Stephan Wilkens - US grants

DESCRIPTION (Adapted from abstract): Vacuolar H+-ATPase represents a ubiquitous class of ATP driven proton pumps found in the membrane of subcellular compartments of eukaryotic cells. The function of the vacuolar ATPase is to acidify the interior and at the same time energize the membranes of organelles such as clathrin coated vesicles, endosomes, lysosomes, chromaffin granules and Golgi derived vesicles. Very little is known about the catalytic mechanism of this multi subunit complex, mainly due to the lack of detailed structural information. This deficit exists despite the fact that a wide variety of human diseases as fundamental as cancer and osteoporosis are associated with an abnormal activity of the human vacuolar type ATPase. The applicant is studying the three-dimensional structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles by electron microscopy in conjunction with image reconstruction methods. His immediate goals are: 1) to obtain a three dimensional model of the vacuolar ATPase, and 2) to define the arrangement of the subunits within the complex by using monoclonal antibodies to tag the corresponding polypeptides. A longer-term objective is to image the complex at different stages in the catalytic cycle in order to resolve the large conformational changes that accompany catalytic turnover and energy coupling.

A grant has been awarded to Dr. James G. Baldwin at The University of California, Riverside (UCR) for a new transmission electron microscope (TEM). The research and teaching needs of this growing campus and scheduling problems on the existing microscope will be met by adding this new TEM. The new microscope is highly complementary with the existing TEM and both instruments will be housed and administered together in the campuswide Central Facility for Advanced Microscopy and Microanalysis (CFAMM) administered within the College of Natural and Agricultural Sciences (CNAGS).

This instrument provides functionality needed by experts, yet simplicity appreciated by students and less experienced microscopists. The new microscope will be scheduled for projects that leverage its ease-of-use for diverse TEM work done by many biology researchers and students that do not require high accelerating voltage nor analytical capabilities. Flexibility and scheduling conflicts will be further addressed by equipping the microscope with an electronically controlled rotating stage (required by many Cell, Molecular and Developmental Biology [CMDB]) investigators, e.g. those using stereo pairs) and digital image capture required for most CMDB scientists and crucial for cost effective printing, storage, 3D reconstruction, and easy interfacing with the adjacent Center for Visual Computing (CVC) with which it will be linked by an existing fiber optics system. A new vacuum evaporator is also awarded and is vital for sample preparation for the electron microscope.

UCR is the most rapidly growing UC campus, with the highest representation of minority students in the UC system and among the highest nationally. New faculty hires and new laboratory construction are underway to accommodate a 100% increase by 2010, with particular growth in life sciences. The new TEM is essential for research and teaching programs to keep pace with the rapidly expanding interdepartmental program in CMDB and to address a worsening scheduling crisis on the only existing modern TEM on campus and shared by both life and materials scientists. For a significant group of Federally-funded UCR researchers in the life sciences, TEM is an indispensable tool, complementary to a range of additional techniques for acquiring structural information at the cellular and sub-cellular level. These include special applications in cell biology that are relevant to basic science as well as agriculture, biomedicine, pathogenesis, mode of action of antibiotics, and exploring character changes in evolution. UCR's existing CFAMM, interfaced with the CVC, provides a unique context for the new TEM dedicated for life science/CMDB research and training. The new TEM will be the primary graduate and undergraduate TEM teaching tool for seven existing courses, and the PIs of this proposal will collaborate to develop a new training course specific to TEM techniques centered around the new instrument at the CFAMM.

