Paul L. Sorgen - US grants

The long term objective is to test whether the high resolution structure of a multi-subunit protein complex can be determined by building up the complex from structures of its individual subunits and subcomplexes. We plan to investigate the membrane protein LH1 in detergent micelles by liquid NMR methods. LH1 is well suited for this study because it easy forms subcomplexes and individual subunits upon increasing the detergent concentration. Also, by comparing the structures of the individual subunits when isolated with those in subcomplexes, we will be able to test the two- stage model of membrane protein folding. To achieve these goals, the structure of the beta subunit, beta--BCh1 (B777) subcomplex, and alpha- beta-2BCh1 (B820) subcomplex will be determined by multidimensional NMR in detergent micelles. In addition, we will investigate the entire LH1 complex (B877) in detergent micelles by TROSY methods. The information from these experiments combined with biochemical constraints will be used to model the LH1 structure and ultimately determine if the structure of a multi-subunit complex can be determined from the sum of its parts. Finally, mapping the structural differences between the different I3 subunit association states will give insight into the two-stage model of protein folding.

? DESCRIPTION (provided by applicant): Gap junctions are integral membrane proteins that enable the direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. They provide a pathway for propagating and/or amplifying the signal transduction cascades triggered by cytokines, growth factors, and other cell signaling molecules involved in growth regulation and development. Dysfunctional intercellular communication via gap junctions has been implicated in causing many human diseases. The objective of this project is to use a multi-disciplinary approach to identify the key intrinsic regulatory mechanisms that are responsible for Cx43 and Cx45 function. The central hypothesis is that unique intermolecular interactions within the divergent CT domain of Cxs affect gap junction regulation. More specifically, we hypothesize that in the failing heart, Cx43CT phosphorylation alters protein partner interactions leading to remodeling of Cx43 from the intercalated disc, and that dimerization of Cx45 CTs is, in part, responsible for the channel properties of Cx45 that distinguish it from Cx43 and for the dominant-negative effect of Cx45 in heteromeric channels with Cx43. It is well-known that the CT domains of Cxs are key regulators of channel properties, and that dimerization of cytosolic domains are key regulators of ion channels. This proposal is significant because discovery of how interactions mediated by the CT domain can be modulated would open the door to strategies to ameliorate the pathological effects of altered Cx regulation in the failing heart. The following Specific Aims are proposed to investigate this concept: 1) Define how tyrosine kinases down regulate Cx43 gap junction intercellular communication, 2) Determine how Cx43 phosphorylation alters protein partner interactions, and 3) Identify the importance and mechanism of Cx45CT dimerization.

Affect; Area; Autoregulation; Binding; Binding (Molecular Function); Biological Models; Body Tissues; Brain; CRISP; Cell Communication and Signaling; Cell Signaling; Cells; Charcot Marie Disorder; Charcot Marie Muscular Atrophy; Charcot Marie Tooth Disorder; Charcot Marie Tooth muscular atrophy; Charcot-Marie Disease; Charcot-Marie-Tooth; Charcot-Marie-Tooth Disease; Charcot-Marie-Tooth neuropathy; Closure; Communicating Junction; Communication; Computer Retrieval of Information on Scientific Projects Database; Condition; Connexin 43; Connexin43; Connexins; Coupling; Cx43; Cytoplasmic Domain; Cytoplasmic Tail; Deafness; Defect; Disease; Disorder; Encephalon; Encephalons; Event; Funding; Gap Junction Proteins; Gap Junctions; Genes; Genetic Diseases, Inborn; Goals; Grant; Heart; Hereditary; Homeostasis; Human; Human, General; Inborn Genetic Diseases; Individual; Inherited; Inherited disorder; Injury; Institution; Intracellular Communication and Signaling; Investigators; Ischemia; Isoforms; Low-resistance Junction; Mammals, Mice; Man (Taxonomy); Man, Modern; Metabolic; Mice; Model System; Models, Biologic; Molecular; Molecular Interaction; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nervous System, Brain; Nexus; Nexus Junction; Organ; Pathway interactions; Peroneal Muscular Atrophy; Phosphorylation; Physiological Homeostasis; Protein Isoforms; Protein Phosphorylation; Proteins; Receptor Protein; Regulation; Research; Research Personnel; Research Resources; Researchers; Resources; Signal Transduction; Signal Transduction Systems; Signaling; Site; Source; System; System, LOINC Axis 4; Testing; Therapeutic; Tissue Preservation; Tissues; United States National Institutes of Health; Work; biological signal transduction; concept; design; designing; disease/disorder; gene product; genetic manipulation; inborn error; intermolecular interaction; particle; pathway; receptor; response

