1985 |
Simon, Sanford M |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Localization and Isolation of Calcium Channels |
1 |
1992 — 1993 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Protein-Conducting Channels in the Er Membrane
The translocation of proteins into and across membranes is an essential step in organelle biogenesis and protein topogenesis. The goal of this project is to understand the molecular mechanisms by which protein are integrated into or across membranes. The experiments focus on the regulation of protein-conducting channels that are observed in endoplasmic reticulum. The specific aims are to understand the regulation of the gating of this channel and to identify, purify and reconstitute the channel and its regulatory proteins. There are three steps of regulation that will be studied: What opens the channel to allow proteins to move across the membrane; what closes it after a protein has moved across; and, when it integrates a transmembrane protein, what opens the channel to the lipid bilayer thereby allowing displacement of latent transmembrane domains from the channel's aqueous environment into the hydrophobic milieu of the bilayer. The experimental approach is a hybrid of two techniques: reconstitution of membrane proteins into planar lipid bilayers and the reconstitution of protein translocation across membranes. Protein translocation is a membrane transport process. Planar lipid bilayers are optimal for studying such processes since they allow both complete control over the lipid and protein components as well as access for experimental manipulation and electrophysiological tools to both sides of the membrane. These tools are used to assay the status (open-close) of the channel. Three approaches are being used for identification and purification of the protein-conducting channels. (i) Protein translocation can be reconstituted after detergent solubilization of either pancreatic endoplasmic reticulum rough microsomes and E. coli inverted plasma membrane vesicles. Proteins can be fractionated prior to dialysis of the detergent to identify those necessary for protein translocation. (ii) Ribosomes are being used to purify integral membrane proteins of the endoplasmic reticulum that are linked to the ribosome via a nascent peptide chain. (iii) Proteins that are known to be essential for protein translocation (such as the six transmembrane proteins of the mammalian ER signal peptidase complex, yeast SEC 62, mitochondrial import receptors, E. coli prlA) are being purified and reconstituted into proteoliposomes to test if any of these (or a combination of these) are sufficient for activity of the protein-conducting channel. These are the first protein-conducting channels to be described. Similar techniques are being applied to the study of other "signal sequence" mediated protein translocations (the E. coli inner membrane, mitochondria, chloroplast). The goal is to identify general principles that may regulate protein topogenesis and organelle biogenesis.
|
1 |
1994 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Protein Conducting Channels in the Er Membrane
The translocation of proteins into and across membranes is an essential step in organelle biogenesis and protein topogenesis. The goal of this project is to understand the molecular mechanisms by which protein are integrated into or across membranes. The experiments focus on the regulation of protein-conducting channels that are observed in endoplasmic reticulum. The specific aims are to understand the regulation of the gating of this channel and to identify, purify and reconstitute the channel and its regulatory proteins. There are three steps of regulation that will be studied: What opens the channel to allow proteins to move across the membrane; what closes it after a protein has moved across; and, when it integrates a transmembrane protein, what opens the channel to the lipid bilayer thereby allowing displacement of latent transmembrane domains from the channel's aqueous environment into the hydrophobic milieu of the bilayer. The experimental approach is a hybrid of two techniques: reconstitution of membrane proteins into planar lipid bilayers and the reconstitution of protein translocation across membranes. Protein translocation is a membrane transport process. Planar lipid bilayers are optimal for studying such processes since they allow both complete control over the lipid and protein components as well as access for experimental manipulation and electrophysiological tools to both sides of the membrane. These tools are used to assay the status (open-close) of the channel. Three approaches are being used for identification and purification of the protein-conducting channels. (i) Protein translocation can be reconstituted after detergent solubilization of either pancreatic endoplasmic reticulum rough microsomes and E. coli inverted plasma membrane vesicles. Proteins can be fractionated prior to dialysis of the detergent to identify those necessary for protein translocation. (ii) Ribosomes are being used to purify integral membrane proteins of the endoplasmic reticulum that are linked to the ribosome via a nascent peptide chain. (iii) Proteins that are known to be essential for protein translocation (such as the six transmembrane proteins of the mammalian ER signal peptidase complex, yeast SEC 62, mitochondrial import receptors, E. coli prlA) are being purified and reconstituted into proteoliposomes to test if any of these (or a combination of these) are sufficient for activity of the protein-conducting channel. These are the first protein-conducting channels to be described. Similar techniques are being applied to the study of other "signal sequence" mediated protein translocations (the E. coli inner membrane, mitochondria, chloroplast). The goal is to identify general principles that may regulate protein topogenesis and organelle biogenesis.
