Search PubMed⌕ Search

Biomedical subjects

Cheryl H Arrowsmith

Publications and source records attributed to Cheryl H Arrowsmith.

At least 19 recordsLinked to original sources
PubMed2026

Comprehensive mass spectrometry screening-derived atlas of HDAC inhibitors reveals histone-specific acetylation changes.

Histone deacetylase inhibitors (HDACis) have emerged as valuable therapeutics for cancer and other diseases; however, their effects on histone post-translational modification remain poorly characterized. Here, we applied quantitative mass spectrometry and high-throughput sequencing to systematically profile site-specific changes in histone modifications in response to a panel of HDACis. This platform enabled mapping of histone modification changes across hundreds of sites, including low-abundance histone marks. Furthermore, an integrative analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) data identified genome-wide binding sites for the low-abundance histone modification of H2A.Z acetylation in HeLa and MDA-MB-231 breast cancer cells, highlighting the role of H2A.Z acetylation in regulating gene expression across diverse biological pathways, including specific genes involved in tumor suppressor pathways. Our findings provide a functional resource for identifying and quantifying histone modification changes and transcriptional regulation of histone H2A.Z acetylation following pharmacological perturbation.

Histone Deacetylase Inhibitors↗
PubMed2025

A covalent chemical probe for Chikungunya nsP2 cysteine protease with antialphaviral activity and proteome-wide selectivity.

Chikungunya is a mosquito-borne viral disease that causes fever and severe joint pain for which there is no direct acting drug treatments. Vinyl sulfone SGC-NSP2PRO-1 (3) was identified as a potent inhibitor of the nsP2 cysteine protease (nsP2pro) that reduced viral titer against infectious isolates of Chikungunya and other alphaviruses. The covalent warhead in 3 captured the active site C478 and inactivated nsP2pro with a kinact/Ki ratio of 5950 M-1 s-1. The vinyl sulfone 3 was inactive across a panel of 23 other cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. A negative control analog SGC-NSP2PRO-1N (4) retained the isoxazole core and covalent warhead but demonstrated > 100-fold decrease in enzyme inhibition. Both 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. Vinyl sulfone 3 and its negative control 4 will find utility as high-quality chemical probes to study the role of the nsP2pro in cellular studies of alphaviral replication and virulence.

Chikungunya virus↗
PubMed2007

NMR structure and binding studies confirm that PA4608 from Pseudomonas aeruginosa is a PilZ domain and a c-di-GMP binding protein.

PA4608 is a 125 residue protein from Pseudomonas aeruginosa with a recent identification as a PilZ domain and putative bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) adaptor protein that plays a role in bacterial second-messenger regulated processes. The nuclear magnetic resonance (NMR) structure of PA4608 has been determined and c-di-GMP binding has been confirmed by NMR titration studies. The monomeric core structure of PA4608 contains a six-stranded anti-parallel beta barrel flanked by three helices. Conserved surface residues among PA4608 homologs suggest the c-di-GMP binding site is at one end of the barrel and includes residues in the helices as well as in the unstructured N-terminus. Chemical shift changes in PA4608 resonances upon titration with c-di-GMP confirm binding. This evidence supports the hypothesis that proteins containing PilZ domains are the long-sought c-di-GMP adaptor proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family.

Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and beta-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases.

Catalysis↗
PubMed2006

Structural basis for molecular recognition and presentation of histone H3 by WDR5.

Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl-lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4-dimethylated histone H3 tail as well as for global and gene-specific K4 trimethylation. Here we report the crystal structures of full-length WDR5, WDR5Delta23 and its complexes with unmodified, mono-, di- and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water-mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes.

Amino Acid Sequence↗
PubMed2006

Solution NMR in structural genomics.

Structural genomics (also known as structural proteomics) aims to generate accurate three-dimensional models for all folded, globular proteins and domains in the protein universe to understand the relationship between protein sequence, structure and function. NMR spectroscopy of small (<20 kDa) proteins has been used successfully within several large-scale structural genomics projects for more than six years now. Recent advances coming from traditional NMR structural biology laboratories as well as large scale centers and consortia using NMR for structural genomics promise to facilitate NMR analysis making it even a more efficient and increasingly automated procedure.

Animals↗
PubMed2006

MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like repeats.

With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339-345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 degrees C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135-7155].

Archaeal Proteins↗
PubMed2006

p53 transcriptional activation domain: a molecular chameleon?

The recent structure of human replication protein A (RPA) bound to residues 38-58 of tumor suppressor p53 exemplifies several important features of protein-protein interactions involved in transcription and DNA repair. First, the N-terminal transcriptional activation domain (TAD) of p53 is multifunctional and dynamic, showing multiple interactions with partner proteins some of which are modulated by phosphorylation. Second, the binding of partner proteins is coupled with a disorder-to-order transition common to many other transcriptional activation domains. Third, the molecular features of p53 residues 47-58 imitate those of single stranded DNA in their interaction with the oligonucleotide oliogsaccharide-binding (OB) fold of the N-terminal domain of RPA70. This regulated association is implicated in transmitting the DNA damage signal to the p53 pathway of stress response. Here we review the recently reported crystal structure of the p53/RPA70N complex and the mechanism by which ssDNA can provide positive feedback to dissociate p53/RPA complexes. The binding mode and regulatory mechanisms of the p53/RPA70N interaction may represent a general paradigm for regulation of the OB folds involved in DNA repair and metabolism.

DNA↗
PubMed2006

Molecular recognition of p53 and MDM2 by USP7/HAUSP.

The ubiquitin-specific protease, USP7, has key roles in the p53 pathway whereby it stabilizes both p53 and MDM2. We show that the N-terminal domain of USP7 binds two closely spaced 4-residue sites in both p53 and MDM2, falling between p53 residues 359-367 and MDM2 residues 147-159. Cocrystal structures with USP7 were determined for both p53 peptides and for one MDM2 peptide. These peptides bind the same surface of USP7 as Epstein-Barr nuclear antigen-1, explaining the competitive nature of the interactions. The structures and mutagenesis data indicate a preference for a P/AXXS motif in peptides that bind USP7. Contacts made by serine are identical and crucial for all peptides, and Trp165 in the peptide-binding pocket of USP7 is also crucial. These results help to elucidate the mechanism of substrate recognition by USP7 and the regulation of the p53 pathway.

Amino Acid Motifs↗
PubMed2005

NMR and X-ray crystallography, complementary tools in structural proteomics of small proteins.

NMR spectroscopy and X-ray crystallography, the two primary experimental methods for protein structure determination at high resolution, have different advantages and disadvantages in terms of sample preparation and data collection and analysis. It is therefore of interest to assess their complementarity when applied to small proteins. Structural genomics/proteomics projects provide an ideal opportunity to make such comparisons as they generate data in a systematic manner for large enough numbers of proteins to allow firm conclusions to be drawn. Here we report a comparison for 263 unique proteins screened by both NMR spectroscopy and X-ray crystallography in our structural proteomics pipeline. Only 21 targets (8%) were deemed amenable to both methods based on an initial 2D 15N-HSQC NMR spectrum and optimized crystallization trials. However, the use of both methods in the pipeline increased the total number of targets amenable to structure determination to 107, with 43 amenable to NMR only and 43 amenable to X-ray crystallographic methods only. We did not observe a correlation between 15N-HSQC spectral quality and the success of the same protein in crystallization screens. Similar results were found for an independent set of 159 proteins as reported in the accompanying paper by Snyder et al. Thus, we conclude that both methods are highly complementary, and in order to increase the number of proteins suited for structure determination, we suggest that both methods be used in parallel in screening of all small proteins for structure determination.

Animals↗