Participating Faculty Research Programs

Ackerman, Steven J., Ph.D.
Dr. Ackerman's research interests center on the molecular biology, biochemistry and hematopoietic development of the human eosinophil leukocyte in health and disease pathogenesis. Ongoing research projects focus on the: (1) transcriptional mechanisms that regulate eosinophil development and lineage-specific gene expression in the process of commitment and terminal differentiation of multipotential myeloid progenitors to the eosinophil granulocyte lineage, (2) molecular biology, biochemistry and biologic actions of granule and cytosolic enzymes and cationic cytotoxins expressed by eosinophils, and their roles in the effector functions of this granulocyte in disease pathogenesis, (3) structural biology (structure-activity relationships) of eosinophil granule-associated cytotoxins and enzyme mediators of inflammation, (4) cytokine and cytokine receptor expression and regulation in terms of the mechanisms of eosinophil terminal differentiation, priming, activation and secretion, including cytokine-activated signal transduction pathways, and (5) the roles of eosinophils and their mediators in normal tissue remodeling and pathological tissue fibrosis. Work has included the cloning, sequencing and characterization of cDNA and genomic clones encoding eosinophil granule-associated proteins, isolation of the regulatory regions (promoters and enhancers) of these eosinophil-specific genes, and functional characterization of the cis-acting DNA elements and transcription factors and transcriptional networks that regulate their expression during eosinophil development and post-mitotic activation. Dr. Ackerman is in the process of characterizing the regulatory regions of the genes encoding the eosinophil-specific alpha (?) subunit of the IL-5 receptor (IL-5R??, the eosinophil granule cationic proteins [major basic protein (MBP) and eosinophil peroxidase (EPO)], and the Charcot-Leyden crystal (CLC) protein (Galectin-10). These eosinophil promoters/enhancers are being analyzed as models for the differential regulation of myeloid specific genes in general, and eosinophil-specific genes in particular, in the process of the commitment and differentiation of stem cells and multipotential bone marrow-derived progenitors to the granulocyte lineages. Transcription factors thus far shown to regulate eosinophil development and/or gene expression that are under investigation include members of the C/EBP family (??????? and ? isoforms), GATA-binding proteins and their coactivators and corepressors [Friend of GATA (FOGs)], members of the ets family of transcriptional regulators including PU.1 and GA-binding protein (GABP), members of the Egr family, and the RFX family of transcriptional regulators. Dr. Ackerman’s group is particularly interested in the functional interactions of transcriptional regulators such as the C/EBPs, GATA-binding proteins (GATA-1 and 2) and PU.1 in terms of their interactions (antagonism versus synergy) on target genes in the eosinophil compared to other myeloid lineages, and the enhancer roles of the RFX and RFX-associated proteins in IL-5 receptor gene expression. The work on eosinophil function focuses primarily on the pro-inflammatory effector roles of eosinophils and their unique granule cationic proteins and lipolytic enzymes in the pathogenesis of asthma, allergic and other eosinophil-associated diseases and hypereosinophilic syndromes. Research on the cytotoxic and inflammatory effector functions of eosinophils includes the expression of recombinant eosinophil proteins (CLC/galectin-10, proMBP and MBP, eosinophil lysophospholipases) and analyses of structure-function relationships for their unique enzymatic and non-enzymatic activities using site-directed mutagenesis and molecular modeling based on determination of crystallographic 3D structure, work done in collaboration with Dr. Ravi Acharya at the University of Bath in the UK. Related projects characterizing eosinophil effector mechanisms in the pathophysiology of asthma and other allergic diseases include studies of the mechanisms by which eosinophils induce airways dysfunction, fibroblast and epithelial cell activation, and the production of inflammatory cytokines and other mediators of tissue remodeling and pathological tissue fibrosis in the lung, gastrointestinal tract and other tissues. In this regard, his laboratory has recently developed a model of eosinophil-fibroblast interactions in which eosinophil products, including IL-1? and TGF-? and other soluble mediators, induce fibroblast signaling, activation and secretion of fibrogenic cytokines such as IL-6, and the upregulation of genes involved in extracellular matrix homeostasis.