|
1 |
1999 — 2002 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Opsin Biogenesis and Retinitis Pigmentosa
DESCRIPTION (Adapted from the applicant's abstract): The objective of this application is to understand how opsin is synthesized into endoplasmic reticulum (ER) membranes. The specific aims are to examine how the amino terminus of opsin crosses the membrane and how opsin integrates into the lipid bilayer. Each of these questions will be addressed both to wild-type opsin as well as some of its point mutants described in autosomal dominant retinitis pigmentosa (RP). Studying the pathologies in these mutants' biogenesis may yield insights into normal physiological pathways of opsin synthesis. Understanding the consequences of these mutations on the biogenesis of opsin may also contribute to our understanding of the pathogenesis of RP. Many of these mutations involve substitutions in regions of the opsin molecule that are critical to its biogenesis. Opsin biogenesis will be studied by trapping incremental stages of synthesis and quantifying: opsin targeting to the ER; translocation across the ER; folding of opsin; interaction with the translocation protein-conducting channels; interactions with ER resident proteins; and integration into the membrane. This application takes a multidisciplinary approach that combines using (1) truncated mRNA to study intermediates in biogenesis; (2) electrophysiology to study the protein-conducting channels; (3) purified reconstituted components of the ER to examine the interactions of opsin with the translocation machinery; (4) photo-activated cross-linkers in nascent opsin to identify ER components interacting with nascent opsin. This work is relevant to the larger class of clinically important polytopic membrane proteins, the G-protein coupled receptors, which share many structural features with opsin. Finally, identification and characterization of the topogenic signals within opsin itself, and a description of how they interact with the translocation machinery, provides a paradigm for understanding general membrane protein biogenesis.
|
1 |
1999 — 2001 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Non-Estrogen Recepetor Tamoxifen Activity
cell component structure /function; drug adverse effect; tamoxifen; organelles; acidity /alkalinity; pathologic bone resorption; protein transport; drug carcinogenesis; drug related neoplasm /cancer; N acylation; liposomes; cell type; lipid bilayer membrane; multidrug resistance; cell line; animal tissue;
|
1 |
2001 — 2002 |
Simon, Sanford |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Sger: Multicolor Total Internal Reflection Fluorescence Microscopy
0110070 Simon The primary goal of this proposal is to develop techniques to image two colors simultaneously at video rates (>30 frames/sec) so that either two different probes, or a single probe with two different emissions, can be imaged synchronously. Further, to develop the techniques for using multiple lasers for excitation. This goal will allow two different kinds of studies: First, it would allow multiple proteins to be studied in a single cell. This is important for addressing questions of protein-protein interactions. Second, it will allow many physiological studies using dual-emission dyes that can be used to quantify important cell signaling molecules such as calcium or pH. The spatial and temporal dynamics of these molecules are key for understanding many aspects of cellular signaling.
|
0.915 |
2001 — 2004 |
Simon, Sanford Muir, Thomas (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Biophotonics: in Situ Optical Imaging of Multiple Proteins
0119468 Simon The objective of the proposed research is to develop a method of optically imaging proteins. This research will adapt newly developed methods for peptide/protein ligation for labeling various proteins and imaging them via improved fluorescence or other molecular imaging probes. The ability to study proteins in situ has come from the introduction of the green Fluorescent protein (GFP). The goal of this research is to generate reporters that share the power of GFP, but without its limitations. The specific goals of this research are to generate reporters that: (1) can be genetically engineered into proteins, (2) are significantly smaller than GFP, (3) have a narrower emission spectra than GFP, (4) emit further in the red range or have a larger Stokes shift than GFP, (5) are more photostable than GFP, (6) can be used to simultaneously label and distinguish different proteins in living cells, and (7) can be used to track proteins or cells as they move throughout an animal's body.