DESCRIPTION (provided by applicant): P-glycoprotein (Pgp; also called multidrug resistance protein) is found in the plasma membrane of higher eukaryotes where it is responsible for ATP-hydrolysis-driven export of hydrophobic molecules. In animals, Pgp plays an important role in excretion of and protection from environmental toxins. When expressed in the plasma membrane of human cancer cells, Pgp can lead to failure of chemotherapy by preventing the hydrophobic chemotherapeutic drugs from reaching their targets inside the cells. Pgp is a member of the superfamily of ATP binding cassette (ABC) transporter proteins. ABC transporters consist typically of four domains, two nucleotide binding domains (NBDs) located in the cytoplasm and two trans-membrane domains (TMDs) responsible for drug binding and transport. Despite its important role in human disease, relatively little is known about the structure of Pgp. We are using electron microscopy of two-dimensional crystals to study the structure of Pgp. The immediate goals of this proposal are 1) to visualize the structural changes Pgp is undergoing during the catalytic cycle, 2) to calculate a three dimensional model of Pgp trapped at the different steps during ATP hydrolysis and drug transport and 3) to optimize the conditions under which we currently generate two dimensional crystals of Pgp. The structural studies will be conducted with Pgp crystallized in its native environment, the lipid bilayer. A three dimensional model of Pgp will serve as a basis for resolving the structural changes which Pgp is expected to undergo during the drug transport cycle. Understanding these structural changes might ultimately aid in the design for specific inhibitors for the protein in order to be able to regulate the activity of Pgp for a more effective chemotherapy.

[unreadable] DESCRIPTION (provided by applicant): Vacuolar ATPases (V-ATPases; V1VO-ATPases) are large, multi subunit protein complexes found in the endomembrane system of eukaryotic organisms where they function to acidify the interior of subcellular compartments. In polarized cells of higher eukaryotes, the vacuolar ATPase can also function in the plasma membrane in order to pump protons to the outside of the cell. The proton pumping action of the vacuolar ATPase is involved in a large number of intra- and inter cellular processes such as receptor mediated endocytosis, protein trafficking, pH homeostasis, storage of metabolites and neurotransmitter release. Given its widespread nature, it is not surprising that more and more diseases as fundamental as diabetes, cancer, osteoporosis and AIDS are found to be associated with a defective human vacuolar ATPase. We are studying the structure of this important enzyme by electron microscopy, X-ray crystallography and solution nuclear magnetic resonance spectroscopy as well as other biophysical techniques. In the first funding period for this project, we have generated three-dimensional structural models of the mammalian- and yeast vacuolar ATPase. Furthermore, by using difference mapping, immuno labeling and fitting of X-ray crystal structures, we have been able to determine the subunit architecture of the V-ATPase complex. We are now proposing to extend our structural studies and to use a variety of independent techniques to obtain high resolution structural data for the mammalian- and yeast vacuolar ATPase and for the vacuolar like ATPase from Archaea. Furthermore, we propose to test a number of hypotheses regarding the mechanism by which the ATPase driven proton pumping activity of the vacuolar ATPase is regulated in vivo. The specific aims for this competing continuation proposal are: 1) to investigate the structure and function of the peripheral stalk(s), 2) to elucidate the mechanism of activity silencing in the V1-ATPase domain 3) to determine the subunit architecture of the yeast VO domain by electron crystallography. From the high resolution structural data for the vacuolar ATPase will hope to better understand the catalytic mechanism of ATP hydrolysis powered proton pumping and the mechanism of reversible dissociation/reassociation by which the enzyme's activity is regulated in vivo. [unreadable] [unreadable] [unreadable]

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 vacuolar ATPase (V-ATPase) is a multi subunit rotary motor enzyme that functions as an ATP hydrolysis driven proton pump in the endomembrane system of eukaryotic cells. The V-ATPase consistes of two motor domains, a cytoplasmic ATPase (V1) and a membrane bound proton channel (V0). The two domains are linked by three peripheral stator proteins that function as a structural link to counteract the rotational torque that is generated during ATP hydrolysis. We have recently obtained a 3-D reconstruction of the intact V-ATPase from yeast (Zhang et al., JBC 283, 35983) and we are now using X-ray crystallography to determine the atomic resolution structures of V-ATPase subunits and subunit domains for fitting into the EM derived map. We have crystallized the peripheral stalk forming subunits of yeast V-ATPase (subunits E&G) in complex with a domain of subunit C. A preliminary diffraction analysis performed at the Chess beamline F1 (Fall 2009;in collaboration with Dr. Edward Berry) resulted in ~5.5 [unreadable] diffraction. The crystals belong to spacegroup P212121 with unit cell parameters of 95.2, 114.1, 133.9 [unreadable] , 90,90.5,90[unreadable][unreadable]. Currently, there is no crystal structure available for the peripheral stalk(s) of the V-ATPase (or any of the related rotary ATPases including the F1F0-ATP synthase or the archaeal A-ATPase). SInce last fall, we have optimized crystallization conditions including a 96 condition additive screen, leading to numerous conditions with varying crystal morphologies. We will use the beam time at Chess, if approved, to screen crystals for high quality diffraction and to collect native data sets if time and crystal quality permits. Subsequent work, for which a full proposal is planned, would include heavy metal soaks and/or SeMet containing protein. Molecular replacement may be possible as a crystal structure for subunit C is available.

