Thomas A. Neubert - US grants

transducin; protein structure function; fatty acylation; biological signal transduction; protein sequence; posttranslational modifications; liquid chromatography; mass spectrometry; transfection;

transducin; protein structure function; fatty acylation; biological signal transduction; protein sequence; posttranslational modifications; liquid chromatography; mass spectrometry; transfection;

The identification and characterization of proteins and peptides involved in causing or preventing many important diseases is the central focus of many NIH-funded research programs at the New York University School of Medicine. Recent developments in mass spectrometry have made it possible to gain essential information about these proteins that would be impractical or impossible to obtain in any other way. The NYU Protein Analysis Facility within the Skirball Institute of Biomolecular Medicine at-the NYU School of Medicine is strongly committed to assuring that scientists here can benefit from this new technology. Because of its versatility, high sensitivity, ease of use and high speed of sample processing, Matrix-Assisted Laser Desorption Time-of-Flight (MALDI- TOF) mass spectrometry is essential for many of our protein analyses. Examples of NIH-funded research projects that will benefit from the requested MALDI-TOF mass spectrometer include the identification of signal transduction proteins involved in nerve cell development, structural characterization of HIV-1 antigens for AIDS vaccine development, identification of signaling proteins involved in intracellular stress responses, structural studies of proteins involved in insulin signal transduction, the study of exotoxin production and virulence responses in pathogenic Staphylococcus aureus, molecular mechanisms that modulate opioid receptor function, the characterization of amyloid peptides involved in Alzheimer's Disease, the pathogenic role of apoptosis in Shigella infection, the pathology of Alzheimer's Disease and aging, signaling proteins involved in neuromuscular synapse formation, and the study of proteins that are important for transcriptional regulation in hepatitis B.

The use of mass spectrometry in functional proteomics studies is a powerful approach for identifying proteins involved in important intracellular signal transduction systems. We propose to develop and use a new functional proteomics method based on isotope-coded affinity tag (ICAT) chemistry and liquid chromatography/mass spectrometry to identify proteins that participate in signal transduction complex formation in response to ligand stimulation. This strategy should improve the accuracy of relative protein quantitation as well as increase the speed, sensitivity, and dynamic range of protein identification over strategies based on the identification of proteins from SDS-PAGE gels. Ephrins and ephrin receptors (EphRs) are involved in patterning of the central and peripheral nervous systems. We will use our method to study signaling through EphRs as well as "reverse signaling" through B ephrin ligands in cultured neuronal cells by identifying and quantitating proteins that associate with previously characterized signal transduction proteins in response to stimulation by ephrinB2 or EphRs. Three groups of proteins will be identified: constitutively binding proteins, those that increase and those that decrease in their association with other signaling proteins in response to ligand stimulation. The goal of the proposed ICAT-based functional proteomics technology is to rapidly discover new protein-protein interactions that depend on ephrin signaling. A comprehensive cataloging of these interactions, especially including quantitative information and dependence upon the signaling state of the cell, will enable focused, hypothesis-driven experiments to elucidate the roles of these proteins in ephrin signaling. The functional proteomics methodology developed for the study of ephrin signaling could also be used to study a variety of other signal transduction systems essential for the functioning of the nervous system. Ultimately, information gained from these experiments may lead to greater understanding of the causes of a variety of cancers and neurodevelopmental diseases.

[unreadable] DESCRIPTION (provided by applicant): The identification and characterization of proteins and peptides involved in causing or preventing many important diseases is the central focus of many NIH-funded research programs here at the NYU School of Medicine. The NYU Protein Analysis Facility within the Skirball Institute at the NYU School of Medicine is strongly committed to assuring that scientists at NYU can benefit from recent developments in biological mass spectrometry. This powerful technology has made it possible to gain very useful information about these proteins that would be impractical or impossible to obtain in any other way. Because of its sensitivity, high mass accuracy, and versatility, quadrupole time of flight (Q-TOF) mass spectrometry is essential for many of our protein analyses. Examples of NIH-funded research that would benefit from the requested QTOF mass spectrometer include studies of intracellular signaling processes involved in neuronal growth and development; development of anticancer vaccines; study of the role of NO signaling in cardiac and other diseases; characterization of immune responses to viral infection; studies of intracellular vesicle trafficking; invasion of host cells by malaria parasites; structure and function of urothelial plaque proteins; characterization of the processes involved in selecting the peripheral T cell repertoire; and studies of intracellular signaling processes involved in diabetes and cancer. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Since its establishment in 1998, the New York University Protein Analysis Facility has provided cutting edge mass spectrometry-based protein analysis to investigators at the New York University School of Medicine. Because of the expensive and sophisticated equipment and expertise required, it is impractical or impossible to perform these analyses in individual labs. As a result, demand for facility services has rapidly increased. The purpose of this grant is to provide funding to enable neuroscientists at NYU to take advantage of sophisticated and involved protein mass spectrometry and proteomics that would otherwise not be available to them, and guarantee them access to the technology. The proposed Protein Mass Spectrometry Core Facility for Neuroscience would enable NINDS- and other NIH funded neuroscientists at New York University to identify from one to many hundreds of proteins of interest, to characterize posttranslational modifications of these proteins such as phosphorylation, glycosylation, proteolysis and modification by lipids, to perform functional proteomics studies to identify the proteins involved in key signal transduction processes in neurons, and to characterize protein-protein interactions by surface plasmon resonance. Research areas that would greatly benefit from this core facility include nerve growth and regeneration, neuromuscular junction formation, cancer metastasis, amyloidosis and cerebral hemorrhagic stroke, axon guidance, axon domain assembly, lesion induced synaptic plasticity, potassium channel function, Alzheimer's and other amyloid diseases, brain development, opioid addiction, aging, and neurotransmitter receptor trafficking.