|
0.915 |
2002 — 2003 |
Simon, Sanford M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Large Scale Chemical Screen Against Pathogenic Bacteria
DESCRIPTION (provided by applicant): Gram negative bacteria are surrounded by two membranes that shield them from the outside world. The viability of the bacteria depends upon the integrity of this membrane barrier. The pathogenicity of many gram-negative bacteria such as Yersinia, Salmonella, Shigella, Erwinia and pathogenic Escherichia coli depend upon their ability to export their toxins across these membranes without compromising the integrity of the membrane barrier. Considerable homology exists between the export pathways used for export of filamentous phage, type II secretion for the export of toxins or degradative enzymes into the extracellular milieu and type III secretion, in which proteacous toxins are secreted and injected directly into the cytosol of eukaryotic host cells, causing cytotoxicity. The export pathway for filamentous phage f1 forms a transmembrane aqueous channel ) through which the phage traverse during biogenesis. The opening of this export channel is tightly regulated. The channel is normally closed and only opens to allow the extrusion of filamentous phage (or in the case of type II or type III secretion, the export of toxin). If this channel opens inappropriately, the viability of the host bacteria is significantly compromised . The permeability of this channel can be studied with a colormetric assay that is compatible with a high-throughput screen for agents that could open these channels. These export channels only exist in pathogenic bacteria. They are encoded on the pathogenicity islands and are not part of the host bacterial genome. This is an application for a large-scale chemical screen for agents that affect the gating of these channels and open them. This could generate an agent that would selectively target a particular pathogenic bacteria and leave the rest of bacterial flora intact. Each of the export proteins from the gram-negative pathogens will be cloned and expressed in an E. coli system that has been optimized for the colorimetric detection of channels that are open. These E. coli will then be screened with a chemical library to detect agents that open these channels.
|
1 |
2003 — 2008 |
Simon, Sanford |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Biophotonics: Multicolor Total Internal Reflection Fluorescence Microscopy
0322867 Simon Some of the most intransigent, yet intriguing biological questions occur at cellular membranes. At the membrane the cell has to take up select substrates while maintaining a permeability barrier that keeps nutrients in and toxins out. The membrane is the site where cells secrete their own signals and detect those of their neighbors. The membrane is not only the site of a dynamic physiology but pathology as well. Many diseases of the nervous system affect either the ability of cells to secrete or detect signals at the cell membrane. For many pathogens our cells are a tempting home. They need to compromise the membranes to enter. However, they also need to maintain membrane integrity so our cells can survive as a hospitable environment. The cell membrane is 4 nm thick. Thus, the wavelength of light constrains our ability to detect changes occurring in the immediate environment of the membrane in the background of signals from the interior of the cell. Total internal reflection fluorescence microscopy (TIF-FM) is a technique that limits the excitation to a plane of 50-70 nm above a coverslip and has been successfully used to image events occurring within this space adjacent to the plasma membrane. This proposal will extend the capabilities of TIR-FM in three ways: 1) Allow multiple fluorophores to be followed simultaneously; 2) Allow simultaneous study of cell surface events by TIF-FM and internal changes by epi-fluorescence; 3) Allow localized photoactivation of signaling by UV while using TIR-FM.
|
0.915 |
2004 |
Simon, Sanford M |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Imaging Single Proteins in Vivo With Quantum Dots (Rmi)
DESCRIPTION (provided by applicant): The long-term goals of this research are to develop robust methods for tracking single proteins in living cells. Recently developed technologies of protein splicing will be used to ligate fluorescent nanocrystals [quantum dot (QD)] derivatives to select proteins in living cells. The availability of such cellular reagents, in combination with modem fluorescence microscopy methods such as to total-internal-reflection microscopy and spectral imaging, will allow insight on protein activity that would be difficult to obtain using macromolecular measurements where protein activities are averaged. There are three specific aims in the proposal: Aim 1: To extend and optimize recently developed in vivo protein trans-splicing and expressed protein ligation approaches to allow the ligation of suitable QD derivatives to either cytosolic and integral membrane proteins. In addition, we will develop a conditional protein trans-splicing approach that will allow probes such as quantum dots to be ligated to proteins following a designated functional interaction. This will allow the cellular fate of "activated" proteins to be monitored. Aim 2: To develop methods for preparing monvalent QDs capable of being ligated to a single copy of a target protein in cells. In addition, to develop a strategy, based on fluorescence quenching, that will allow the fluorescence properties of a QD to be linked to the in vivo trans-splicing reaction. This will allow the fluorescence of a protein-QD ligation product to be distinguishable from unreacted QDs. Aim 3: To apply in vivo protein ligation and quantum dots to single proteins in vivo. The technology will be established in the context of two systems of biological interest: exocytosis and transport through the nuclear pore. For each system there is a set of questions that require the tracking of individual proteins. For example, exploring whether protein movement through the pore is primarily diffusive, driven by thermal fluctuations, or deterministic, driven by a molecular motor. Longer-term strategic directions will include trying to develop the technology for chemically tagging interacting proteins and technologies for tracking proteins for longer time periods as they move in three dimensions.
