Delgoshaie N.,University of Montréal |
Celic I.,High Throughput Biology Center |
Dai J.,High Throughput Biology Center |
Dai J.,Tsinghua University |
And 7 more authors.
Genetics | Year: 2015
In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3Δ hst4Δ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3Δ hst4Δ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage–induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3Δ hst4Δ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3Δ hst4Δ cells. Our data further suggest that elevated DNA damage–induced signaling significantly contributes to the phenotypes of hst3D hst4Δ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage. © 2015 by the Genetics Society of America.
Sutandy F.X.R.,National Central University |
Qian J.,The Sidney Kimmel Comprehensive Cancer Center |
Chen C.-S.,National Central University |
Zhu H.,The Sidney Kimmel Comprehensive Cancer Center |
Zhu H.,High Throughput Biology Center
Current Protocols in Protein Science | Year: 2013
Protein microarray technology is an emerging field that provides a versatile platform for the characterization of hundreds of thousands of proteins in a highly parallel and high-throughput manner. Protein microarrays are composed of two major classes: analytical and functional. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reversephase protein microarray. Applications of protein microarrays, especially functional protein microarrays, have flourished over the past decade as the fabrication technology has matured. In this unit, advances in protein microarray technologies are reviewed, and then a series of examples are presented to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. Relevant areas of research include the detection of various binding properties of proteins, the study of protein post-translational modifications, the analysis of host-microbe interactions, profiling antibody specificity, and the identification of biomarkers in autoimmune diseases. © 2013 John Wiley & Sons, Inc.
Zhu J.,High Throughput Biology Center |
Chen M.-R.,National Taiwan University |
Hu S.,High Throughput Biology Center |
Woodard C.,High Throughput Biology Center |
And 7 more authors.
Cell Host and Microbe | Year: 2011
Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase that is critical for virus replication and spread. The extent of substrate conservation and the key common cell-signaling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach, we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched, and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective antiviral strategies. © 2011 Elsevier Inc.
Lee Y.-I.,Institute for Cell Engineering |
Lee Y.-I.,Samsung |
Giovinazzo D.,Samsung |
Kang H.C.,Institute for Cell Engineering |
And 15 more authors.
Molecular and Cellular Proteomics | Year: 2014
Nitric oxide (NO) mediates a substantial part of its physiologic functions via S-nitrosylation, however the cellular substrates for NO-mediated S-nitrosylation are largely unknown. Here we describe the S-nitrosoproteome using a high-density protein microarray chip containing 16,368 unique human proteins. We identified 834 potentially Snitrosylated human proteins. Using a unique and highly specific labeling and affinity capture of S-nitrosylated proteins, 138 cysteine residues on 131 peptides in 95 proteins were determined, defining critical sites of NO's actions. Of these cysteine residues 113 are novel sites of S-nitrosylation. A consensus sequence motif from these 834 proteins for S-nitrosylation was identified, suggesting that the residues flanking the S-nitrosylated cysteine are likely to be the critical determinant of whether the cysteine is S-nitrosylated. We identify eight ubiquitin E3 ligases, RNF10, RNF11, RNF41, RNF141, RNF181, RNF208, WWP2, and UBE3A, whose activities are modulated by S-nitrosylation, providing a unique regulatory mechanism of the ubiquitin proteasome system. These results define a new and extensive set of proteins that are susceptible to NO regulation via S-nitrosylation. Similar approaches could be used to identify other post-translational modification proteomes. Molecular & Cellular Proteomics 13: 10.1074/ mcp.M113.032235, 63-72, 2014. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Yin Z.,Johns Hopkins University |
Yin Z.,The DNA Medicine Institute |
Tao S.-C.,High Throughput Biology Center |
Tao S.-C.,Shanghai JiaoTong University |
And 3 more authors.
Integrative Biology | Year: 2010
Living cells have evolved sophisticated signaling networks allowing them to respond to a wide array of external stimuli. Microfluidic devices, facilitating the analysis of signaling networks through precise definition of the cellular microenvironment often lack the capacity of delivering multiple combinations of different signaling cues, thus limiting the throughput of the analysis. To address this limitation, we developed a microfabricated platform combining microfluidic definition of the cell medium composition with dielectrophoretic definition of cell positions and protein microarray-based presentation of diverse signaling inputs. Ligands combined at different concentrations were spotted along with an extracellular matrix protein onto a glass substratum in alignment with an electrode array. This substratum was combined with a polydimethylsiloxane chip for microfluidic control of the soluble medium components, in alignment with the electrode and protein arrays. Endothelial cells were captured by dielectrophoretic force, allowed to attach and spread on the protein spots; and the signaling output of the NF-κB pathway in response to diverse combinations of IGF1 and TNF was investigated, in the absence and presence of variable dose of the pathway inhibitor. The results suggested that cells can be potently activated by immobilized TNF with IGF1 having a modulating effect, and the response could be abolished to different degrees by the inhibitor. This study demonstrates considerable potential of combining precise cell patterning and liquid medium control with protein microarray technology for complex cell signaling studies in a high-throughput manner. © 2010 The Royal Society of Chemistry.
