The Experts below are selected from a list of 669 Experts worldwide ranked by ideXlab platform
Fraser Hof - One of the best experts on this subject based on the ideXlab platform.
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Aza‐amino acid scanning of chromobox homolog 7 (CBX7) ligands
Journal of Peptide Science, 2017Co-Authors: Mariam Traoré, Fraser Hof, Michael C. Gignac, Ngoc-duc Doan, William D. LubellAbstract:An aza-amino acid scan of peptide inhibitors of the chromobox homolog 7 (CBX7) was performed to study the conformational requirements for affinity to the Methyllysine reader protein. Twelve azapeptide analogues were prepared using three different approaches employing respectively N-(Fmoc)aza-amino acid chlorides and submonomer azapeptide synthesis to install systematically aza-residues at the first four residues of the peptide, as well as to provide aza-lysine residues possessing saturated and unsaturated side chains. The aza-peptide ligands were evaluated in a chromobox homolog 7 binding assay, providing useful insight into structural requirements for affinity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
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Structural aspects of small-molecule inhibition of Methyllysine reader proteins.
Future Medicinal Chemistry, 2016Co-Authors: Natalia Milosevich, Zoey Warmerdam, Fraser HofAbstract:Methyl reader proteins recognize and bind to post-translationally methylated residues. They execute the commands issued by protein methyltransferases and play functional roles in diverse cellular processes including gene regulation, development and oncogenesis. Efforts to inhibit these proteins are relatively new. Only a small number of methyl reader proteins belonging to the chromodomain, malignant brain tumor domain, plant homeodomain finger and Tudor domain families have been targeted by chemical inhibitors. This review summarizes inhibitors that have been reported to date, and provides a perspective for future progress. Structural determinants for methyl reader inhibition will be presented, along with an analysis of the molecular interactions that control potency and selectivity for inhibitors of each family.
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Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics
Analytical Chemistry, 2016Co-Authors: Graham A.e. Garnett, Melissa J. Starke, Alok Shaurya, Fraser HofAbstract:Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host–guest complexes selectively with Methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation—a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of m...
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Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics
2016Co-Authors: Graham A.e. Garnett, Melissa J. Starke, Alok Shaurya, Fraser HofAbstract:Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host–guest complexes selectively with Methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylationa feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of Methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods
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Chemical Inhibitors of Epigenetic Methyllysine Reader Proteins.
Biochemistry, 2015Co-Authors: Natalia Milosevich, Fraser HofAbstract:Protein methylation is a common post-translational modification with diverse biological functions. Methyllysine reader proteins are increasingly a focus of epigenetics research and play important roles in regulating many cellular processes. These reader proteins are vital players in development, cell cycle regulation, stress responses, oncogenesis, and other disease pathways. The recent emergence of a small number of chemical inhibitors for Methyllysine reader proteins supports the viability of these proteins as targets for drug development. This article introduces the biochemistry and biology of Methyllysine reader proteins, provides an overview of functions for those families of readers that have been targeted to date (MBT, PHD, tudor, and chromodomains), and reviews the development of synthetic agents that directly block their Methyllysine reading functions.
Ruibing Wang - One of the best experts on this subject based on the ideXlab platform.
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Host–Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes
Analytical Chemistry, 2020Co-Authors: Min Liu, Rui Wang, Liang Zhang, Ludan Yue, Ning Zhang, Yujie Liang, Lixin Cheng, Jiang Xia, Ruibing WangAbstract:Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A Methyllysine-binding protein HP1β chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture Methyllysine proteomes from cancer cells. The captured "fish"-Methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel Methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1β CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of Methyllysine proteins in coupling to mass spectrometry.
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host guest protein assembly for affinity purification of Methyllysine proteomes
Analytical Chemistry, 2020Co-Authors: Min Liu, Rui Wang, Liang Zhang, Ludan Yue, Ning Zhang, Yujie Liang, Lixin Cheng, Jiang Xia, Ruibing WangAbstract:Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A Methyllysine-binding protein HP1β chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture Methyllysine proteomes from cancer cells. The captured "fish"-Methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel Methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1β CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of Methyllysine proteins in coupling to mass spectrometry.