DESCRIPTION (provided by applicant): This application requests funding for an 800 MHz nuclear magnetic resonance (NMR) spectrometer dedicated for life science research in the Upstate New York area. The proposed instrument will support the research programs of 9 NIH funded major users (holding a total of 11 NIH research grants in support of the described projects) as well as 7 significant minor users (holding a total of 7 grants from the NIH and other federal agencies). The group of users includes researchers from SUNY Upstate Medical University, Syracuse University and SUNY ESF, all located in Syracuse, NY as well as researchers from Cornell University, Ithaca, NY, University of Rochester, Rochester, NY and the Hauptman Woodward Institute, Buffalo, NY. Research areas supported by the proposed instrument include, but are not limited to, protein and RNA structure, folding and interactions. The biomolecules studied in the user's laboratories are involved in a variety of processes relevant to human health and disease such as energy transduction, RNA structure and mRNA splicing, development of protein and RNA based biosensors, oncogenesis, neuronal function and neurodegeneration, endocrine and exocrine signaling, regulation of histone gene transcription, and membrane transport. There is currently no 800 MHz instrument near the participating institutions so that researchers with a need for very high field NMR applications have to bring or send samples to one of the national facilities such as NMRFAM in Madison, WI or the Pacific Northwest National Labs in Richland, WA. The relatively long turnaround times as well as the cost for traveling and the difficulties with shipping sensitive samples limit the efficiency of these offsite alternatives, creating an ever-greater need for a local facility with esy access. The instrument for which funding is requested is forefront both regarding sensitivity and spectral resolution, thus allowing the analysis of structure, dynamics and interactions of proteins and nucleic acids beyond a size range of what is currently possible with the available instrumentation. Funding is also requested for a 5 mm cryogenically cooled probe for ultimate sensitivity allowing the analysis of samples with low abundance or poor solubility. The requested instrument is equipped with four channels for conducting triple resonance experiments with simultaneous decoupling of deuterium for analyzing (partially) deuterated samples. The instrument will be housed in the existing NMR facility at SUNY College of Environmental Science and Forestry (SUNY ESF), where it will be managed and maintained by the facility manager, Mr. David Kiemle. Mr. Kiemle has managed the SUNY ESF NMR facility for the past 25 years and has extensive experience with NMR analysis of bio macromolecules as well as maintenance and repair of modern NMR spectrometers. Acquisition of an 800 MHz NMR spectrometer will provide the Upstate New York region with a state-of-the-art instrument that will accelerate NIH funded biomedical research in the areas of structural biology and drug design, research areas that are essential if we want to find ways to fight human disease on a molecular level.