Caffrey, Michael, Ph.D.
The general interests of our laboratory concern biochemical and NMR studies of protein-protein interactions. We are especially interested in the mechanisms of viral entry and the mechanisms of toxin entry. Current viral systems under study include HIV, SARS Coronavirus, Ebola and Influenza. Current toxins under study include HIV tat and Bacillus anthracis anthrax toxin. Our general approach consists of 5 stages: (i) generation of protein samples suitable for biochemical and NMR studies; (ii) determination of the protein structural properties by NMR spectroscopy; (iii) characterization of the protein intermolecular interactions and dynamic properties by NMR spectroscopy; (iv) correlation of the protein structural and dynamic properties to protein activity in vivo by biochemical studies; (v) NMR-based drug discovery studies. We feel that our general approach, which includes classic biochemical and state of the art NMR studies, will lead to detailed structure-function information on our current projects, as well as other future projects of large and physiologically relevant protein systems. In what follows, I will discuss 4 systems that are currently being studied in our laboratory.

Chishti, Athar, Ph.D.
Our primary research interest is in the assembly and regulation of the cytoskeleton. We are currently studying MAGUKs (Membrane Associated GUanylate Kinase homologues), a family of multidomain peripheral membrane proteins that play important roles in cell proliferation and tumor suppression pathways. MAGUKs are composed of PDZ, SH3, and guanylate kinase-like domains, and our overall objective is to elucidate the cellular mechanism of subcellular targeting, assembly, and trafficking of MAGUKs by utilizing genetically altered mouse models. Another aspect of our research program is the exploration of the mechanism of malaria parasite pathogenesis in red blood cells. We are investigating the basis of ligand-receptor interactions for merozoite invasion, adhesion of infected erythrocytes to the endothelium, and the role of cysteine proteases during the intracellular development of blood stage malaria. Our current interest in cysteine proteases originated from the development of mu-calpain knock out mice in our laboratory. This mouse model is helping us delineate the role of limited proteolysis in the remodeling of the cytoskeleton by identifying physiological substrates of mu-calpain in a variety of mammalian cells. Together, these studies are designed to elucidate the mechanism of intracellular signaling in the regulation of mammalian cytoskeleton.

Christman, John, M.D.
Dr. John W. Christman received his M.D from Indiana University in 1978. He trained in Internal Medicine at Indiana University from 1978-1981, and completed a Pulmonary Fellowship at the University of Vermont in 1983 and a Critical Care Fellowship at the University of Pittsburgh in 1984. His research interests include investigation of transcription factors that regulate macrophage gene expression and mediate their role in the evolution of neutrophilic lung inflammation. The overall goal of the lab is to investigate the pre-transcriptional events in macrophages that lead to lung and systemic inflammation. There is a focus on the PU.1, C/EBP beta, and NF-kappa B activation pathway intitiated by endotoxin and mediated by the proximal cytokines, TNF alpha and IL-1 beta. A comprehesive approach is used to address clinically relevant questions that employ murine models of acute lung and systemic inflammation. Molecular approaches include generation and characterization of novel inducible transgenic mice, employment of bioluminescent technology, and use of bone marrow chymerics. Supportive studies employing various macrophage cell lines and bone marrow derived macrophages are also employed. There is a particular emphasis on the mechanism of COX-2 gene expression and the influences of COX-2 gene expression and COX-2 products on the initiation of acute inflammation through interactions with the NF-kappa B activation pathway.

Colamonici, Oscar, M.D.
The main research focus of my lab has been the regulation of cell proliferation by interferons. Recently, our interest has centered on a protein originally discover due to its interaction with the type I interferon receptor and that has interferon independent growth inhibitory effects. The two programs underway in my lab focus on the characterization on this novel proteins and can be summarize as follows:

1) Regulation of G1 by Mip/LIN-9
Understanding the mechanisms that govern cell cycle progression is important for the development of novel therapeutic agents against cancer. The G1 phase is a critical crossroad where positive and negative regulatory signals converge to control cell cycle progression. The family of pocket proteins is responsible for restricting cell cycle progression via the formation of repressor complexes with E2F and DP family members, which result in the inhibition of E2F target genes. We cloned a novel gene, originally named BARA, currently termed human Mip/LIN-9, which regulates cell cycle progression. It has been previously reported that Mip/LIN-9 collaborates with pRB in the regulation of transformation. Our studies demonstrate that deletion of the first 84 amino acids of Mip/LIN-9 (Mip/LIN-9?84) corrects the CDK4 null phenotype. Therefore, Mip/LIN-9, like the pocket proteins pRB, p107 and p130, is negatively regulated by CDK4. Interestingly, the correction of the CDK4 null phenotype is accompanied by a restoration of the expression of genes such as E2F1, E2F3, and cyclin E suggesting that Mip/LIN-9 participates in the regulation of E2F target genes required for the G1/S transition. This is further supported by the finding that Mip/LIN-9 interacts with two members of the pocket family, p107 and p130. The objectives of this project are: 1) To characterize the mechanism that leads to the correction of the CDK4 null phenotype by the mutation ?84 of Mip/LIN-9. 2) To test the hypothesis that BARA is part of the transcriptional repressor complex formed by p107,130/E2F4,5/DPs and that its interaction with other members of the complex is required for the regulation of the expression of E2F target genes responsible for cell cycle progression. 3) To determine if the expression of E2F-regulated genes observed in CDK4–/–Mip/LIN-9?84/?84 MEFs is also responsible for the rescue of the phenotype in affected tissues.

2) Regulation of S phase and mitosis by Mip/LIN-9.
In Drosophila, the homolog of Mip/LIN-9, Mip130, is part of a large complex termed dREAM (drosophila RB, E2F and Myb) that includes pocket proteins, repressor forms of E2F, B-Myb and B-Myb-interacting proteins termed Mip(s). This complex inhibits transcription of specific genes and duplication of specific genomic regions. Our preliminary data suggest that the mammalian equivalent of Mip130, Mip/LIN-9, forms a complex with pocket proteins, E2Fs and B-Myb; however, unlike the Drosophila counterpart, not all proteins are in the same complex simultaneously. For example, Mip/LIN-9 interacts with p107, p130 and E2F4 in G0 and early G1, and with B-Myb in late G1 and S phase. Moreover, while the complex that Mip/LIN-9 forms with E2F4 and p107 or p130 has a repressor effect, the interaction with B-Myb is responsible for the induction of critical S-phase and mitotic genes such as cyclin A, cyclin B and CDK1. This project focuses on the role of Mip/LIN-9 within the context of B-Myb and test the hypothesis that Mip/LIN-9 is responsible for the regulation of the expression of B-Myb and B-Myb-regulated genes. Finally, we will develop a conditional KO for the Mip/LIN-9 gene to determine its role in development and in the adult animal.

Colley, Karen, Ph.D.
Major Interests: The control and organization of protein glycosylation and the mechanism of protein specific polysialylation.

The overall goal of my research program is to understand how the process of glycoprotein glycosylation is controlled and how attached oligosaccharide structures influence the biological function of the proteins that they modify. We have three projects in the laboratory. The first involves evaluating the biosynthesis and function of ?2, 8-polysialic acid, a developmentally regulated, anti-adhesive glycan that is critical for brain development, neuronal regeneration, and is found on the surface of highly metastatic cancer cells. Polysialic acid is predominantly found on the oligosaccharides of the neural cell adhesion molecule (NCAM). The limited number of substrates for the polysialyltransferases suggests that the addition of polysialic acid is a protein specific event and is likely to involve an initial protein-protein contact. We determined that the first fibronectin type III repeat of NCAM is required for polysialyltransferase recognition. In collaboration with the Lavie laboratory, we have identified features of this domain that are critical for polysialyltransferase recognition and N-glycan positioning. Using this information we have been able to reconstitute polysialylation in a typically unpolysialylated protein. Our goal is to precisely characterize the interactions between the polysialyltransferases and NCAM so that we can devise approaches to diminish or enhance polysialylation and its negative effects on cell adhesion during development, in repair/regeneration and in cancer.

The second project involves elucidating the signals and mechanisms involved in the localization of glycosyltransferases in the Golgi. We have used ST6Gal I, the ?2, 6-sialyltransferase of Asn-linked protein glycosylation, as our model system. We have found that several regions (cytosolic tail, transmembrane region, and luminal sequences) play a role in Golgi localization, and correlated the formation of insoluble oligomers with the stable Golgi localization of one isoform of this enzyme. Currently, we are pursuing studies to evaluate other enzymes and whether they too have multiple Golgi localization signals and to determine the role of oligomerization in their stable localization.