2-dimensional; Amino Acids; Asparagine; Auricle Helix; Binding; Binding (Molecular Function); C-terminal; CRISP; Calcium Binding; Cell membrane; Cells; Charge; Computer Retrieval of Information on Scientific Projects Database; Cytoplasmic Membrane; Endosomes; Funding; Grant; Helix; Helix (Snails); Helix of ear; Inositide Phospholipids; Inositol Phosphoglycerides; Inositol Phospholipids; Institution; Investigators; L-Asparagine; L-Phenylalanine; L-Proline; Lipids; Localized; Mediating; Membrane; Modeling; Molecular Interaction; N-terminal; NH2-terminal; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nuclear Magnetic Resonance; Phenylalanine; Phenylalanine, L-Isomer; Phosphates; Phosphatides; Phosphatidyl Inositol; Phosphatidylinositols; Phosphoinositides; Phospholipids; Plasma Membrane; Position; Positioning Attribute; Proline; Property; Property, LOINC Axis 2; Proteins; PtdIns; Receptosomes; Recycling; Research; Research Personnel; Research Resources; Researchers; Resources; Side; Source; Stretching; Tubular; Tubular formation; United States National Institutes of Health; aminoacid; ear helix; gene product; inorganic phosphate; membrane structure; plasmalemma; receptor recycling; two-dimensional