An award is made to State University of New York College of Environmental Science and Forestry (ESF) to acquire a field emission scanning/transmission electron microscope (FES/TEM) with cryo-capabilities and elemental analysis using Energy Dispersive Spectroscopy (EDS). This instrument will replace a 30 year-old failing TEM in the shared-core N.C. Brown Center for Ultrastructure Studies at ESF. This project is a joint partnership of three adjacent universities, ESF, SUNY Upstate Medical University (UMU) and Syracuse University (SU). This new FES/TEM will provide these institutions with capabilities that are currently not available in central New York, and will expand the research capabilities of faculty and graduate students at these institutions, and provide support for competitive extramural funding. The NC Brown Center at ESF offers a unique academic program, a "Microscopy Minor" in central New York with graduate and undergraduate coursework and comprehensive formal training in the theory and application of microscopy such as: sample preparation, instrumentation and interpretation of results. This acquisition will enhance these academic offerings. In addition to its academic program, the Center routinely provides light, scanning and transmission microscopy demonstrations to community and industrial groups including outreach activities and demonstrations. This project will support such activities. The new FES/TEM will facilitate interactive, online high school and client access, with potential to provide interactive information with online demos. The new TEM enables the asbestos testing lab at ESF to become the only local laboratory to offer both phase contrast and TEM asbestos analysis for the asbestos remediation industry. Societal benefits of the project include raising scientific literacy of students and the public, providing students with skills for employment, providing potential for research that can result in wide-ranging impacts such as new vaccines or drug delivery systems for disease prevention, as well as environmental and industrial impact from the development of novel nanomaterials.

Acquisition of this cryo-capable field emission scanning and transmission (S/TEM) will permit advances within a wide-range of research groups at ESF, SU and UMU. Among these, at ESF: better tracking of measurable chemical changes and ultrastructure in fish ear stones due to environmental conditions, chemistry of both inorganic and organic nanoparticles, with potential use in targeted drug delivery, studies of wood cell wall degradation by fungi affecting forest trees and wood products, studies of insect vectors of human and plant diseases; at SU: this microscope will determine size and shape of semi-conductive quantum rods and alloy nanoparticles, atomic imaging and elemental inspection of atomic derived lattice planes and compositional gradients in alloy nanoparticles, S/TEM and EDS analysis of compositional changes in hetero-structured nanoparticles, S/TEM of stacking faults and extended defects in quantum dots, and analysis of protein and DNA modified nanomaterials; and at UMU: the proposed field emission electron source with its superior brilliance and beam coherence will produce high contrast images from frozen hydrated biological macromolecules at much better resolution than is now possible with the older machine. Such data will allow 3-D structure determination of ATP molecular motors. The high resolution 4K CCD camera will enable high throughput situations especially when thousands of images are collected for single particle or 3Dimensional reconstruction. The motorized goniometer will enable generating tomographic or tilted reconstructions of cryo sections of animal or plant cells revealing the internal structure and location of organelles, virus or nanoparticles as well as large drug molecules without the confusing artifacts created by chemical fixation. This microscope features a 2 nanometer resolution for examination of unstained samples, bacteria, viruses, proteins, nanoparticles and at the same time, map and locate elements. This new generation S/TEM will have a field emission gun, cryo-capable stage, Energy Dispersive X-ray Spectrometer, 4K CCD digital camera, electron diffraction, tomography software, and remote access. This instrument permits entry into the fields of protein folding, molecular motors, materials research, 3D reconstruction of single particles, cryo-imaging and elemental analysis. The FES/TEM with these options will enable visualization and reconstruction of subcellular particles, drug delivery vehicles, identification of elements in biological samples and nanoparticles, and localization of pharmaceuticals in target tissues by cryo-sections; all things that cannot be achieved with present instrumentation.

Project Summary The proton-pumping vacuolar ATPase (V-ATPase; V1Vo-ATPase) is an essential enzyme complex found in the endomembrane system of all eukaryotic organisms and in the plasma membrane of some animal cells. The V-ATPase functions in ATP hydrolysis-driven acidification of subcellular compartments or the extracellular space, a process vital for basic cellular processes including endocytosis, protein trafficking, bone remodeling, urine acidification, sperm maturation, and neurotransmitter release. Loss of V-ATPase in animal cells is embryonic lethal, while partial loss of function (or hyperactivity) has been linked to numerous human diseases, such as renal tubular acidosis, osteoporosis, diabetes, male infertility, neurodegeneration, cancer, and AIDS. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism, and regulation of the eukaryotic V-ATPase complex. In cells, V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, a condition under which the complex dissociates into V1-ATPase and Vo proton channel sectors that are both functionally silenced. Despite its important role in V-ATPase function, the molecular mechanism of activity regulation by reversible disassembly is poorly understood, a gap in knowledge that is largely due to the lack of high-resolution structural information and a defined in vitro model system to study the process under controlled conditions. The immediate goal of this project is to obtain high-resolution structural and mechanistic information aimed at a better understanding of V-ATPase regulation and to uncover non-canonical functions of the V- ATPase Vo membrane sector that may play a role in neuronal communication. We will address these questions with the following specific aims: (1) Molecular determinants of V-ATPase assembly and disassembly (2) Non-canonical functions of the V-ATPase Vo membrane sector. We study the structure and regulation of the yeast V-ATPase, a powerful model system for the mammalian enzyme due to the high level of conservation of the enzyme's structure and mechanism across species, the ease of genetic manipulation, and the ability to obtain highly purified enzyme with a defined subunit composition. The long-term objective of the project is to develop strategies aimed at either promoting or inhibiting the process of reversible disassembly of the human V-ATPase that will allow modulation of the activity of the enzyme in a tissue and subunit isoform dependent manner for therapeutic purposes.