Evan R Williams - One of the best experts on this subject based on the ideXlab platform.
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proton affinity and zwitterion stability new results from infrared spectroscopy and theory of cationized lysine and analogues in the gas phase
Journal of Physical Chemistry A, 2009Co-Authors: Matthew F Bush, Jos Oomens, Evan R WilliamsAbstract:The gas-phase structures of alkali metal cationized lysine (Lys), α-N-Methyllysine (NMeLys), and ϵ-N,N-diMethyllysine (Lys(Me)2) are investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser and ab initio calculations. The proton affinities of the compounds span a range of ∼20 kJ/mol. For NMeLys•M+, experiment and theory indicate that NMeLys is nonzwitterionic for M = Li and zwitterionic for M = Na and K. For Lys(Me)2•M+, experiment and theory indicate that Lys(Me)2 is zwitterionic for M = Li, Na, and K. This is the first spectroscopic observation of the zwitterionic form of an amino acid complexed with Li+. The results are compared with IRMPD spectra reported previously for Lys and ϵ-N-Methyllysine (Lys(Me)) complexed with Li, Na, and K, and new calculations performed at higher levels of theory for those ions. The combined experimental and theoretical results indicate that protonation in the zwitterionic forms of the these amino acids...
Tatiana G. Kutateladze - One of the best experts on this subject based on the ideXlab platform.
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Structural Insight into Recognition of Methylated Histone H3K4 by Set3.
Journal of Molecular Biology, 2016Co-Authors: Jovylyn Gatchalian, Forest H. Andrews, Muzaffar Ali, Yi Zhang, Alexander S. Barrett, Tatiana G. KutateladzeAbstract:The plant homeodomain (PHD) finger of Set3 binds methylated lysine 4 of histone H3 in vitro and in vivo; however, precise selectivity of this domain has not been fully characterized. Here, we explore the determinants of Methyllysine recognition by the PHD fingers of Set3 and its orthologs. We use X-ray crystallographic and spectroscopic approaches to show that the Set3 PHD finger binds di- and trimethylated states of H3K4 with comparable affinities and employs similar molecular mechanisms to form complexes with either mark. Composition of the Methyllysine-binding pocket plays an essential role in determining the selectivity of the PHD fingers. The finding that the histone-binding activity is not conserved in the PHD finger of Set4 suggests different functions for the Set3 and Set4 paralogs.
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Preparation, Biochemical Analysis, and Structure Determination of Methyllysine Readers.
Methods in Enzymology, 2016Co-Authors: Catherine A. Musselman, Tatiana G. KutateladzeAbstract:In-depth in vitro characterization of Methyllysine reader domains and their association with cognate Methyllysine substrates is essential to better understand fundamental mechanisms of chromatin regulation and to design targeted therapeutics that disrupt these interactions. In this chapter, we summarize commonly used methods for preparation, biochemical characterization, and determination of structures of Methyllysine reader domains. We provide a detailed protocol for the preparation of a GST-tagged Methyllysine reader domain and for analysis of histone-binding activities using a combination of pull-down, tryptophan fluorescence, and NMR assays, and describe initial steps toward crystallization of the complexes.
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Regulation of Methyllysine Readers through Phosphorylation
ACS Chemical Biology, 2016Co-Authors: Forest H. Andrews, Jovylyn Gatchalian, Krzysztof Krajewski, Brian D. Strahl, Tatiana G. KutateladzeAbstract:Methyllysine post-translational modifications (PTMs) of histones create binding sites for evolutionarily conserved reader domains that link nuclear host proteins and chromatin-modifying complexes to specific genomic regions. In the context of these events, adjacent histone PTMs are capable of altering the binding activity of readers toward their target marks. This provides a mechanism of “combinatorial readout” of PTMs that can enhance, decrease, or eliminate the association of readers with chromatin. In this Perspective, we focus on recent studies describing the impact of dynamic phospho-serine/threonine/tyrosine marks on the interaction of Methyllysine readers with histones, summarize mechanistic aspects of the phospho/methyl readout, and highlight the significance of crosstalk between these PTMs. We also demonstrate that in addition to inhibiting binding and serving as a true switch, promoting dissociation of the Methyllysine readers from chromatin, the phospho/methyl combination can act together in a ...
