Rajini Rao, Ph.D. - US grants

Na/H exchangers (NHE) control the movement of salt, water and acid-base equivalents, and play a critical role in pH regulation, cell proliferation, volume control and ion homeostasis. A wide range of clinical conditions are associated with derangement's in Na/H exchange, including hypertension, cardiac ischemia and various acid-base disorders. Although a number of plasma membrane NHE isoforms have been described in higher vertebrates, endosomal Na/H exchangers represent a physiological important sub-type that have yet to be identified at the molecular level. The-yeast NHE homologue, Nhx1, has been cloned and localized to a unique, late endosomal compartment, thus providing a starting point to explore the cellular and physiological role of intracellular Na/H exchange. Using a fully functional, epitope-tagged transporter and an array of secretory and trafficking mutants the cellular routing of Nhx1 from synthesis to degradation will be characterized. Further clues on function will derive from localization studies following exposure of cells to salt, osmotic stress and pH perturbations. Ion transport activity will be characterized in purified vesicles with respect to the reversibility of Na/H exchange, selectivity and affinity for ions, and sensitivity to a range of inhibitors, including the diuretic amiloride and its derivatives. These studies will form the foundation of studies designed to map the structure and function of the exchanger. In addition, NHX1 gene expression in response to a range of physiological signals and search for protein factors interacting with putative regulatory domains in promoter regions, and at the C-terminus of the exchanger. These approaches may lead to a better understanding of the signal transduction pathways involved in cell volume regulation, hypertonic response and pH homeostasis. Expression and localization of the human homologue of the yeast Nhx1 will serve as a starting point for understanding the function of a putative endosomal mammalian NHE.

Many important cellular and physiological events, including nutrient uptake, signal transduction and cell cycle progression are mediated by transmembrane ion gradients. An extensive, multigene family of cation pumps, the P-ATPases, have evolved to transport a wide variety of different ions (Ca2+, Na+, K+, H+, Mg2+, Cu2+, to name a few). In keeping with their essential roles, the P-ATPases are a target for pharmacological intervention in disease (such as congestive heart failure and stomach ulcers), and are defective in various inherited disorders (Menkes, Wilson, Brody and Hailey-Hailey disease). Despite the similarities in sequence, structure and mechanism within this family, individual members differ strikingly in ion selectivity. The molecular basis of selectivity in ion pumps remains one of the fundamental unanswered problems in the field of membrane bioenergetics. To approach this problem, we will: focus on the Golgi Ca2+, Mn2+-ATPase, Pmrl, in the genetically tractable organism yeast, apply simple and powerful phenotypic screens that will identify loss of function or selectivity mutations, develop rigorous biochemical tools to analyze the defective pumps. In Aim 1, we will identify the molecular determinants of divalent cation selectivity in yeast Pmrl, a founding member of the newly- defined subgroup of Golgi/secretory pathway Ca2+-ATPases. Specifically, we will focus on selectivity for Mn2+ versus Ca2+ ions. In one approach, we will use directed and random mutagenesis techniques in conjunction with biological assays for Ca2+ chelator and Mn2+ toxicity to identify mutations that alter ion selectivity. In a second approach, we will use homology modeling of yeast Pmrl, based on the known crystal structure of the SERCA pump, to design rational targets for mutagenesis. Target residues will include those predicted to line the ion conducting pathway, stabilize adjacent membrane helices or form domain interfaces. In Aim 2, loss-of-function mutants with interesting properties such as alterations in ion selectivity or uncoupling of ATPase hydrolysis from ion transport, will be further mutagenized and subjected to phenotypic selection in order to identify intragenic suppressor mutations. These will provide unique insight on critical interactions between domains, and within or between membrane helices, that will complement structural information on ion pumps. In Aim 3, large-scale purification of Pmrl from fermentor-grown Pichia pastoris cultures will be undertaken for structural studies on cation binding and the concomitant conformational changes. Taken together, these aims constitute a powerful approach toward deciphering the molecular basis of selectivity and transport in ion pumps.