Project Summary The vacuolar H+-ATPase (V-ATPase) is an essential proton pump that is exploited by cancer cells to promote proliferation, migration and drug resistance. In normal cells, this pump creates a defined pH in subcellular organelles that is essential for organelle communication and function, and thus, inextricably ties the V-ATPase to diverse fundamental cellular processes. In normal acid secreting cells, the V-ATPase is found on the plasma membrane where it pumps protons out of the cell, a process required for e.g. urinary acidification and bone resorption. The impact of V-ATPase function on various cellular processes is determined by the membrane on which the enzyme resides, therefore, in normal cells, its abundance and localization are tightly controlled. In many cancers, however, the V-ATPase is upregulated and mislocalized, an essential adaptation for cancer survival. Indeed, inhibition of the V-ATPase leads to suppression of metastasis, increased drug sensitivity and ultimately, cancer cell death. However, total loss of V-ATPase function is embryonic lethal and most of the enzyme's ~15 different subunits are expressed as multiple isoforms, imposing significant barriers to both the study and therapeutic targeting of the enzyme. Importantly, subunit a exists as four isoforms (a1-4), with differential tissue and (sub)cellular localization and recently, specific isoforms have been shown to be overexpressed and mislocalized in breast (a3, a4) and ovarian cancers (a2). Further, our preliminary data indicates that a4 is highly upregulated in renal cancers. The long term objectives of this work are to improve cancer patient outcomes by revealing novel targets for therapeutic development, namely subunit isoforms of the human V-ATPase. The immediate goal of the here proposed work is to generate a powerful new tool, single domain antibodies or Nanobodies (Nbs), for the study of specific V-ATPase populations. Nbs are derived from the unique heavy chain antibodies found in Camelidae and have many advantages over traditional antibodies. For example, Nbs are small, highly stable, and can be used intracellularly. We will use biochemical and biophysical methods, live cell imaging and cell based assays in the following Specific Aims: 1.) Generation and characterization of Nanobodies against subunit a isoforms of human V-ATPase and 2.) Nb mediated characterization and ablation of V-ATPase isoform a4 in kidney cancer. This project is aimed at overcoming the current limitations in the study of V-ATPase isoforms by developing novel and innovative tools, which will have highly transformative potential for the understanding of specific V-ATPase populations in human health and disease. At the end of the proposed research, we expect to have established Nbs as a powerful means to study isoforms of the V-ATPase and that implementation of these Nbs will illuminate the specific role of isoform a4 in promoting kidney cancer survival and malignant phenotypes. The information generated as a result of these studies will provide a firm foundation for developing novel therapeutics for subunit isoform specific targeting of V-ATPase populations in cancer.