The third project in the laboratory focuses on the CMP-sialic acid transporter. The sialyltransferases in the Golgi require the nucleotide sugar donor, CMP-sialic acid, to function. This molecule is transported from the cytoplasm into the Golgi lumen via the CMP-sialic acid transporter. We have found that this transporter exhibits a restricted Golgi localization that does not involve complex formation with sialyltransferases, and that it can even function, albeit at a lower efficiency when trapped in the endoplasmic reticulum. It also possesses two redundant ER export signals in its C-terminal cytoplasmic tail. Current studies are focused on elucidating the mechanism of the transporter and the residues in helix 7 involved in substrate recognition.

Cook, James L., M.D.
One project involves studies of viral gene regulation of mammalian cell responses to injuries inflicted by cellular components of the innate immune response. Mammalian cells expressing the E1A gene of human adenovirus types 2 or 5 are phenotypically changed from being resistant to immunological injuries to becoming highly susceptible to apoptotic death triggered by natural killer (NK) cells, cytotoxic T lymphocytes (CTL), activated macrophages, tumor necrosis factor alpha (TNF) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The in vivo correlate of this observation is that E1A expression in neoplastic cells converts them from highly tumorigenic cells to cells that are easily rejected by immunocompetent (but not immunodeficient) animals. E1A-induced cellular sensitization to apoptotic injury is not unique to immunological injuries, but is also observed with selected chemical and physical injuries. This suggests that the mechanisms by which E1A sensitizes cells to apoptosis are not unique to interactions between immunological mediators and E1A-expressing cells but involve one or more common apoptosis pathways through which cells respond to diverse injuries. The objective of these studies is to define the molecular mechanisms and cellular pathways through which this viral transcriptional regulatory gene mediates cellular conversion to the apoptosis-sensitive phenotype.

The other project area involved studies of the role of macrophages in the host response to anthrax infection and anthrax lethal toxin-induced shock syndrome. Bacillus anthracis infection and toxin production during systemic infection are associated with a shock syndrome that appears to be triggered through cellular mechanisms that involve activated macrophages, among other host cells. Studies in this project include evaluation of infection-induced and toxin-triggered macrophage activation and cytotoxicity and development of animal models of the anthrax-induced shock syndrome. The effects of anthrax infection and related toxin production on endothelial cell function are also being evaluated to better understand the mechanisms of anthrax-induced changes in vascular permeability as part of the pathogenesis of systemic infection. A third area of anthrax-related study involves development of cell and animal models to assess novel therapeutics aimed at controlling anthrax infection and blocking anthrax toxin-mediated pathogenesis. This therapeutics project involves a multi-laboratory collaborative interaction.

De Lanerolle, Primal, Ph.D.
The research in my lab focuses on the role of myosin I and II in signal transduction in the cytoplasm and the nucleus. Myosin I and II are members of a superfamily of actin-activated molecular motors that convert chemical energy into mechanical work. Most members of this superfamily do not form filaments. Myosin I is the best studied of these "unconventional" myosins. We have demonstrated the presence of a nuclear isoform of myosin I that contains an unique 16 amino acid NH2-terminal extension. We have also shown that nuclear myosin I forms a complex with RNA polymerase II and that it is required for RNA polymerase II activity. We are actively pursuing how nuclear myosin I interacts with RNA polymerase II, how it gets into the nucleus and how it interacts with cellular signalling pathways. We are also investigating GTPase-myosin II interactions. The activation of protein kinases by the GTP-bound form of small G proteins (GTPases) results in highly regulated changes in the actin cytoskeleton. Actin usually works in concert with myosin II. The actin-myosin II interaction in smooth muscle and non-muscle cells is regulated by the phosphorylation of ser 19 of the 20 kDa light chain of myosin II by the calcium-calmodulin dependent enzyme myosin light chain kinase (MLCK). We, and others, have shown that myosin II light chain phosphorylation is modulated by GTPases. Therefore, we proposed the hypothesis that the level of myosin II light chain phosphorylation is important in determining the integrated cellular response to GTPase activation. To test this hypothesis, we are studying the regulation of myosin light chain kinase in hypertension and other proliferative diseases