Jeong J.S.,High Throughput Biology Center
Methods in molecular biology (Clifton, N.J.) | Year: 2011
Functional protein microarrays offer a versatile platform to address diverse biological questions. Printing individually purified proteins in a spatially addressable format makes it straightforward to investigating binary interactions. To connect substrates to their upstream modifying enzymes, such as kinases, ubiqutin (Ub) ligases, SUMOylation E3 ligases, and acetyltransferases, is an especially daunting task using traditional methodologies. In recent years, regulation via various types of posttranslational modifications (PTMs) on lysine residues is emerging as an important mechanism(s) underlining diverse biological -processes. Our group has been developing and applying functional protein microarrays constructed for different model organisms to globally identify enzyme-substrate interactions with a focus on lysine PTMs. In particular, we have characterized the pleiotropic functions of a ubiquitin E3 ligase, Rsp5, via identification of its downstream substrates using a yeast proteome chip. Also, we have identified nonhistone substrates of the acetyltransferase NuA4 complex in yeast, and revealed that reversible acetylation on a metabolic enzyme affects a glucose metabolism and contributes to life span. In this chapter, we will provide detailed protocols for the investigation of ubiquitylation and acetylation. These protocols are generally applicable for different organisms.
Discovery of a series of 2-phenyl- N -(2-(pyrrolidin-1-yl)phenyl)acetamides as novel molecular switches that modulate modes of K v7.2 (KCNQ2) channel pharmacology: Identification of (S)-2-phenyl- N -(2-(pyrrolidin-1-yl) phenyl)butanamide (ML252) as a potent, brain penetrant K v7.2 channel inhibitor
Cheung Y.-Y.,Vanderbilt University |
Yu H.,High Throughput Biology Center |
Xu K.,High Throughput Biology Center |
Zou B.,High Throughput Biology Center |
And 8 more authors.
Journal of Medicinal Chemistry | Year: 2012
A potent and selective inhibitor of KCNQ2, (S)-5 (ML252, IC 50 = 69 nM), was discovered after a high-throughput screen of the MLPCN library was performed. SAR studies revealed a small structural change (ethyl group to hydrogen) caused a functional shift from antagonist to agonist activity (37, EC 50 = 170 nM), suggesting an interaction at a critical site for controlling gating of KCNQ2 channels. © 2012 American Chemical Society.
Rivera C.G.,Johns Hopkins University |
Rivera C.G.,High Throughput Biology Center |
Rosca E.V.,Johns Hopkins University |
Pandey N.B.,Johns Hopkins University |
And 4 more authors.
Journal of Medicinal Chemistry | Year: 2011
Angiogenesis is the growth of new blood vessels from existing vasculature. Excessive vascularization is associated with a number of diseases including cancer. Antiangiogenic therapies have the potential to stunt cancer progression. Peptides derived from type IV collagen are potent inhibitors of angiogenesis. We wanted to gain a better understanding of collagen IV structure-activity relationships using a ligand-based approach. We developed novel peptide-specific QSAR models to study the activity of the peptides in endothelial cell proliferation, migration, and adhesion inhibition assays. We found that the models produced quantitatively accurate predictions of activity and provided insight into collagen IV derived peptide structure-activity relationships. © 2011 American Chemical Society.
Rivera C.G.,Johns Hopkins University |
Rivera C.G.,High Throughput Biology Center |
Mellberg S.,Uppsala University |
Claesson-Welsh L.,Uppsala University |
And 3 more authors.
PLoS ONE | Year: 2011
Angiogenesis is important for many physiological processes, diseases, and also regenerative medicine. Therapies that inhibit the vascular endothelial growth factor (VEGF) pathway have been used in the clinic for cancer and macular degeneration. In cancer applications, these treatments suffer from a "tumor escape phenomenon" where alternative pathways are upregulated and angiogenesis continues. The redundancy of angiogenesis regulation indicates the need for additional studies and new drug targets. We aimed to (i) identify novel and missing angiogenesis annotations and (ii) verify their significance to angiogenesis. To achieve these goals, we integrated the human interactome with known angiogenesis-annotated proteins to identify a set of 202 angiogenesis-associated proteins. Across endothelial cell lines, we found that a significant fraction of these proteins had highly perturbed gene expression during angiogenesis. After treatment with VEGF-A, we found increasing expression of HIF-1α, APP, HIV-1 tat interactive protein 2, and MEF2C, while endoglin, liprin β1 and HIF-2α had decreasing expression across three endothelial cell lines. The analysis showed differential regulation of HIF-1α and HIF-2α. The data also provided additional evidence for the role of endothelial cells in Alzheimer's disease. © 2011 Rivera et al.
Mitchell L.A.,High Throughput Biology Center |
Cai Y.,High Throughput Biology Center |
Taylor M.,High Throughput Biology Center |
Taylor M.,Johns Hopkins University |
And 5 more authors.
ACS Synthetic Biology | Year: 2013
Multichange ISOthermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis. © 2013 American Chemical Society.