[unreadable] DESCRIPTION (provided by applicant): The NYU Protein Analysis Facility within the Skirball Institute at the NYU School of Medicine is dedicated to ensuring that biomedical scientists have access to cutting edge mass spectrometry technology to enable their research. We are requesting funds for a triple quadrupole (Q TRAP) mass spectrometer to make possible the specific and sensitive measurements of quantities of target proteins and peptides involved in many important diseases. These targeted multiple reaction monitoring (MRM) experiments would allow for the discovery and verification of protein and peptide biomarkers for the early detection of diseases such as lung, ovarian, breast, and prostate cancers, as well as cardiovascular and neurodegenerative diseases. In addition, the requested instrument would enable time course MRM experiments that would greatly extend the findings of preliminary stable isotope labeling (SILAC) experiments for the study of intracellular signal transduction in neurons and other cells. These experiments would greatly increase our understanding of the causes and mechanisms of a number of neurodegenerative, developmental, and neuromuscular diseases, as well as diabetes and HBV infection. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): ATP-sensitive potassium [KATP] channels in the heart muscle and coronary myocytes couple cellular metabolic status to membrane excitability, thereby contributing to the regulation of tissue responses to physiological and pathophysiological stimuli. In the heart muscle, opening of KATP channels participate in the stress response and protect against ischemic episodes. In the coronary vasculature, K(ATP/NDP) channels contribute to the regulation of basal flow as well as responses to metabolic impairment (hypoxic dilatation and ischemic reactive hyperemia). We found glycolytic enzymes to associate with KATP channel subunits, We hypothesize that glycolytic enzymes are integral components of the KATP channel macromolecular complex and that glycolytic enzymes regulate KATP channel activity under physiological and pathophysiological conditions, both in the cardiac myocyte as well as in the coronary smooth muscle and endothelium. In a first Specific Aim, we will investigate the hypothesis that enzymes of the glycolytic complex are associated with the KATP channel. Using co-immunoprecipitation assays we will investigate the specificity of interaction of glycolytic enzymes with individual KATP channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B). Co-immunoprecipitation assays of native proteins will be performed to investigate interactions under physiologically relevant conditions. Protein interactions will also be investigated using advanced proteomic approaches (ICAT & ITRAQ), which has the potential to uncover additional novel KATP channel interacting proteins. In a second Aim, we will examine the hypothesis that physical interaction of glycolytic enzymes with KATP channel subunits is required and that channel modulation occurs because of altered nucleotide levels in the microenvironment of the channel complex. This will be accomplished using mutant KATP channel subunits (lacking interaction with glycolytic enzymes or altered nucleotide sensitivity). In a final Aim, we will investigate the interaction of glycolysis and KATP channels in the context of ischemic protection in cardiac myocytes and the coronary vasculature. To this end, we will utilize our genetic mouse models that express dominant-negative K(ATP) channel subunits specifically in the cardiac myocyte, smooth muscle or endothelium. Our findings may have important implications for understanding the role of KATP channels in the heart and coronary vasculature under physiological and non-pathophysiological conditions.

The NYU Cancer Institute Proteomics Core Facility provides state-of the-art biological mass spectrometry analyses to cancer researchers at NYU to help them identify and characterize proteins of medical importance. The facility has identified many tens of thousands of proteins from SDS-PAGE gels, often at subpicomolar levels, by LC-MS/MS of proteins followed by MASCOT or BLAST homology database searching. The facility has also characterized posttranslational modifications such as phosphorylation, acylation, and glycosylation. In addition to its technical expertise, one of the strengths of the facility is its ability to advise clients in the design and interpretation of experiments so that useful data can be obtained and meaningful information can be had from these data. Reliance on cutting edge technology and its expert and dedicated staff allow these services to be performed in a cost effective manner. Indeed, many experiments by investigators at NYU and the NYU Cancer Institute could not have been done without the assistance of the facility. Recently the Facility has established a Clinical Proteomics Core for the detection of protein and peptide biomarkers for the early detection of cancer.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this program is to asses if proteomics technology is sufficiently robust and reproducible to be useful for biomarker discovery and verification. In the first phase of the project the focus was to develop metrics and SOP's to assess and maximize inter-lab reproducibility. In the second phase, a large number of clinical samples will be analyzed to discover and verify cancer biomarkers.