Garcia-Martinez, Jesus, M.D., Ph.D.
The García-Martínez laboratory studies signal transduction mechanisms involving calcium channels in striated muscle. Work from this laboratory has shown that the intracellular calcium release channel 1,4,5-trisphosphate receptor (IP3R) is found in the nuclear region of cardiac cells and that it is involved in modulation of gene expression. Ongoing experiments seek to characterize the properties of calcium release in nuclei of ventricular myocytes mediated by IP3Rs in response to hypertrophic agonists and identify the transcription factors involved in the hypertrophic response. Other experiments in this laboratory are examining a potentially new mechanism of signal transduction in skeletal and cardiac cells involving the membrane protein a2/d1. The a2/d1 subunit forms part of the L-type calcium channel or dihydropyridine receptor complex (DHPR), a vital plasma membrane component necessary for excitation-contraction coupling (ecc). The role of the a2/d1 subunit in normal muscle functioning is largely unknown and its participation in muscle pathology is tenuous. Only a genetic linkage has been established between the locus of this subunit and malignant hyperthermia susceptibility (MHS). No mutations have been identified in the a2/d1 subunit and, furthermore, little is known about its involvement in controlling calcium release in normal or MHS patients. Recent evidence suggests that the a2/d1 subunit might regulate calcium release, the L-type current (ICa-L) and charge movement in skeletal myotubes. In addition to modifying calcium release, preliminary data from this laboratory blocking the expression of a2/d1 indicate that this subunit is important for migration of myoblasts, a previously unidentified role of a2/d1 in muscle. The idea of the a2/d1 subunit having a role in a mechanism other than calcium release is both novel and important for understanding muscle formation and regeneration. This project will determine the role of the a2/d1 subunit in ecc and whether the properties of charge movement, calcium release, and ICa-L change as the a1 and a2/d1 subunits co-localize in muscle cells. It also seeks to determine the role of the a2/d1 subunit in cell adhesion, migration, and differentiation of muscle cells in culture. The projects are approached from different, complementary angles with a variety of precedures and techniques. Trainees are given the opportunity to use and become proficient at these procedures and integrate the information derived from his/her experiments. Most of the experiments are performed at the level of single cells. Dr. García-Martínez uses molecular biological and biochemical techniques (site-directed mutagenesis, coning, sequencing, different variants of PCR, blotting, immunochemistry, siRNA), electrophysiological techniques (variants of patch clamp and vaseline gap), measurements of calcium levels with fluorescent dyes under conventional and confocal microscopy, and immunolocalization of proteins with confocal microscopy.

Gettins, Peter G.W., D. Phil.
There are three main projects in the Gettins laboratory. All involve use of biophysical approaches to study the structure and function of the system of interest. The approaches include NMR spectroscopy, x-ray crystallography, fluorescence, especially FRET, calorimetry and kinetics. These are coupled with site-directed mutagenesis.

A longstanding interest has been elucidation of the mechanism of action of the family of proteins known as serpins (serine proteinase inhibitors). This is a specific family of proteins of 40-60 kDa that possess a hallmark fold that is extremely unusual in being a metastable conformation. As part of this metastable conformation the reactive center loop (the region recognized by target proteinases) is an exposed loop that can undergo a dramatic conformational change involving insertion into the center of one of the three main beta-sheets of the protein. The resulting conformation is very much more stable than the original metastable state. Studies in the Gettins lab have shown that inhibition of proteinase results when, after initial association of the proteinase with the reactive center loop and partial completion of the proteolysis reaction to the point of formation of the acyl interemediate and release of the first product, the now-cleaved reactive center loop inserts into the central beta sheet and drags the covalently-bound proteinase with it to the distal end of the serpin, a movement of over 70Å. In this location, the apposition of proteinase and serpin results in gross distortion of the proteinase active site rendering it kinetically incompetent. This compromise of catalytic competence represents a kinetic trapping of the proteinase and hence its inhibition. This translocation was first demonstrated by FRET and subsequently by NMR and x-ray approaches. Currently, the lab is attempting to delineate the nature of intermediate stages of the process of translocation and distortion to determine how the energy available from loop insertion is coupled to proteinase distortion and hence trapping.