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long term goal of our work is to gain a structural and functional understanding of the mechanisms of gap junction regulation. Gap junctions, formed of proteins called connexins (Cxs), provide an intercellular pathway for the propagation of electrical/molecular signals, which are necessary for cellular differentiation, metabolic homeostasis, and in excitable tissue, electrical coupling. This type of communication permits individual cell events to synchronize into the functional response of an entire organ. Defects in human Cx genes that affect cell coupling are associated with a variety of inherited disorders (e.g. Charcot-Marie-Tooth disease and hereditary non-syndromic deafness). Genetic manipulations in mice have demonstrated the functional importance of Cxs in a variety of organs. Moreover, not only the presence but also the proper regulation of gap junctions is critical for homeostasis. For example, intracellular acidification leads to closure of gap junctions in all native tissues and exogenous expression systems tested. The study of pH-dependent regulation of gap junctions becomes even more relevant given that intracellular acidification is a major consequence of tissue ischemia. Acidification-induced uncoupling has an impact on the preservation of tissue surrounding the ischemic area. Therefore, we have chosen Cx43, the most widely expressed junction protein in the heart, brain, and other tissues, as our model system to study the structural regulation of Cxs. Our objective is to apply biophysical approaches to investigate intra- and intermolecular interactions that define the structural regulation of Cx43 during pH gating. We hypothesize that the Cx43 carboxyl terminal domain (CT) acts as a gating "particle" that, under the appropriate conditions (e.g. intracellular acidification or phosphorylation), binds to a "receptor" (i.e. Cx43 cytoplasmic loop;CL) affiliated with the pore and closes the channel. The following Specific Aims are proposed to investigate this concept: 1) To establish how the CT interacts with molecular partners that are involved in gap junction regulation;2) To assess the structural effect of pH on the CT and CL domains;3) To characterize cytoplasmic domain interactions between Cx isoforms. These Aims are designed to identify the functional consequences resulting from CT interactions with molecular partners and the CL in an effort to develop site-directed, specific modulators of gap junction communication with potential implications in therapeutic treatment of disease and ischemic injury.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Endocytic transport is a key mechanism for controlling the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its transport. Rab11, which regulates transport via the ERC, regulates the exit of cholesterol from this organelle. EHD1, a member of the C-terminal EH-domain family of proteins that regulates recycling and coordinates transport via the Rab11 pathway, has also been reported to play a role in free cholesterol transport through the ERC. To directly assess the role of EHD1 on cholesterol homeostasis and cellular distribution, we utilized mouse embryonic fibroblasts derived from EHD1 knockout mice (MEF -/-). Surprisingly, these cells displayed reduced levels of cellular free and esterified cholesterol, an effect that could be rescued by overexpression of wild-type EHD1. To understand the reduction in intracellular cholesterol in the absence of EHD1, we turned our focus to low density lipoprotein (LDL) and its receptor, LDLR, a major source of cellular cholesterol intake. We observed higher levels of LDLR on the plasma membrane of MEF -/- cells, yet LDL itself was internalized at a slower rate in these cells. Furthermore, in cells lacking EHD1, lipid droplets appeared greatly reduced in size, suggesting that less esterified cholesterol and triglycerides were packaged in lipid droplets. Two-hybrid binding assays and NMR spectroscopy suggest that the EH-domain of EHD1 interacts with Epsin, a well-characterized component of the endocytic pathway that contains NPF motifs and regulates receptor internalization. Our data indicates EHD1 affects cholesterol homeostasis and lipid droplet biogenesis by controlling internalization of LDL receptor, possibly through interactions with NPF-containing endocytic regulators such as epsin.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The main objective is to solve the structure of the EH-domain of the C-terminal Eps15 homology domain(EHD) protein, EHD1. We shall thus determine how it regulates endocytic transport and recycling of integrinand signaling receptors at the plasma membrane, and assess the impact of EHD1 on mitogenesis.A key cause of aberrant cell proliferation is the unchecked transduction of mitogenic signals by receptorslocalized to the plasma membrane. The internalization of growth factor receptors leads to attenuation ofstimulatory signals and receptors defective in endocytosis induce prolonged mitogenic effects. Subversion ofthe endocytic trafficking machinery is predicted to affect cell proliferation and lead to cancer. Recent studieshave also begun to address the endocytic itineraries of beta1 integrins, key receptors that interact with theextracellular matrix and coordinately transduce growth factor receptor signals that culminate in geneexpression, proliferation, and motility, thereby directly influencing cancer and metastatic potential.EHD1 is a central player in endocytic transport and recycling and we have recently demonstrated impairedb1 integrin recycling and localization to the plasma membrane as well as downstream events including cellspreading and migration in the absence of EHD1 (Jovic et al, manuscript submitted, Appendix I).Accordingly, the loss of EHD1 and/or its interactions with the network of proteins that regulate the endocyticmachinery disrupts normal cell function, and understanding the molecular basis by which the EH-domain ofEHD1 interacts with these regulatory proteins is a key goal of this proposal. Research in our laboratory hasidentified a novel function for EH-domains: direct binding to inositol phospholipids. However, the mode bywhich EH-domains interaction partners has yet to be determined. The proposed collaborative study will allowus to elucidate the molecular mechanisms of EHD1 function by NMR-based solution of the EHD1 EH-domainstructure, and provide novel insights into the functioning of this key protein and its regulation of integrins.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. TRAINING IN THE USE OF BRUKER AND VARIAN SPECTROMETERS AND NMR

DESCRIPTION (provided by applicant): Control of receptor localization to the plasma membrane is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. Receptor levels on the plasma membrane are dependent upon the rates of internalization and recycling back to the cell surface. We and others have recently identified a novel Eps15 homology (EH)-domain containing protein, EHD1, as a critical component of the endocytic recycling pathway. The physiologic importance of EHD-mediated function has been clearly demonstrated, as loss of EHD1 function leads to impaired recycling of many critical receptors. The long- term goals of this project are to understand the fundamental mechanisms controlling intracellular trafficking and transport of proteins to the plasma membrane, with emphasis on understanding the mode by which EHD structure impacts its function. The central hypothesis of this proposal is that the electrostatic surface charge of the EH-domain of EHD1 (EH-1) and its C-terminal EHD paralogs is responsible for the selectivity of interactions with NPF-containing protein partners and phosphoinositides, and that these interactions are important for the regulation of endocytic transport. We provide compelling evidence for our hypothesis based on our strong preliminary data that EH-1 selectively interacts with proteins containing NPF motifs followed by a cluster of negatively charged residues. In addition, our new preliminary data also reflect the functional importance of the EHD/phosphoinositide interactions by demonstrating that EH-1 is capable of interacting directly with phosphatidylinositol moieties, and that this interaction is required to allow localization of EHD1 to tubular and vesicular membrane structures. Our Specific Aims for the proposal are: 1) To identify the molecular and structural basis for the selectivity of C-terminal EH-domains for specific NPF-containing proteins, and 2) To elucidate the specific phosphoinositide(s) that are enriched/comprise EHD tubular membranes and determine the significance of EHD/phosphoinositide binding in the regulation of endocytic trafficking. The knowledge to be obtained from this study will provide critical new information on the mode by which the C-terminal EHD proteins associate with endosomal membranes and coordinate control of recycling. This will lead to a significantly enhanced understanding of the endocytic mechanisms that regulate receptor recycling, and will have an important bearing on health and disease. PUBLIC HEALTH RELEVANCE: Control of receptor localization to the plasma membrane is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. The knowledge to be gained from undertaking this proposal will lead to a significantly enhanced understanding of the endocytic mechanisms that regulate receptor recycling, and will have an important bearing on health and disease.