|
1 |
2005 — 2008 |
Simon, Sanford M |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Imaging Single Proteins in Vivo With Quantum Dots
Th.e long-term goals of this research are to develop robust methods for tracking single proteins in living cells. Recently developed technologies of protein splicing will be used to ligate fluorescent nanocrystals [quantum dot (QD)] derivatives to select proteins in living cells. The availability of such cellular reagents, in combination with modem fluorescence microscopy methods such as to total-internal-reflection microscopy and spectral imaging, will allow insight on protein activity that would be difficult to obtain using macromolecular measurements where protein activities are averaged. There are three specific aims in the proposal: Aim 1" To extend and optimize recently developed in vivo protein trans-splicing and expressed protein ligation approaches to allow the ligation of suitable QD derivatives to either cytosolic and integral membrane proteins. In addition, we will develop a conditional protein trans-splicing approach that will allow probes such as quantum dots to be ligated to proteins following a dcsignatcd functional interaction. This will allow the cellular fate of "activated" proteins to be monitored. Aim 2" To develop methods for preparing monvalent QDs capable of being ligated to a single copy of a target protein in cells. In addition, to develop a strategy, based on fluorescence quenching, that will allow the fluorescence properties of a QD to be linked to the in vivo trans-splicing reaction. This will allow the fluorescence of a protein-QD ligation product to be distinguishable from unreacted QDs. Aim 3: To apply in vivo protein ligation and quantum dots to single proteins in vivo. The technology will be established in the context of two systems of biological interest: exocytosis and transport through the nuclear pore. For each system there is a set of questions that require the tracking of individual proteins. For example, exploring whether protein movement through the pore is primarily diffusive, driven by thermal fluctuations, or deterministic, driven by a molecular motor. Longer-term strategic directions will include trying to develop the technology for chemically tagging interacting proteins and technologies for tracking proteins for longer time periods as they move in three dimensions.
|
1 |
2006 — 2010 |
Simon, Sanford |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Biophotonics: Quantum Dots For Single Molecule Imaging in Live Cells
0620813 Simon The work in this proposal will optimize fluorophores for following single proteins in cells. This could significantly enhance the ability to study the roles of proteins in many different cellular reactions. There are some organic fluorophores that can be monovalently linked to proteins. Unfortunately, they are not photostable enough to be used for many biological studies. Quantum dots have significantly greater brightness and photostability. However, they aggregate and are multivalent in their linkages. This project will optimize the imaging techniques and designs of quantum dots to reduce aggregation and ensure that their linkages to proteins are monovalent.