Summary The proton-pumping vacuolar ATPase (V-ATPase; V1Vo-ATPase) is an essential enzyme complex found in the endomembrane system of all eukaryotic organisms and in the plasma membrane of some animal cells. The V- ATPase functions in ATP hydrolysis-driven acidification of subcellular compartments or the extracellular space, a process vital for basic cellular processes including endocytosis, protein trafficking, bone remodeling, urine acidification, sperm maturation, and neurotransmitter release. Loss of V-ATPase in animal cells is embryonic lethal, while partial loss of function (or hyperactivity) has been linked to numerous human diseases such as renal tubular acidosis, osteoporosis, diabetes, male infertility, neurodegeneration, cancer, and AIDS. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism, and regulation of the eukaryotic V-ATPase complex. In cells, V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, a condition under which the complex dissociates into V1- ATPase and Vo proton channel sectors that are both functionally silenced. Despite its important role in V- ATPase function, the molecular mechanism of activity regulation by reversible disassembly is poorly understood, a gap in knowledge that is largely due to the lack of high-resolution structural information and a defined in vitro model system to study the process under controlled conditions. The immediate goal of this project is to obtain high-resolution structural and mechanistic information aimed at a better understanding of V- ATPase's catalytic mechanism and unique mode of regulation. We will address these aspects of V-ATPase structure and regulatory mechanisms with the following Specific Aims: (1) Atomic structures of lipid nanodisc reconstituted Vo and V1Vo and mechanism of proton pumping, and (2), Molecular determinants of V-ATPase regulation by reversible disassembly. We study the structure and regulation of the V-ATPase from the yeast Saccharomyces cerevisiae, a powerful model system for the mammalian enzyme due to the high level of conservation of the enzyme's structure and mechanism across species, the ease of genetic manipulation, and the ability to obtain highly purified enzyme with a defined subunit composition. The long-term objective of the project is to develop strategies aimed at either promoting or inhibiting the process of reversible disassembly of the human V-ATPase that will allow modulation of the activity of the enzyme in a tissue and subunit isoform dependent manner for therapeutic purposes. 1

Project Summary Our laboratory has a long standing interest in understanding the catalytic and regulatory mechanism of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase), a dynamic multisubunit membrane integral rotary motor enzyme found in all eukaryotic cells. The V-ATPase acidifies the lumen of organelles and, in professional acid secreting cells, the extracellular space. Enzyme function is required for fundamental cellular processes such as endocytosis, bone remodeling, protein trafficking, acid-base balance, sperm maturation, and neurotransmitter release. While complete loss of V-ATPase function is embryonic lethal, partial loss or hyperactivity is associated with numerous human diseases such as osteopetrosis, diabetes, male infertility, neurodegeneration, and cancer. Moreover, some viruses such as influenza rely on the acidic environment created by the V-ATPase for infection. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism and regulation of the eukaryotic V-ATPase. In cells, V- ATPase activity is regulated by a unique mechanism referred to as ?reversible disassembly?, wherein the complex reversibly dissociates into V1-ATPase and Vo proton channel, with both sub-complexes becoming autoinhibited. Despite its important role in V-ATPase physiology, the molecular mechanism of reversible disassembly is poorly understood. This gap in knowledge is largely due to a lack of both high-resolution structural information and an in vitro model system to study the process under defined conditions, aspects that we are working to address. An interesting, and technically challenging feature of the mammalian V-ATPase is that most of its subunits are expressed as multiple isoforms. However, as such isoforms display differential tissue enrichment, they may provide opportunities for targeted therapeutics. Indeed, several diseases have been linked to malfunction or upregulation of specific isoform containing V-ATPase. However, how different isoform combinations determine tissue localization, and whether these isoform specific complexes have unique biochemical or regulatory properties, is currently unknown. We have started to develop a system to purify wild type and mutant forms of human V-ATPase in an isoform specific fashion for biochemical and structural analyses. Further, we are developing single-domain antibodies (Nanobodies) against specific subunit isoforms to serve as research tools, and to explore isoform specific modulation of V-ATPase activity in disease. Our research program employs the tools of structural biology, cell biology, biochemistry and biophysics to address broad questions of V-ATPase catalytic and regulatory mechanisms. For some fundamental aspects of V- ATPase structure and regulation, we study the enzyme from yeast, a well documented model system for the human V-ATPase. We use human tissue culture for questions that cannot be addressed in yeast, such as structure and biochemical properties of specific isoform containing enzymes. The long term goal of our research is to find ways to modulate the activity of disease causing V-ATPases in an isoform specific way.