? DESCRIPTION (provided by applicant): Gap junction channels (GJCh) comprise the pathway for intercellular propagation of the electrical signals responsible for coordinated activation of cardiac contraction; heterogeneity and failure of GJCh function lead to heterogeneous slowing of conduction and consequent micro- or macro-reentry circuits that set the stage for tachycardia and fibrillation in the atria and ventricles. Heterogeneity and failure of GJCh function reflect acute, phosphorylation-dependent regulation of GJCh gating and conductance. This application focuses on the GJCh gating and conductance changes that are antiarrhythmic (preconditioning) and proarrhythmic, contributing to sudden cardiac death. The long-term goals of the project are to: 1) understand the mechanisms underlying phosphorylation-dependent regulation of Cx43 GJCh and hemichannel (HCh) function, and 2) develop from that knowledge peptidomimetics that will target or induce specific functional properties of Cx43 channels that will preserve cardiac rhythmicity and prevent irreversible injury. Two key issues limit our ability to implement Cx43 based therapies. First, we do not understand the consequences of differential Cx43 phosphorylation on GJCh function. Second, the impact of heterogeneous Cx43 phosphorylation on impulse propagation in the heart is unknown. Because these issues cannot be addressed in pairs of adult ventricular myocytes (due to heterogeneity of Cx43 phosphorylation state and the large number of functioning GJChs that preclude study of gating and single channel behavior by the necessary whole-cell voltage clamp methods), we address our aims using site-mutants of Cx43 that mimic the phosphorylation state of Cx43 in normally functioning, preconditioned and injured heart. We express these Cx43 site mutants in Cx-deficient rat insulinoma cells, Cx43-deficient cardiomyocytes, and wild-type cardiomyocytes, where their gating, conductance and antiarrhythmic properties can be studied. The results of our studies will guide design and testing of function- specific, high-affinity peptidomimetics to interfere with or mimic the interactions tht underlie anti-arrhythmic GJCh and HCh function. The proposed combination of molecular, structural and functional approaches can be expected to provide new mechanistic insight on regulation of the dynamic function of Cx43 GJChs as they support the extraordinary dynamic range of cardiac function. In addition, we expect to develop Cx43- and function-specific, experimentally and therapeutically useful tools that will protect coordinated function of the heart despite ongoing disease and pharmacologic therapies.

Overall Component Project Summary The mission of the Nebraska IDeA Networks of Biomedical Research Excellence (NE-INBRE) is to stimulate and develop biomedical research capacity at institutions of higher education in Nebraska. The NE-INBRE is structured around two major components: primarily undergraduate institutions (PUIs) and PhD granting research institutions (RIs). Support for each PUI consists of: 1) campus research capacity development through support for faculty research and infrastructure enhancement, and 2) development of the undergraduate research pipeline of students through the NE-INBRE Scholars Program. Expanding research capacity at the RIs includes providing significant support to multi-user core facilities in order to allow investigators from PUIs and RIs access to cutting- edge technology. The nine participating PUIs in the NE-INBRE research network include two publically supported State Colleges, three campuses of the University of Nebraska system, and four private institutions. The three participating RIs in the research network include two campuses of the University of Nebraska system and one privately supported medical center. Cutting-edge multiuser core facilities include cores in genomics, bioinformatics, and advanced microscopy. The three themes reflect the scientific foci of the NE-INBRE, infectious diseases, cancer biology, and cell signalling. These themes serve to link faculty and students at the separate institutions into productive networks based on their areas of expertise and interest. Throughout the tenure of the NE-INBRE, its primary objective at the undergraduate level has been to provide and expand research opportunities for students and create a pipeline of students to enter into biomedical research and other health professions. NE-INBRE investments in faculty research projects and infrastructure at the PUIs have created opportunities for both NE-INBRE Scholars and other undergraduate students to become involved in advanced biomedical research.