[unreadable] DESCRIPTION (provided by applicant): Na+/H+ exchangers control the movement of salt, water and acid-base equivalents, and play a critical role in pH regulation, cell proliferation, volume control and ion homeostasis. A wide range of clinical conditions are associated with derangements in Na+/H+ exchange, including hypertension, cardiac ischemia and various acid base disorders. Although a number of plasma membrane NHE isoforms have been described in higher vertebrates, endosomal Na+/H+ exchangers represent a physiologically important sub-type that have only recently been identified at a molecular level. We have cloned, expressed, and localized novel endosomal exchangers from yeast, Nhx1, and from human, NHE6, as a starting point to explore the cellular and physiological role of intracellular Na+/H+ exchange. We will begin by determining the transport characteristics of human NHE6, including ion selectivity, pH sensitivity and pharmacological profile, using 22Na transport and pH-sensitive fluorescent indicators targeted to the endosomal lumen (Aim 1). These studies will establish whether NHE6 serves as a H+ leak pathway, to limit vesicular acidification and sequester osmotically active ions, as has been reported for endosomal exchangers from yeast and plants. The molecular determinants of ion binding and transport in exchangers remain largely unknown. We will examine structure-function relations in Nhx1, as a model of the NHE family, using phenotype screening in the genetically amenable model organism, yeast (Aim 2). A combination of directed and localized random mutagenesis will be used to target membrane helices believed to be important for transport. Loss-of-function in plasmid-encoded mutants will be identified by assessing salt or hygromycin sensitivity in yeast strains engineered to lack chromosomal Nhx 1. Second site mutations that confer regain-of-function are likely to reveal inter- and intra-domain interactions between side chains. These studies will help define the ion transport pathway. The C-terminal domains of Nhx1 and NHE6 are smaller and more divergent than those of other NHE, suggesting that they may interact with distinct regulatory proteins for endosome-specific functions. We have identified several candidate proteins by yeast 2-hybrid approaches and will pursue detailed studies or them to determine a functional role (Aim 3). In summary, we propose to elucidate cellular function and mode of regulation of two novel members of a physiologically important, clinically relevant and mechanistically interesting family of transport proteins by drawing on the combined and complementary strengths of yeast and mammalian cells in culture.

The goal of this proposal is to elucidate the pathway of programmed cell death by a novel antifungal agent and explore its use as an antimycotic adjunct. Amiodarone is an effective antiarrhythmic drug that was recently discovered to have potent and broad range fungicidal activity. We have shown that amiodarone toxicity in the yeast Saccharomyces cerevisiae is mediated by disruption of calcium homeostasis, followed by the appearance of apoptosis markers and cell death. In Aim 1, we propose to use a combination of biochemical and cell biological approaches to determine the identity and temporal order of events leading from the initial burst of cytosolic calcium to cell death. We will seek to validate key findings in the pathogenic yeast Candida albicans as proof-of-principle for the universality of the fungicidal mechanism of amiodarone. In Aim 2, we will use a variety of genome-wide approaches to identify the genes and signaling pathways that contribute to amiodarone-induced cell death. Genes identified by these studies will be organized into biomodules and placed in cellular pathways by integrating experimental data from high-throughput biochemical assays with information from online databases to give a global view of drug toxicity. We have shown that low doses of amiodarone exhibit potent synergism with existing antifungals against pathogenic fungal species of Candida, Cryptococcus and Aspergillus. Drug synergy from these in vitro studies will form the basis for combination therapy that will be explored in a murine model of systemic Candidiasis (Aim3). Taken together these studies will develop the calcium-mediated cell death pathway as a major new drug discovery target opportunity. Amiodarone will serve as a model test compound for targeting this pathway and for validating the potential of a new class of antifungal potentiating agents. The public health relevance of this project arises from the emergence of new fungal pathogens and drug resistant fungi, and the urgent need for alternative strategies in the management of fungal infections.