While most of the studies on the serpin mechanism have been limited to inhibition of serine proteinases of the chymotrypsin family, it is known that serpins can inhibit serine proteinases with the subtilisin fold as well as cysteine proteinases of two different families (caspases and papain-like proteinases). Comparable NMR and fluorescence studies are being carried out on complexes of such proteinases with serpins to determine if a common mechanism of proteinase translocation and distortion is employed in each case.

A second project is the elucidation of the mechanism of conformational change in the very high molecular weight pan-proteinase inhibitor alpha-2-macroglobulin. Human alpha-2-macroglobulin is a 720 kDa homotetramer that inhibits proteinases by a Venus fly trap-like closure of the inhibitor around the proteinase. This is initiated by cleavage of the proteinase within a region termed the bait region. This massive conformational change is thought to involve the reorganization and internal conformational change of discrete domains within the protein. The Gettins lab is currently attempting to define and characterize the domains involved in these changes and examine the alteration in pairwise interactions that represent the conformational reorganization. These studies involve extensive use of calorimetry to examine protein-protein interactions and fluorescence and CD to monitor conformational changes within isolated domains.

The third project involves characterizing the specificity of the mosaic receptor LRP for its wide range of protein ligands. This receptor is a member of the LDL receptor family of proteins and so is composed of clusters of ~40 residue domains (termed complement-like repeats) that constitute the protein ligand binding sites, flanked by EGF-like domains and interspersed by YWTD-containing propellor domains. The complement-like repeats contain three disulfides and a calcium binding site, the latter required for structural rigidity and ligand binding. Amongst the proteins that LRP binds and internalizes are the alpha-2-macroglobulin-proteinase complex and various serpin-proteinase complexes. Currently NMR is being used to determine the solution structure of the receptor binding domain from alpha-2-macroglobulin bound to a two-repeat complement domain fragment from LRP. In addition, NMR is also being used to examine binding interactions and conformation of the complex of a serpin-proteinase pair with a longer binding fragment from LRP. The goal of such studies is to understand both the types of determinants used by LRP to bind its target protein ligands, as well as how it can bind so many structurally diverse proteins.

Gibori, Geula, Ph.D.
The major research effort of Dr. Gibori’s laboratory deals with the expression and mechanism of action of steroid and peptide hormones in ovarian and placental cells. Presently, members of Dr. Gibori’s laboratory are actively investigating the molecular mechanisms(s) by which prolactin, ProstaglandinF2a and estradiol signals are transduced, and how these hormones affect such different phenomenon in the ovary as cell hypertrophy and steroidogenesis allowing pregnancy to either be maintained or be terminated. One approach has been to examine the effect of these hormones on gene expression, and on calcium-dependent phosphorylation of key proteins involved in signaling and steroidogenesis. Another aspect of their research focuses on the secretion and action of decidual proteins. Dr. Gibori and her colleagues in the laboratory has discovered and cloned a hormone expressed by discrete cell population of the decidua. They are presently investigating its role in pregnancy.

Hay, Nissim Ph.D.
We are utilizing genetics, molecular biology and cell biology approaches, as well as biochemistry to elucidate mechanisms of cell survival, proliferation, differentiation, and energy homeostasis, and how they are related to diseases such as cancer, aging and diabetes. These topics are investigated at both the cellular and organismal levels utilizing mouse genetics. The major focus is on the role of the PI3K/Akt/mTOR signaling pathway in these processes. We are investigating the mechanisms by which the serine/threonine kinase Akt is promoting cell survival and how it is related to the role of Akt in energy metabolisms. Since Akt is frequently activated in human cancers we are delineating the mechanism by which activation of Akt contributes to the genesis of cancer. At the cellular level we are determining the roles of Akt in cell cycle progression, cell cycle checkpoints and susceptibility to oncogenic transformation. At the organismal level we are using mice deficient for the different Akt isoforms. By crossing these mice with mice models of cancer we can determine the roles of the different Akt isoforms in the genesis of different types of cancer. On another front we are investigating the phenotypes of mice null for the individual Akt isoforms and compound Akt knockout mice in relation to embryonic development, diabetes, and lifespan.