|
0.915 |
2009 — 2012 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Selective Fluorescent Labeling of Proteins in Living Cells
DESCRIPTION (provided by applicant): Studies of molecular events inside of cells have provided novel and important insights into many questions in physiology and pathology: How do cellular motors work? How does a proton pump generate ATP? How is cargo secreted from cells? How do viruses enter a cell and, once in, how do they assemble. This application has three aims that will significantly advance ability to follow single molecular events in living cells. The first specific aim develops the technology for loading probes into cells. The technique is efficient and directly delivers probe into the cytosol rather than into the endocytic pathway. Of particular interest for us is the ability to deliver inteins. The second specific aim will generate inteins that will function inside of cells at physiological temperatures at faster rates and higher efficiencies. The third specific aim uses two biological questions to test the strengths and limitations of the results from the first three specific aims. The two questions relate to transport in and out of the nucleus: Can we follow the conformational changes of proteins in single nuclear pores and can we follow the transport of single molecules as they interact with the nuclear pore components and move through the pore. These questions are significant for understanding a fundamental process in cell biology. They will also provide insight as to further modifications that are needed in our technology for studying single molecular events in living cells. PUBLIC HEALTH RELEVANCE: Optical imaging has contributed very significant advances to our understanding of biology in the last few years. This is the consequence of a number of advantages of imaging: Imaging allows us to study biological systems that are still alive;Imaging allows us to study individual molecules, individual cells rather than the average behavior;Imaging allows us to study many different size scales from single molecules to whole individuals. Often a defect that occurs at the level of a single molecule represents itself as pathology at the level of the whole organism. Imaging allows us to follow the disease from the single molecule, to the molecular machine, to the whole cell, to the whole organ, to the individual patient. A major limitation of optical imaging has been the capability to get probes into cells to specifically and selectively label individual molecules. This application will contribute to our ability to study individual molecular events in the cell by giving us new methods of putting our probes into cells, without disturbing the cells and new methods of labeling cellular components with minimal, often no, effect on the cell.
|
1 |
2010 — 2013 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Biogenesis of Hiv 1 in Living Cells
DESCRIPTION (provided by applicant): By developing the ability to follow single viruses as they assemble, it is possible to identify and characterize discrete steps in viral assembly. The earliest stages of this work established the criteria to be used in determining if a single virion, in this case of HIV-1, is being observed. This was followed by establishing criteria for determining and characterizing the packing of various components at the plasma membrane, for example the packing of Gag, the recruitment of the genome. This current project carries the work to the next level of identifying many more partial reactions in the process of viral assembly: When is the genome recruited? What is the interaction between the genome and Rev for packing, between the genome and Gag for packing? What determines the ability of the membrane to bend outward - is it just packing of Gag or are there additional factors? When are does the protease become active, what determines the apparent specificity of cleavage steps? When does the virion septate from the cell and, how do each of these steps inter-relate? Must the membrane bend to a certain degree to attain a sufficient proximity for the protease to activate or is it sufficient for Gag to pack to a critical spacing? What are the role(s) of the host ESCRT proteins? Do they simply facilitate budding or do they accelerate the assembly rate? With these assays of assembly at the level of the single virion, it becomes possible to final describe and define the process of assembly. PUBLIC HEALTH RELEVANCE: Viruses are a major threat to human health. This work has developed the ability to follow single viruses assembling in living cells. By elucidating the procedure of assembly, in this case for HIV-1, it opens the possibility of targeting these steps for disruption.
|
1 |
2011 — 2014 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Analysis of Force Developed by a Aaa Atpase
DESCRIPTION (provided by applicant): The AAA ATPases family includes molecules whose roles include, but are far from limited to, biogenesis of mitochondria and multivesicular bodies, proteins in involved in gene regulation and protein transport. This project will focus on studying the activity of these machines at the single molecule level. The model protein will be ClpX - it is the part of the proteosome, a key effect of protein degradation that unfolds the proteins to prepare them for degradation. . Cellular proteins differ widely in their liability, from half-lives of minutes to days. Regulated degradation, by allowing rapid changes in the levels of cellular proteins, helps control signal transduction pathways, the cell- cycle, transcription, apoptosis, antigen processing, biological clock control, differentiation and surface receptor desensitization. The questions to be addressed are: How is work partitioned between alternative outcomes? What is the maximum work that can be performed by the system? What factors limit its efficiency in performing work? These questions have health implications: human pathological conditions are associated with failures of the degradation system and its regulation offers the potential for therapeutic intervention. Furthermore, an inhibitor of proteasome catalytic activity is in use for treatment of recurrent multiple myeloma, and proteasome inhibitors are in clinical trial for treatment of a broad spectrum of human malignancies. Thus, understanding the regulation of the half-life of proteins should provide critical insights into cell physiology and pathology. The mishandling of aberrant proteins incurs penalties throughout biology: the survival of bacteria subjected to stress depends on the effective performance of systems which deal with misfolded and structurally aberrant proteins- to either fold them properly or destroy them. The specific questions to be addressed are: How hard can the device pull to cause unfolding? How many pulls are needed to commit irreversibly? What is the limit of pulling power, and the statistical distribution of pulling power? Answers to these questions will begin to reveal not just what these machines do but the decision tree that describes how outcomes are controlled and when machine capacity may be exceeded. We want to know not just how the machine works, but how its decision tree yields alternative outcomes.
