Marc Jay Glucksman, PhD - US grants

Mammalian zinc metalloendopeptidase EC 3.4.24.15 [EP 24.15] activity is crucial to the formation and degradation of many bioactive peptides. This enzyme has been localized in vivo, and its cDNA cloned and sequenced. The importance of EP 24.15 is demonstrated with the decapeptide gonadotropin releasing hormone [GnRH] (also known as luteinizing hormone releasing hormone), the pivotal neuropeptide regulating mammalian reproduction. Cleavage by EP 24.15, renders GnRH inactive, and is the rate limiting step in its extracellular processing and degradation. EP 24.15 also metabolizes small peptide substrates such as bradykinin, substance P and neurotensin and generates enkephalins from precursor proteins. Recently, this enzyme has also been implicated in the physiology of nociception, blood pressure regulation and pulmonary responsiveness. Therefore, elucidating the function and structure of EP24.15 may yield clues to the pathophysiology of certain diseases, and act as a paradigm for understanding the regulation of neuropeptides and other peptide hormones by a peptidase. Specific objective addressed in this project are: Which critical residues are involved in the enzyme's catalytic mechanisms, active site and substrate specificity? What is responsible for a small substrate preference, and can this be altered? Site-directed mutagenesis and enzyme assays will be performed. Circular dichroism analysis of wild type/mutants as well as intrinsic fluorescence in the presence and absence of the denaturing agent urea will confirm that attenuated activity is not due to changes in global protein conformation (non-native folding). What are the physiological effects of clinically relevant analogues of GnRH on EP 24.15 activity and can a well characterized EP24.15 inhibitor be found, and what is the effect of these EP 24.15 inhibitors upon GnRH degradation? Can structural information in EP 24.15 aid in designing new pharmacologically active agents? The pharmacopoeia defined could be used to develop nonsteroidal managed male and female contraceptives and be used in the treatment of such diverse disorders as sterility, endometriosis, sex-steroid dependent mammary and prostate cancers, and precocious puberty. What is the atomic structure of EP 24.15? How will this information direct future rational drug design? Structural determination will use homologous metalloprotease modelling, X-ray diffraction experiments, simulated annealing analysis and macromolecular simulations of EP 24. 15, inhibitors and GnRH analogues. The realized goals of this research proposal will substantially contribute to the strategy of an integrated approach of biochemical, theoretical, and structural methods to study enzyme-ligand interactions of EP24.15 in neuroendocrinology. While aiding future rational drug design, these results should also be applicable to other macromolecules.

DESCRIPTION(Adapted from applicant's abstract): The overall objective of this application is to elucidate the biochemical functions of E.C.3.4.24.15 [EP24.15], the prototypical member of the mammalian family of endopeptidases that process/modulate neuropeptides. EP24.15 is present in brain, pituitary, and gonads, and has been directly implicated in critical physiologic pathways and neuroendocrine/neurodegenerative disorders and cancer. This enzyme processes neuropeptides extracellularly and thus is crucial in regulating signal transduction events essential to cell growth and differentiation. Understanding the function of EP24. 15 and its interaction with substrates and inhibitors will provide unique insight into aspects of normal and disease states and will aid in the design of new therapeutic agents for a variety of disorders. Experiments in this application will integrate biochemical, genetic, theoretical, structural and cell biological approaches to studying the enzyme-ligand interactions of this metalloenzyme. These strategies are also applicable to other macromolecules. Therefore, the interrelated specific aims of this project are: Specific Aim 1. Determinatlon of elements responsible for substrate binding and catalvsis in EP24.15 EP24. 15 protein will be functionally characterized utilizing structural genetics and kinetic determinations. Aim la. What amino acid deterrninants are responsible for the catalysis of neuropeptides? Aim lb. Which residues comprise the substrate [and inhibitor] binding subsites of the enzyme? Specific Aim 2. Define components modulatingr EP24. 15 activitv. localization. and substrate size selectivity. 4.15 is phosphorylated in vitro/in vivo, present in the nucleus, and though an endooligopeptidase, can cleave some large substrates. Aim 2a. Determine the effect of phosphorylation upon EP24. 15 enzyme activity. Aim 2b. What are the structural determinants of the enzyme which translocate it to the nucleus? Aim 2c. EP24. 15 is defined as an 'endooligopeptidase'. What is the mechanism of size selectivity? Specific Aim3. Modeling and higher resolution studies of EP24. 15 Structural biological approaches: molecular modeling of homologous domains and diffraction analyses. Aim 3a. Build a prototype from bacterial homologues for this class of mammalian enzymes . Aim 3b. Execute multiwavelength anomalous diffraction analyses of EP24. 15.