Kanteti, Prasad V.S., Ph.D.
We previously cloned a pro-apoptotic molecule, Siva, in the context of CD27, a member of the TNFR (Tumor Necrosis Factor Receptor) family. A role for Siva has been demonstrated in various cell death pathways however underlying mechanisms are not known. In humans, the Siva gene is localized to chromosome 14 (14q32), close to TRAF3 and AKT1. In both humans and mice, it has 4 exons and generates two transcripts. The predominant full-length form, Siva-1, is coded by all 4 exons and is apoptotic. Its alternate splice form Siva-2, lacks the second exon and is much less toxic. Although Siva-1 has no true BH (BCL-2 homology) domains, primary sequence analysis of the exon-2 region of Siva-1 predicted the presence of a strong 20 amino acid long amphipathic helix (SAH, residues 36-55), almost the size of the BH3 domain seen in the pro-apoptotic BCL-2 family members. In addition, a significant amount of the Siva-1 protein was co-localized to mitochondria despite the absence of a mitochondrial targeting sequence. This raised the possibility that Siva-1 could specifically interact with the anti-apoptotic members of the BCL-2 family such as BCL-XL. Using multiple approaches, we were able to demonstrate specific and direct binding between Siva-1 and BCL-XL. Also, colocalization of Siva-1 and BCL-XL could be visualized in transfected cells using confocal microscopy and natural complexes of Siva-1/BCL-XL could be detected in murine thymocytes, where both Siva-1 and BCL-XL are both highly expressed. Using various mutants forms of Siva-1, we mapped the binding site to the SAH region and confirmed it by successfully competing off BCL-XL bound to Siva-1 using synthetic SAH but not control peptide. The functional importance of the association between Siva-1 and BCL-XL was evaluated using MCF7 cells permanently transfected with BCL-XL, which unlike parent cells are highly resistant to UV induced apoptosis. Transient transfection of Siva-1 into these cells abrogated the protection offered by BCL-XL against UV radiation mediated cell death. In comparison, Siva-1 mutants (including Siva-2) with altered or partially deleted SAH region were less effective. Significant leakage of cytochrome c into the cytosol in Siva-1/BCL-XL double transfectants upon UV radiation in comparison to single transfectants suggested attenuation of mitochondrial function. Taken together the work clearly supports a physiological role for Siva-1 in the regulation of BCL-XL mediated cell survival and is principally mediated by the unique SAH region. We plan understand the significance of the interaction between Siva-1 and BCL-XL in the context of cancer using various drug induced apoptosis models, develop transgenic mice that have targeted expression of a dominant negative mutant of Siva-1 in T cells and study the signaling pathways mediated through Siva in vivo.

Kaplan, Jack, Ph. D.
Structure-function studies, mechanism, biosynthesis, assembly and cellular trafficking of P-type ATPases or Ion pumps. Mechanism and Regulation of Copper transport systems in Human Cells.

The active transport of ions across cell membranes is performed by P-type ATPases. These are integral membrane proteins which use the energy of hydrolysis of ATP to pump ions across biological membranes. The mechanism by which these proteins couple ATP hydrolysis to ion transport is one of the central interests of the laboratory. These proteins form phosphorylated intermediates with ATP and transport Na+, K+, H+, Ca2+, Cu2+, Cd2+ etc. across animal, bacterial, and plant cell membranes.

Using baculovirus-infected insect cells to express mutant Na pump molecules we are now studying protein conformational changes using site directed fluorescence and how the sub-units assemble in the ER and are trafficked via the Golgi to the plasma membrane using cell fractionation, immunological and metabolic labelling techniques. We are also interested in studying how Cu ions enter cells and we are carrying out the molecular analysis and characterization of hCTR1 a protein which mediates the entry of Cu ions into human cells. In this program we are employing the baculovirus-infected insect cell lines to generate recombinant transporter protein for structure-function and regulation studies.

During the last twenty years, we have been responsible for the introduction and development of caged compounds for biophysical and physiological studies. These are novel photolabile compounds which on u-v illumination release the caged substrate (ATP, ADP, Pi, Ca2+, Mg2+, etc.) in the msec-microsec time range and thus synchronously and rapidly initiate biological processes. We plan to continue to develop this strategy to provide novel photorelease techniques for cellular and molecular studies.