|
1 |
2011 — 2013 |
Simon, Sanford |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mri: Development of Polarization Control For Total Internal Reflection Fluorescence Microscopy
1126312 Simon
Total internal reflection (TIR) fluorescence microscopy allows high resolution imaging of membrane processes with very low background. Unique properties of TIR can lend themselves to interesting and unique quantifications. However there are obstacles in the way of robust quantification of TIR data. First, the excitation field created with TIR is non-homogenous due to interference fringes. These fringes, which are often of larger magnitude than the biological signals, result from interferences in the light source, the delivery optics, the objectives and the sample. This makes quantification of fluorescence intensities difficult. It is also difficult to control the excitation field polarization, particularly while correcting for interference fringes discussed above. Polarization based TIR allows for quantitative measurements of the levels, orientation and dynamics of proteins as well as membrane orientations. This group has shown that polarization can be a powerful tool for exploring the dynamics of molecules. The goal of this proposal is to advance quantitative TIR imaging by redesigning the illumination of the evanescent field and building a new microscope to allow: Improved uniformity of the TIR excitation field, control of the excitation beam, and through the image acquisition software control of the polarization of the excitation beam.
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0.915 |
2011 — 2016 |
Simon, Sanford |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Dynamics of the Nuclear Pore Proteins and the Mechanism of Transport
Intellectual merit. Nanoscale molecular machines found in a cell exceed any human creations in their intricacy and vastly exceed anything humans have created for efficiency. These are responsible for cellular functions that range from transporting objects across membranes to machines that synthesize, to motors that move cargo from one location to another. For a few of the smaller machines we have been able to merge our studies on the structure of these machines, with our observations on what the machine is doing and, as result, provide insight to how the machine works. Alas, our ability to analyze, and therefore understand, the larger, more sophistical biological machines. This project develops the computational tools to test how these machines work. The first model to be studied is the pore that regulates transport in and out of the nucleus across the nuclear envelope, the central part of the cell that guards our genome. Preliminary studies demonstrate that advances in computation can allow us to understand how these machines function at a molecular level with such great efficiency and speed.
Broader impacts. These results have impacts on several important levels. First, understanding the basic operation of the pores that guard the nucleus is an essential goal of cell biology. Which genes are turned on and off often depends upon which molecules are allowed to enter through these pores that surround the nucleus. Second, we need to fully understand how malfunctions of these pores can affect the normal state and integrity of the cell. Third, practical application of the engineering design principles used by cellular machines at a nanoscale can have tremendous economic and technological potential when applied and scaled to other problems. Nature has created nanomachines that vastly exceed in efficiency anything humans have created. Understanding how these nanomachines work can provide insights into our own technology. Fourth, this work makes science research accessible to a much broader community. The investigators have been working in the New York public school system for 35 years. Current involvement includes integrating high school teachers and their students in the laboratory over the summer, participating as a board member of the education committee of the New York Academy of Sciences, running monthly meetings for New York regional science teachers, as well as placing graduate students and postdoctoral fellows in middle and high schools in the New York area. Even though most high school students do not have access to high-end experimental tools, work such as this can be done on a home personal computer, thus making this science accessible to a broad population, which might not otherwise have the chance to pursue scientific research.