DESCRIPTION (provided by applicant): Mass spectrometry (MS) is the choice method for complex protein sample analysis. This proposal requests the ABI QSTAR XL hybrid mass spectrometer. This vastly improved instrument contains the capabilities for both Matrix- Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) as well as an Electrospray Ionization Tandem Mass Spectrometer [ESI-MSn] to assist researchers at the Finch University of Health Sciences/Chicago Medical School (FUHS/CMS) in the areas of protein identification/characterization and proteomics/functional genomics. Currently, there is no MS capability at FUHS/CMS to satisfy the growing and urgent need for analysis of complex protein samples. The purchase of the hybrid mass spectrometer is being requested in conjunction with the establishment of the "Mass Spectrometry Core Facility", part of the Midwest Proteome Center, a unique infrastructure in the Chicago/Milwaukee region. Dr. Marc J. Glucksman, a recent recruit to CMS to establish a proteomics center, will head the MS facility. As described in the application, 14 investigators from 6 different departments on campus, funded by 7 different institutes at the NIH have a specific need for this equipment. Indeed, due to the lack of an instrument on campus, several investigators are currently forced to collaborate or contract with other institutions/companies to pursue their research goals, a situation often resulting in long delays, lower resolution data and hindered productivity. Thus, the purchase of the QSTAR XL mass spectrometer, in conjunction with the recruitment of additional faculty to establish the core facility, will have a major impact on biomedical research at CMS. A detailed organizational plan is described for efficient management and use of the instrument. The University has committed cost-sharing support for five years including the costs of setup, installation, equipment maintenance, support personnel, and a facility manager. The Principal Investigator has garnered world experts as members of the External Advisory Board of the facility, and an Internal Advisory Committee, as specified in the proposal. The establishment of this versatile MS core facility and the purchase of the instrument are essential to the continuing expansion and enhancement of the basic research programs on the FUHS/CMS campus. This proposed grant will allow these investigators and others to enhance their research in structural biology, neuro degenerative diseases, drug addiction, cardiovascular disorders, and infectious diseases with an in-house core analytical facility. The resulting work has practical application towards diagnosis, prevention, gene product discovery and validation.

The objective of this proposal is to investigate the substrate binding, isoforms, associated proteins, and substrate size specificity of E.C.3.4.24.15 (EP24.15), with the goal of availing design of new therapeutic drugs and small molecules, to modulate development of neuroendocrine/neurodegenerative diseases and endocrine cancer(s). EP24.15 is the prototypic member of the metalloendopeptidase family, and is widely expressed in the brain, pituitary, and reproductive organs. Extracellularly, EP24.15 cleaves and modulates neuropeptides, and thus EP24.15 plays a significant role in neuroendocrine hormone regulation, pain perception, development of neurodegenerative diseases, and prostate cancer cell growth. The goal of this proposal is to further the understanding of the functions of EP24.15, of its isoforms, and of closely related metalloendopeptidases, with the goal of understandingthe prominence of EP24.15 in neuroendocrine/neurodegenerative diseases and cancer. Therefore, this proposal will integrate biochemical, genetic, structural, and proteomic approaches to study substrate binding, isoforms, associated proteins, and substrate size specificity of EP24.15. Therefore, the specific aims are: SPECIFIC AIM 1. Elucidate functional sites conferring substrate binding and catalysis. Aim la. Determine which residues comprise the substrate (inhibitor) binding site of EP24.15. Aim Ib. Determine which residues are crucial for substrate catalysis and specificity. SPECIFIC AIM 2. Determine isoforms and protein binding partners of EP24.15 and of related enzymes. Aim 2a. Identify the extracellular and intracellular isoforms of EP24.15 and EP24.16. Aim 2b. Characterize complexes of EP24.15 with associated protein-binding partners. SPECIFIC AIM 3. Unveil new substrates and mechanism of substrate size selectivity. Aim 3a. Determine what other (neuro)peptide substrates exist for EP24.15. Aim 3b. Elucidate structural changes upon substrate binding, focusing on domain movements.

