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Baltimore Highlands, MD, United States

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. Source

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. Source

Delgoshaie N.,University of Montreal | 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. Source

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. Source

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. Source

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