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0.915 |
2012 — 2016 |
Simon, Sanford M |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
The Center For Hiv Rna Studies (Crna) |
0.943 |
2016 — 2017 |
Simon, Sanford M |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Cellular Pathogenesis of Fibrolamellar Hepatocellular Carcinoma
PROJECT SUMMARY Fibrolamellar hepatocellular carcinoma (FLHCC) is an often-lethal disease affecting primarily children and young adults. The poor prognosis stems from both the lack of sensitive non-invasive diagnostic tests and the lack of a targeted systemic therapy. We have recently demonstrated a single, consistent deletion in one copy of chromosome 19 of FLHCC patients that results in a chimeric gene. This encodes a chimeric protein comprised of the amino terminal portion of the heat shock protein DNAJB1 fused to the active catalytic subunit of protein kinase A. This project will determine if this chimera is sufficient for transforming cells. It will then determine the extent of cellular changes upon induction of the chimera and begin to elucidate the pathways and mechanisms for these changes.
|
1 |
2016 — 2019 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single Virions to Study Assembly of Hiv-1
? DESCRIPTION (provided by applicant): The productive site of HIV-1 assembly is the plasma membrane, both in cultured cell lines and primary cells. This conclusion is based both on the observation that newly synthesized Gag first appears at the plasma membrane, and is only detected later in endosomes, and the demonstration that inhibiting endocytosis blocks the appearance of virions in the internal organelles without affecting the yield of extracellular particles This assembly of HIV-1 at the plasma membrane make the assembly accessible to imaging using total internal reflection fluorescence microscopy (TIR-FM) a technique on which a large fraction of the experiments in this proposal are based. This technique has been used to visualize individual virions of HIV-1 as they assemble as well as the movement and packaging of individual molecules or dimers of HIV-1 genome. The technique has been used to demonstrate that the virions accumulate at the plasma membrane over a period of 6-20 minutes. The genome is recruited to the plasma membrane immediately before recruitment of Gag. In contrast, ESCRTs are recruited for only tens of seconds and tens of molecules transiently at the very end of the recruitment of Gag. The AAA-ATPase, Vps4, is recruited just seconds later. Super-resolution optical microscopy has added the information that ESCRT recruitment is to the neck that links the nascent virion to the mother cell. The long-term goal of this project is to identify, characterize and ultimately understand the steps in the biogenesis of HIV-1 and related retroviruses. We want to understand the dynamics of viral components as they interact with each other and with host components. Imaging based approaches allow the examination of both the dynamics and localization of molecules during which may be inaccessible through biochemical techniques. The main foci will be on the dynamics and localization of the viral protein Gag, the dynamics and localization of host molecules and then the relative dynamics and localization of the viral and host molecules.
|
1 |
2019 — 2020 |
Simon, Sanford M |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Center For Therapeutic Targeting of the Fusion Oncoprotein of Fibrolamellar Hepatocellular Carcinoma
Project Summary / Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary tumor in children, adolescents and young adults. The primary tumor is initiated and driven by a single alteration in the DNA: A deletion of ~400kb that results in a fusion gene between the heat shock co-chaperone DNAJB1 and the catalytic subunit of protein kinase A, PRKACA. If the tumor is limited to the liver, then surgery is the accepted therapy. However, if the tumor has metastasized, there is no accepted therapy. This project will determine how the fusion oncoprotein leads to pathogenesis and will develop therapeutics targeted to the fusion oncoprotein. The first two projects will explore the pathogenesis: In project 1, what is different about the fusion oncoprotein that causes changes in the cell; In Project 2, how its expression in human organoids or in mouse tissue leads to a specific pathology in the liver. The next two projects will explore the therapeutics: In project 3 targeting to the fusion mRNA transcript with shRNA and anti-sense oligonucleotides and in Project 4, targeting the fusion oncoprotein through small molecules to block activity, or small molecules to send it to the proteasome, or small molecules for allosteric inhibition.
|
1 |
2019 |
Simon, Sanford M |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Targeting the Oncoprotein That Drives Flc
Project Summary / Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary tumor in children, adolescents and young adults. The primary tumor is initiated and driven by a single alteration in the DNA: A deletion of ~400kb that results in a fusion gene between the heat shock co-chaperone DNAJB1 and the catalytic subunit of protein kinase A, PRKACA. If the tumor is limited to the liver, then surgery is the accepted therapy. However, if the tumor has metastasized, there is no accepted therapy. Project 4 will develop therapeutics targeted to the fusion oncoprotein. It will use therapeutics to block the kinase activity of the oncoprotein and therapeutics to target the oncoprotein to the proteasome for destruction. The therapeutics will be tested on isolated protein, in dissociated cells from patient-derived xenografts, in human liver organoids of FLC and in mice with patient derived xenografts and in genetically engineered mouse models.