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Towards understanding Methyllysine readout.
Biochimica et Biophysica Acta, 2014Co-Authors: Catherine A. Musselman, Sepideh Khorasanizadeh, Tatiana G. KutateladzeAbstract:Abstract Background Lysine methylation is the most versatile covalent posttranslational modification (PTM) found in histones and non-histone proteins. Over the past decade a number of Methyllysine-specific readers have been discovered and their interactions with histone tails have been structurally and biochemically characterized. More recently innovative experimental approaches have emerged that allow for studying reader interactions in the context of the full nucleosome and nucleosomal arrays. Scope of review In this review we give a brief overview of the known mechanisms of histone lysine methylation readout, summarize progress recently made in exploring interactions with methylated nucleosomes, and discuss the latest advances in the development of small molecule inhibitors of the Methyllysine-specific readers. Major conclusions New studies reveal various reader-nucleosome contacts outside the methylated histone tail, thus offering a better model for association of histone readers to chromatin and broadening our understanding of the functional implications of these interactions. In addition, some progress has been made in the design of antagonists of these interactions. General significance Specific lysine methylation patterns are commonly associated with certain chromatin states and genomic elements, and are linked to distinct biological outcomes such as transcription activation or repression. Disruption of patterns of histone modifications is associated with a number of diseases, and there is tremendous therapeutic potential in targeting histone modification pathways. Thus, investigating binding of readers of these modifications is not only important for elucidating fundamental mechanisms of chromatin regulation, but also necessary for the design of targeted therapeutics. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.
Rui Wang - One of the best experts on this subject based on the ideXlab platform.
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host guest protein assembly for affinity purification of Methyllysine proteomes
Analytical Chemistry, 2020Co-Authors: Min Liu, Rui Wang, Liang Zhang, Ludan Yue, Ning Zhang, Yujie Liang, Lixin Cheng, Jiang Xia, Ruibing WangAbstract:Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A Methyllysine-binding protein HP1β chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture Methyllysine proteomes from cancer cells. The captured "fish"-Methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel Methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1β CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of Methyllysine proteins in coupling to mass spectrometry.
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Host–Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes
Analytical Chemistry, 2020Co-Authors: Min Liu, Rui Wang, Liang Zhang, Ludan Yue, Ning Zhang, Yujie Liang, Lixin Cheng, Jiang Xia, Ruibing WangAbstract:Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A Methyllysine-binding protein HP1β chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture Methyllysine proteomes from cancer cells. The captured "fish"-Methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel Methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1β CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of Methyllysine proteins in coupling to mass spectrometry.
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Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation
Analytical Chemistry, 2018Co-Authors: Rui Wang, Linting Li, Courtney Voss, Tomonori Kaneko, Liang Zhang, Shawn S C LiAbstract:A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a Methyllysine-binding protein HP1β chromodomain on the agarose resin was used to purify the Methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag–nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified pr...
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Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation.
Analytical Chemistry, 2018Co-Authors: Rui Wang, Courtney Voss, Tomonori Kaneko, Liang Zhang, Mei Huang, Jiang XiaAbstract:A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a Methyllysine-binding protein HP1β chromodomain on the agarose resin was used to purify the Methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag-nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified proteome identified 275 lysine methylated sites and 184 methylated proteins from 332 HP1β CD-binding proteins, including 30 novel methylated proteins. This work demonstrates that a robust site-specific covalent protein immobilization method is well-suited for proteomic analysis of low-abundance proteins. This method also enables the identification of new methylated proteins and methylation sites in the Methyllysine proteome.