[unreadable] DESCRIPTION (provided by applicant): Secretory Pathway Calcium-ATPases (SPCA) are a newly defined family of ion pumps that transport calcium and manganese into the lumen of the Golgi apparatus where they are essential for sorting, processing, and glycosylation of proteins. The first member of this family, named PMR1, was described in Saccharomyces cerevisae, and more recently, two mammalian homologues of PMR1, SPCA1 and SPCA2, have been identified. Mutations in hSPCAl cause Hailey Hailey disease, a debilitating disorder characterized by severe ulceration of the skin, thought to result from dysregulation of cellular calcium. Excess manganese is deposited in the brain and leads to Parkinsonism. The SPCA have been implicated in diverse physiological processes, ranging from manganese detoxification in the liver, calcium transport across intestinal epithelia, and milk production by the mammary glands, although there is little molecular evidence for their specific roles. This proposal combines three parallel approaches to investigate the SPCA: biochemical studies using purified proteins or Golgi membranes, cell biological studies in polarized cultured mammalian cells, and large scale phenomic analysis of ion homeostasis in a model organism. In previous studies, we have defined the transmembrane helices and residues critical for ion transport and have identified a role for helix packing in determining ion selectivity. In Aim 1 of this proposal, we will shift our focus to understanding the ion binding and modulatory role of EF motifs in the cytoplasmic N-terminal domain. We have new evidence for trafficking of the pumps between the Golgi stacks and a novel, vesicular compartment in polarized cell models of hepatocytes and enterocytes. In Aim 2, we will determine if trafficking is ion-dependent and related to transcellular transport, and whether specific retrieval or PDZ-binding motifs at the C-terminus are important for localization. Gene knockdown approaches will be used to evaluate the isoform-specific contributions of the two SPCA pumps in the enterocyte model. Finally, we propose to use the yeast model organism for high resolution phenomic analysis that will identify new genes and pathways associated with the cellular function of the SPCA pumps (Aim 3). Taken together, this proposal lays the foundation for understanding the role of this novel family of transporters at the molecular, cellular and physiological level. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Na+/H+ exchangers of the NHE superfamily mediate the transmembrane exchange of cations with protons to regulate salt, pH and water homeostasis. We have uncovered an evolutionarily ancient subgroup of endosomal NHE that includes yeast Nhx1 and mammalian NHE6, 7 and 9. In yeast, Nhx1 localizes to the late endosome where it regulates luminal pH to control vesicle trafficking and delivery of the multivesicular body (MVB) to the vacuole for degradation. In mammals, the MVB pathway is important in HIV biogenesis, drug detoxification, erythrocyte maturation, and protein degradation. Defects in this pathway are likely to lead to lysosomal storage disorders and concomitant neurological and kidney dysfunction. Inhibitors of endosomal NHE offer a therapeutic potential to offset defects in endosome acidification seen in Dent's and Fanconi disease, and as antiviral agents. The goal of this proposal is to extend our understanding of yeast Nhx1 function and extrapolate our findings to mammalian cells. In Aim 1, we will use a combination of yeast genetics, biochemical assays of trafficking, and electron microscopy to define the precise pH-dependent step in lysosomal biogenesis. In parallel, we will test the hypothesis that NHE6 and/or NHE9 localize and function in MVB bodies in a mammalian cell culture model. In Aim 2, we will evaluate synthetic variants of exoporide, a novel amiloride analog, to find a selective inhibitor of intracellular NHE. An immediate goal of this proposal is to complete ongoing studies that seek to derive a global view of the role of cation/proton exchange by Nhx1 (Aim 3). To this end, we will continue our analysis of the genetic basis for pH regulation (pHome) and identify genes and cellular pathways that interact with Nhx1 (phenome). In Aim 4, we will assess an emerging homology model of NHE, based on the crystal structure of E. coli NhaA, using structure- bioinformatics driven mutagenesis in conjunction with phenotype screening in yeast. These studies will focus on defining the molecular basis for differences in ion selectivity and inhibitor sensitivity between the intracellular and plasma membrane subtypes of NHE, provide insight into the mechanism of transport by the NHE superfamily, and serve as a template for the design of novel NHE inhibitors. In summary, we propose a multidisciplinary approach that targets the function and mechanism of a clinically and physiologically important family of membrane transport proteins. PUBLIC HEALTH RELEVANCE This proposal targets a newly discovered but evolutionarily ancient family of ion transporters that regulate the movement of salt, water and acid equivalents across the boundaries and compartments of all cells. We plan to define the function of these proteins using parallel approaches in yeast and cultured mammalian cells, and identify new drugs using a novel screening strategy. These drugs may offer therapeutic benefits in kidney storage diseases (Dent's and Fanconi), and against envelope viruses such as HIV.

DESCRIPTION (provided by applicant): The Graduate Program in Cellular and Molecular Medicine (CMM) was created in 1993 and offers highly qualified Ph.D. candidates the opportunity to learn the theory and practice of modern cellular and molecular biology while conducting laboratory research on problems with direct clinical relevance. CMM is an independent Graduate Program at the Johns Hopkins School of Medicine with separate admissions, a special curriculum, and is certified to award the degree of Doctor of Philosophy (PhD). Now in its 16th year, CMM matriculates -18-25 incoming students selected from ~200 applicants every year. A total of 98 Students (as of May 2008) have graduated from the Program. CMM faculty members are from clinical departments and basic science departments. They were selected because of their successful independent laboratory programs, NIH funding, and their suitability to serve as mentors for young scientists. Importantly, CMM faculty pursues research at cellular and molecular levels on human diseases including: cancer;cardiopulmonary and vascular disorders;neurobiology and neurological disorders;immunological and infectious diseases;metabolic, developmental, and genetic defects. The goal of CMM is to train young scientists for careers studying human diseases at cellular and molecular levels. We expect that most CMM graduates will take academic positions in medical schools pursuing research in clinical departments, while some may choose to pursue research in industry. RELEVANCE: Rapid progress in cellular and molecular biology has strongly impacted on clinical medicine, offering insights about the fundamental causes of many diseases. Now new discoveries in the laboratory can be applied rapidly to the diagnosis, treatment and prevention of disease. The trainees in this program are working precisely at this interface between science and medicine to contribute to the long term well being of society.

