally, as tools to alter chromatin state with promise of a block of tumor progression.
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http://dx.doi.org/10.1021/ja101557kDOI Listing
May 2010

A novel mammalian flavin-dependent histone demethylase.

J Biol Chem 2009 Jun 30;284(26):17775-82. Epub 2009 Apr 30.

Dipartimento di Genetica e Microbiologia, Università di Pavia, Via Ferrata 1, 27100 Pavia, Italy.

Methylation of Lys residues on histone proteins is a well known and extensively characterized epigenetic mark. The recent discovery of lysine-specific demethylase 1 (LSD1) demonstrated that lysine methylation can be dynamically controlled. Among the histone demethylases so far identified, LSD1 has the unique feature of functioning through a flavin-dependent amine oxidation reaction. Data base analysis reveals that mammalian genomes contain a gene (AOF1, for amine-oxidase flavin-containing domain 1) that is homologous to the LSD1-coding gene. Here, we demonstrate that the protein encoded by AOF1 represents a second mammalian flavin-dependent histone demethylase, named LSD2. The new demethylase is strictly specific for mono- and dimethylated Lys4 of histone H3, recognizes a long stretch of the H3 N-terminal tail, senses the presence of additional epigenetic marks on the histone substrate, and is covalently inhibited by tranylcypromine. As opposed to LSD1, LSD2 does not form a biochemically stable complex with the C-terminal domain of the corepressor protein CoREST. Furthermore, LSD2 contains a CW-type zinc finger motif with potential zinc-binding sites that are not present in LSD1. We conclude that mammalian LSD2 represents a new flavin-dependent H3-Lys4 demethylase that features substrate specificity properties highly similar to those of LSD1 but is very likely to be part of chromatin-remodeling complexes that are distinct from those involving LSD1.
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http://dx.doi.org/10.1074/jbc.M109.003087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719416PMC
June 2009