DESCRIPTION (provided by applicant): The NYU Mass Spectrometry Core Facility for Neuroscience, the 100 Women in Hedge Funds Clinical Proteomics Facility and the NYU Protein Analysis Facility within the Kimmel Center for Molecular Biomedicine at the Skirball Institute at the NYU School of Medicine are dedicated to ensuring that biomedical scientists have access to cutting edge mass spectrometry technology to enable their research. We are requesting funds for a Thermo Electron LTQ-Orbitrap Velos mass spectrometer to make possible the accurate and sensitive detection, identification, characterization and relative quantitation of proteins and peptides involved in many important diseases for 11 NIH-funded major users and 5 minor users. In addition, the requested instrument would enable stable isotope labeling (SILAC) experiments for the study of intracellular signal transduction in neurons and other cells. These experiments would greatly increase our understanding of the causes and mechanisms of a number of neurodegenerative, developmental, infectious, and neuromuscular diseases and cancer.

? DESCRIPTION (provided by applicant): Low-grade gliomas (LGGs) are primary brain tumors affecting young adults. Although they initially grow relatively slowly, they eventually transform t more aggressive high-grade gliomas, leading to neurologic deterioration and death. The questions of cell of origin and significance of known mutations in early low-grade gliomagenesis remain unanswered, thus limiting the ability to develop sensitive detection methods and new therapies. In up to 80% of LGGs, gain-of-function mutations are found in the gene encoding the cytosolic isoform of Isocitrate DeHydrogenase (IDH), usually due to an arginine to histidine substitution at position 132 (R132H). While the wild-type enzyme normally functions to convert isocitrate to a-ketoglutarate (aKG), R132H-IDH1 catalyzes the production of R-2-hydroxyglutarate (2HG), which leads to genome-wide epigenetic modifications that may be related to tumorigenesis. The putative role of IDH1 mutations in gliomagenesis has been supported by in vitro observations in human astrocytes and glioma cells, as well as the fact that patients with Ollier disease, which is due to mosaic germline mutations of IDH1, occasionally develop gliomas. LGGs bearing IDH1 mutations are predominantly located in the frontal lobes in close proximity to the frontal horns of the ventricular system. Because the subventricular zone around the lateral ventricles is an area of active neurogenesis, we postulate that the cell of origin in IDH1-mutated LGGs is a component of the neurogenic niche in the subventricular zone. Mouse models of brain-specific mutant IDH1 expression suffer from perinatal lethality and fail to show tumorigenesis. To test the hypothesis that mutant IDH1 represents a driver alteration in LGG initiation and to investigate which brain cells are predisposed by mutant IDH1 to undergo oncogenic transformation, we propose a new approach that overcomes limitations associated with in vitro and in vivo mouse models. Our strategy makes use of human embryonic stem cells to inducibly express R132H-IDH1 in specific cell types within the human neural lineage: neural stem cells, neuroblasts, astrocytes and oligodendrocytes. We propose to test the hypothesis that inducible expression of R132H-IDH1 alters the self-renewal, differentiation potential, proliferation rate, metabolome and epigenetic/transcriptional profile of neural stem cells or other components of the human neural lineage in vitro. Furthermore, to test the hypothesis that mutant IDH1 expression in specific human neural cell types contributes to initiation of LGG formation, we will transplant these target cells expressing R132H-IDH1 into the mouse brain and assess their ability to form invasive tumors. The proposed research will address the important question of whether IDH1 mutations in human embryonic stem cell-derived neural lineages alter cellular physiology and facilitate oncogenic transformation. Successful completion of this project will lead to a disease model, which will allow detailed analysis of the metabolome, epigenome and transcriptome of early human LGGs. Furthermore, such a model can be used for translational applications, such as high-throughput drug screening and biomarker identification.

PROJECT SUMMARY/ABSTRACT The biomedical research community is becoming increasingly aware of the importance of small molecule metabolites in the control of cellular activity in health and disease. Acquisition of the requested ThermoFisher Scientific Q Exactive Plus quadrupole-Orbitrap mass spectrometer would bring for the first time to the New York University School of Medicine the ability to perform cutting- edge LC-MS/MS analyses of small molecules for biomedical research. The requested instrument would be indispensible for the proposed research projects of 13 NIH-funded major users and several NIH-funded additional users. These projects include studies of the effects of microbial metabolites on intestinal diseases, the link between exercise-induced metabolites and human brain, studies of cardiac protection during stress, the role of catecholamines in communication between the brain and bone marrow in the regulation of hematopoiesis, studies on neurotransmitter function in modifying neural circuits to influence perception and especially on the role of oxytocin and its metabolites on maternal behavior, studies of the role of glia in neural development and disease, studies on acyl CoA, acyl carntitines, ceramides, diacylglycerides, and triglycerides in the heart and kidney as influenced by various interventions used to treat heart disease, for studies on the metabolism of pancreatic cancer, and studies into the molecular causes of other cancers, retinal degeneration, immune disorders, Barth Syndrome and diabetes. !