DESCRIPTION (provided by applicant): The 2014 Gordon Research Conference on Membrane Transport Proteins will bring together approximately 150 investigators from diverse scientific disciplines, including structural and computational biology, genetics and medicine, cell biology, biochemistry, pharmacology, and bioengineering, to focus on the newest developments and challenges in the field relating to Structure, Function, Physiology and Targets in Disease. The GRC has a unique tradition of bringing together top notch established and junior investigators whose fields do not normally intersect, in a collegial and informal setting that maximizes exchange of unpublished and cutting edge science. Many interdisciplinary and inter-continental collaborations have stemmed from these meetings. The conference, to be held for the 9th time on July 13-18, 2014 at Mount Snow Resort, West Dover (VT), has a highly interactive format with ample time for poster viewing, informal gatherings at meals and social excursions that foster a collaborative and stimulating environment. A major goal of this conference will be to integrate exciting new technological advances with a wealth of emerging functional information for a deep, mechanistic understanding of human pathologies including neurological disorders ranging from Autism to Wilson Disease. The conference, to be chaired by Professors Poul Nissen (Aarhus University, Denmark) and Rajini Rao (Johns Hopkins University, Baltimore), will cover the spectrum of membrane transporters, from anion channels to cation/proton exchangers, transient receptor type channels, ion pumps, neurotransmitter transporters and lipid flippases and will incorporate molecular structure, physiology and disease relevance into each session. Included in the highlights will be the role of disease associated genetic variants in transporters, translational research using clinical studies or transgenic anima models, optogenetics and photoswitchable ligands controlling membrane transporters, and new atomic structures of important transporter families both from X-ray crystallography and electron microscopy. Each session will include both invited speakers as well as junior investigators selected from the abstracts. An opening session on hot topics will feature the most recent findings and a final keynote session will highlight the newest developments of optogenetics that at the same time summarizes the conference and the field and points to the future. A concerted effort will be made to be inclusive of women, minorities and persons with disabilities. To support the international aspect of the conference it has unanimously been voted earlier to alternate between European and U.S. venues. This generates an ideal platform for new interactions and collaborations at an international level as well as the exploration of possibilities for both mentos and trainees.

Project Summary Hypertension is a major health problem affecting more than 1 in 4 adults in the United States and is an independent risk factor for heart and kidney failure. Approximately 95% of cases are idiopathic and classified as essential hypertension. Increased Na+-Li+ countertransport (SLC) is a well-characterized inheritable trait and known marker for essential hypertension and diabetic nephropathy. SLC represents an alternative mode of Na+/H+ exchange and correlates with sodium transport in the renal tubule. Recently, our laboratory identified NHA2, a novel sodium proton (Na+/H+) antiporter that mediates SLC, and is expressed in the distal nephron of the kidney. NHA2 is a member of a phylogenetically distinct and uncharacterized branch of the superfamily of metazoan cation proton transporters that includes the well-known NHE family of transporters. In Aim 1, we will generate and test model structures of NHA2 based on similarity with bacterial and archaeal orthologs of known structures in outward and inward facing conformations. The predictive power of the structural models, combined with functional screening of transport phenotypes in yeast, will be tested against a database of human variants and patient electronic medical records in an innovative PheWAS approach to link genotypes to disease phenotypes. Our preliminary observations implicate NHA2 in renal cyst formation and point to a potential role in nephrogenic diabetes insipidus, a major complication in patients prescribed lithium for bipolar and other neurological disorders. Therefore, in Aim 2 we will use renal epithelial cell models and 3-dimensional cysts to determine the role of NHA2 in salt and pH homeostasis. Modern Western diet is high in sodium and is known to be associated with hypertension. In preliminary experiments, we have observed elevation of NHA2 transcript and protein in mice fed a high salt diet. Experiments in Aim 3 will investigate the salt induction of NHA2 in the kidney and the role of NHA2 in salt and pH homeostasis by extrapolation to animal models. We will directly test whether NHA2 is responsible for SLC activity in red cells from mouse and human. This proposal will provide the first mechanistic and functional insights on a novel human Na+ transporter and its relevance to hypertension and human disease.