DESCRIPTION (provided by applicant): The Midwest Proteome Center was created at the Rosalind Franklin University of Medicine and Science (RFUMS)/Chicago Medical School in 2003 to bolster the proteomics initiatives centered upon mass spectrometry. As a targeted technology to update our research capabilities in structural biology and proteomics, and to insure state-of-the-art instrumentation for a major expansion already underway, RFUMS has targeted the acquisition of a ThermoFisher LTQ Orbitrap Velos with Electron Transfer Dissociation (ETD) mass spectrometer. With the exception of FT/ICR instruments, the Velos has the highest mass accuracy, resolution and dynamic range on the market, coupled with multiple fragmentation methods. Mass spectrometry is now a routine structure analysis and identification tool with the Midwest Proteome Center the nexus of many biomedical research initiatives identified by the University. This proposal requests the LTQ Orbitrap Velos ETD mass spectrometer to assist fifteen major users, seven of which have recently received New/Early stage NIH funding, in all five of the basic science research departments, funded by ten Institutes of the NIH requiring protein characterization, identification and proteomics. Currently, our NIH supported users exceed the capacity and specifications of our much older equipment, some about which are to lose manufacturer support; the fundamental reason for this request. While several universities 1.5 hours south in Chicago (the Chicago Biomedical Consortium comprising U. of Chicago, Northwestern U., and U. of Illinois in Chicago only) have older machines with an instrument similar to this request, in Core Facilities or within individual labs, researchers at RFUMS have no access to this crucial technology. As detailed in the NIH project descriptions, the rapidly expanding demands of our NIH supported researchers, and newly hired faculty, require the increased mass accuracy, resolution, throughput and capabilities afforded by the Orbitrap Velos ETD to assist studies of infectious diseases, cocaine addiction, muscle development, pulmonary insufficiency, neural cell signaling, nuclear cell biology and structural biology, with adjunct proteomic approaches to many of these areas. The Midwest Proteome Center core facility obtained a QSTAR XL (QqTOF) in 2003 under the auspices of the S10 program, and provided substantial bang for the buck with supplemented funds from the institution. The seed planted by the NCRR 8 years ago yielded a bountiful harvest with respect to expansion of the instrument repertoire, increased user base, faculty hires obtaining New/Early Stage NIH funding and our established investigators availing themselves of cutting edge technology. The wide breadth of disciplines supported by this submission is at the crossroads of the NIH Roadmap. The proposed LTQ Orbitrap Velos ETD is designed to meet the diverse and realized research needs of users in virtually every basic science department and research center at RFUMS. PUBLIC HEALTH RELEVANCE: This submission will augment sponsored research by providing mass spectrometry support in diverse areas funded by ten Institutes of the NIH. Fifteen major users have been enrolled, half of them New/Early Stage Investigators with their first grants. Crucial areas of health research to be explored are: infectious diseases, cocaine addiction, muscle development, pulmonary insufficiency, cell signaling in the brain, nuclear cell biology and membrane structural biology.