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2019 |
Simon, Sanford M |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Admin Core
Project Summary / Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary tumor in children, adolescents and young adults. The primary tumor is initiated and driven by a single alteration in the DNA: A deletion of ~400kb that results in a fusion gene between the heat shock co-chaperone DNAJB1 and the catalytic subunit of protein kinase A, PRKACA. If the tumor is limited to the liver, then surgery is the accepted therapy. However, if the tumor has metastasized, there is no accepted therapy. This center is focused on advancing our knowledge of how the fusion oncoprotein drives the cancer, the pathogenesis of the tumor and therapeutics that target the fusion oncoprotein. The Administrative Core is dedicated to facilitating communication and sharing of resources between the four projects, between the different investigators. Additionally, it will facilitate the flow of information and resources with the rest of the FusOnC2 consortium and the NIH.
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2019 |
Simon, Sanford M |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
What Differences Between the Chimeric Fusion Oncoprotein Dnajb1-Prkaca and Native Kinase Prkaca Lead to Transformation in the Childhood Cancer, Fibrolamellar Hepatocellular Carcinoma?
Project Summary / Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary tumor in children, adolescents and young adults. The primary tumor is initiated and driven by a single alteration in the DNA: A deletion of ~400kb that results in a fusion gene between the heat shock co-chaperone DNAJB1 and the catalytic subunit of protein kinase A, PRKACA. If the tumor is limited to the liver, then surgery is the accepted therapy. However, if the tumor has metastasized, there is no accepted therapy. Project 1 of the center will determine how the fusion oncoprotein leads to pathogenesis. Specifically what is different about the fusion oncoprotein that causes changes in the cell. The fusion oncoprotein that drives this childhood cancer is a kinase and this project will determine the direct targets for this enzyme as well as explore how changes in the structure of the fusion oncoprotein may affect its localization and its targets.
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2020 — 2021 |
Simon, Sanford M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Aso and Shrna For Targeting the Oncogenic Transcript Driving Fibrolamellar Hepatocellular Carcinoma
Fibrolamellar hepatocellular carcinoma (FLC) is a liver cancer that primarily affects adolescents and young adults. There are no known successful therapies for this disease and surgery is the only potential path to a cure. Once the disease has grown or metastasized to a point where surgery is no longer an option, a patient?s chance for survival approaches zero. Unfortunately, 65% of patients are diagnosed at stage IV. Our lab identified a recurrent genetic deletion in FLC cells, which has been found in almost all FLC tumor samples sequenced to date, but not in normal liver tissue from the same patients. The deletion encompasses ~400kb on chromosome 19 beginning after the first exon of DNAJB1, which codes for a member of the heat shock protein 40 (HSP40/DNAJ) family, and ends before the second exon of PRKACA, which codes for the catalytic subunit of protein kinase A (PRKACA). This results in a functioning chimeric kinase with exon 1 of DNAJB1 and exons two through ten of PRKACA (DNAJB1-PRKACA). We have shown that expression of this chimeric protein, but not the native kinase, in the liver of mice results in the formation of phenotypic FLC and lethal tumors. This strongly supports the notion that the DNAJB1-PRKACA chimera is the primary driver for this cancer. We have shown that the structure of the catalytic site of the native and fusion kinases are almost identical and it has been difficult to find blockers that selectively inhibit the fusion kinase. The goal of this research proposal is to develop a therapeutic for this devastating disease utilizing antisense and shRNA technology. This approach will allow us to specifically target the nucleotide sequence encompassing the junction of the fusion transcript, without affecting any of the native transcripts. Our approach is to 1) screen antisense oligonucleotides (ASOs) and shRNA with sequences that span this junction in an attempt to find the ASO or shRNA that results in the greatest knockdown of chimeric protein; 2) assess the effects of these ASOs and shRNA on the viability of FLC cells in vitro; 3) assess the efficacy of the ASO and shRNA to cause knockdown of the protein in the tumor cells in FLC patient-derived xenografts growing in mice; 4) assess the effects of the ASO and shRNA on the health of the mice, with a particular attention to liver toxicity, and 5) assess the efficacy of the ASO and shRNA to reduce tumor burden in mice.
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