? DESCRIPTION (provided by applicant): The mission of the training program in Cellular and Molecular Medicine is to prepare PhD scientists for laboratory research at the cellular and molecular level on topics with a direct impact on the understanding, diagnosis, treatment and prevention of human diseases. Through a series of tailored courses, small group discussions, clinical Grand Rounds and laboratory research, PhD graduates of this program are provided a rigorous training in scientific research and a thorough knowledge of human biology and human diseases. Consistent with the emphasis on translational research, most CMM faculty work in clinical settings, distributed over 23 clinical and basic science departments and 4 institutes within the Johns Hopkins School of Medicine and School of Public Health. Trainers are rigorously evaluated according to criteria of merit, mentorship ability and program fit, and the faculty: student ratio of 1 is maintained by an active process of mentor recruitment, renewal and turnover to ensure the best training environment for our PhD candidates. The first year curriculum begins with the intensive Introduction to Human Body course that combines hands-on dissection of the human cadaver, virtual histology labs, in-class and e-lectures, and small group presentations by students. Following a rigorous curriculum in principles in molecular biology, genetics, biochemistry and cell biology, accompanied by three 10-week laboratory rotations, the year ends with Cellular and Molecular Basis of Disease that covers a spectrum of clinically and/or socially relevant disorders of the human body, also arranged by organ systems, completing the cycle of the year from book to bench to bedside. Other program- specific activities include a practical Grant Writing course and clinical Grand Rounds in the second year. The program sponsors an Annual Retreat and career training opportunities in teaching and non-academic tracks. Most trainees graduate pursue research careers in academia (43%), medicine (15%) or industry (27%). Currently in its 20th year, CMM supports trainees in their first year of graduate training. In this renewal, funds are requested 2 additiona training slots for a total of 17, to accommodate increased applications from an outstanding pool of training grant eligible candidates, including underrepresented groups.

The 2016 Gordon Research Conference on Membrane Transporters titled ?Translating Molecules to Medicine? will bring an urgently needed translational focus to research on clinically relevant and highly druggable membrane transporters. Although they account for 10% of the human genome and contribute to a gamut of highly prevalent (autism, diabetes) as well as orphan and rare diseases (Mucolipidosis type 4, Christianson syndrome), membrane transporters remain largely untapped in their potential as therapeutic targets. The nine scientific sessions are highly interdisciplinary, bringing together topics and techniques that are not conventionally related: these include stem cell technology, 3D organoids, virtual mining of the chemical universe for inhibitors, and single molecule dynamics, as examples. Ample discussion time after each talk, daily poster sessions and afternoon free time allow plenty of opportunity for informal discussion, networking and forming new collaborations. A special Pharma session will help build consortia between industry and academics to tackle challenges in the drug development pipeline. The Keynote session will feature the GRC Alexander M. Cruickshank Lecture awardee, Dr. Nieng Yan, whose recent landmark high-resolution structures of human glucose transporters have captured the challenges and opportunities of this membrane transporter field. Approximately 150 participants will be drawn from industry, academia, research institutions and government and will span all career levels, ranging from early career investigators (graduate students, postdoctoral fellows) projected to comprise 50% of attendees, to mid-career and senior scientists and professors. This mix offers an ideal opportunity for vertical and peer mentoring, and networking, essential for the future health and productivity of this field. For the first time, we will host a pre-conference Graduate Research Seminar organized by, and exclusively for, young investigators. The GRS will include poster sessions, short talks and a Careers Panel with invited leaders from pharma, academia and publishing sectors, with a goal of fostering scientific and professional development and instilling confidence and leadership skills in student trainees and fellows. The organizers are committed to promote diversity and inclusion of women, minorities and persons of disability. To this end, our goal is to achieve gender parity (50%) in speakers and discussion leaders, and to increase the visibility of all underrepresented groups. Another innovative feature of this conference, the Power Hour, is a forum designed to address challenges that women face in science. To foster international reach, participants have elected to alternate the location of the conference between the US and Europe. Most recently, the conference was held in Vermont, and in 2016 the venue is Italy. The GRC provides seed money and negotiates costs directly with the conference site, to keep expenses at a minimum. This application seeks partial funding to cover registration costs for invited speakers, including women and minorities, and young investigators.