Connexins are integral membrane proteins that oligomerize to form gap junction channels. Gap junctions composed of Cx43 mediate electrical coupling and impulse propagation in the normal working myocardium. In the failing heart, Cx43 remodeling (decreased expression, loss at intercalated discs, increased presence at lateral membranes) contributes to ventricular arrhythmias. However, the failing heart also aberrantly upregulates expression of Cx45 in ventricles, where it is normally at very low levels. This greatly enhances the propensity for arrhythmias, logically due to the low conductance and high voltage-sensitivity of Cx45 channels relative to Cx43. Crucially, the deleterious effect of Cx45 at the intercalated discs is likely amplified by the propensity of Cx45 to form heteromeric channels with Cx43, in which it has a dominant effect on the function of the resulting channels. Unfortunately, little is known about the mechanisms that drive Cx45 presence at intercalated discs or about the determinants of the functional properties of Cx45 that make its presence at ventricular intercalated discs dangerous. Studies proposed in Specific Aims 1 and 2 address novel mechanisms by which phosphorylation of the Cx45 carboxyl terminal (Cx45CT) domain modulates Cx45 protein partner interactions to increase or decrease gap junction intercellular communication in vitro and in vivo (and the differences from effects on Cx43). Specific Aim 3 focuses on determining how a recently discovered high-affinity protein-protein interaction of the Cx45CT, dimerization, affects the channel functional properties. The significance of this proposal is that discovery of how phosphorylations and interactions of the CT domain can be modulated would enable strategies to ameliorate pathological alterations of connexins the failing heart and elsewhere.

PROJECT SUMMARY: Neuronal sterol synthesis can be impacted by genetic mutations in cholesterol synthesis genes or by psychopharmaceuticals that inhibit one of the sterol synthesis enzymes (shown in our ongoing studies). Epidemiological studies and our ongoing studies have shown that the use of psychopharmaceuticals during pregnancy is ubiquitous. Many commonly used medications have not been evaluated for safety during pregnancy and the long-term consequences of fetal exposure remain unknown. Testing the effects of commonly prescribed psychopharmaceuticals will provide insight into the role of cholesterol and fatty acids for the formation and maturation of the zebrafish nervous system. We hypothesize that exposure to psychotropic pharmaceuticals that inhibit cholesterol synthesis enzymes will alter neuronal cholesterol and acylcarnitine levels disrupt nervous system development. Zebrafish larvae will be exposed to pharmaceuticals at several distinct developmental stages and analyzed at 5 days post fertilization. LC-MS/MS will be used to measure sterol and oxysterol levels, acylcarnitines, acetylated tubulin, as well as neuronal and glial specific markers. These studies will elucidate the effects of commonly used psychotropic medications on sterol synthesis and acylcarnitine modification and utilization as well as whole body and nervous system development and function in zebrafish larvae. Whole zebrafish and brain immunohistochemistry will be used to visualize drug effects on brain development. In vivo lateral line neurosensory cells and brain neural network electrical activity will be assessed using state-of-the -art technologies available through the COBRE collaboration. Locomotor behavior of drug and control treated zebrafish will be compared using ViewPoint Behavioral Technology. The outcome are threefold: 1. Our results will identify drugs that should be used with extreme caution during pregnancy and provide a framework for future preclinical testing of new therapeutic agents for sterol biosynthesis inhibition; 2. Our results will also provide important information about the role of cholesterol homeostasis and acylcarnitine levels during nervous system development and behavior; and 3. Our results will provide excellent training for graduate and undergraduate research students in the fields of lipid biochemistry, sensory and central nervous system development, physiology, and function by an experienced, dedicated working group of NE-INBRE and COBRE funded scientists.