Katzen, Alisa, Ph.D.
Appropriate cell cycle regulation is a critical component in determining whether cells should divide, terminally differentiate, or die during development, and for avoiding cancerous growth. We use the powerful genetic and developmental model system, Drosophila melanogaster, to study highly conserved biochemical pathways that regulate cellular division and differentiation during development

Kozasa, Tohru M.D.,Ph.D.
We are conducting biochemical investigations on G protein-mediated signal transduction pathways. The major goal is to understand the regulatory mechanism of activation of Rho family GTPases (Rho, Rac, Cdc42) by heterotrimeric G proteins. Rho family GTPases are involved in a variety of cellular functions by controlling the organization of actin cytoskeleton or gene expression. We have recently demonstrated that Ga12 and Ga13, whose effectors were previously unknown, interact with and regulate the activity of a novel Rho specific GEF (guanine nucleotide exchange factor), p115RhoGEF. This result was the first demonstration of the biochemical link between Rho family monomeric GTPases and heterotrimeric G proteins. We are characterizing the regulation of p115RhoGEF by G12/G13 in detail by reconstituting purified components and also using cultured cell lines. The involvement of this signaling pathway in cellular functions such as neurite extension, angiogenesis, or cell-cell adhesion will be pursued in future. We are also trying to find new effectors for G protein signaling pathways. We have recently identified a brain specific effector candidate GRIN1 for Gao. Gao is extremely abundant protein in brain but its physiological function is unknown. Both Gao and GRIN1 are highly enriched at growth cone of neurons. In addition, we demonstrated that activation of Gao-GRIN1 pathway stimulates neurite formation in cultured cells. The physiological significance of Gao-GRIN1 signaling pathway in brain function will be further investigated.

Lau, Lester F., Ph.D.
Proteins of the extracellular matrix are capable of regulating a variety of biological processes, including cell adhesion, migration, differentiation, and proliferation. Although cell adhesion to the matrix has long been known to provide a pro-survival function, we have recently shown that the matrix proteins CCN1 and CCN2 can induce apoptosis as adhesion substrates in specific cell types. Functioning through direct binding to integrin receptors, these proteins play important roles in development and regulate angiogenesis and matrix remodeling associated with inflammation and tissue response to injury. Our current work aims to understand the roles of CCN1 and CCN2 in embryonic development and in pathological conditions, including inflammation, wound healing, fibrosis, and cancer. In a separate project, we are interested in how ribosome biogenesis and cell cycle progression are coordinated. Dysfunction in ribosome biogenesis can lead to derailment of the cell cycle, suggesting a crosstalk between these two processes. We have recently identified a novel nucleolar protein, Bop1, that is involved in processing of the 28S and 5.8S ribosomal RNA as well as in cell cycle progression. Furthermore, we have shown that p53 serves as a surveillance mechanism for ribosome biogenesis, defects in which induces a cell cycle arrest through p53. Current work focuses on the mechanism of p53 action in this novel ribosome biogenesis check point.

Lavie, Arnon Ph.D.
Antimetabolites, which are compounds that are administered to inhibit DNA replication, are often administered as prodrugs. Such prodrug molecules such as AraC, a potent agent for the treatment of acute myeloid leukemia, or AZT, used to treat HIV infection, are active in their tri-phosphorylated form, AraC-triphosphate and AZT-triphosphate, respectively. However, they are administered as the uncharged nucleoside due to the inability of charged molecules to traverse biological membranes. Conversion of the uncharged nucleoside analog (NA) prodrugs to an active form is mediated and dependent on the activity of cellular enzymes. For most prodrugs it is this activation that limits their therapeutic efficacy. My laboratory is concerned with understanding the factors that limit the activation of therapeutically important nucleoside analogs and aim at developing alternative strategies to circumvent the reliance on endogenous enzymes.
To achieve our goals we have set out to structurally characterize the interactions between NAs and the kinases that phosphorylate them. The structural results obtained from such studies can explain the relative activity observed with various NAs, and importantly can be used to design novel NAs that are more suitable to act as substrates for these enzymes. Additionally, our structural characterization of enzyme/substrate states along the reaction trajectory allows for a fuller understanding of the catalytic mechanism.

Another project in the laboratory focuses on the recently discovered family of proteins called MAGUKs (Membrane Associated Guanylate-kinase like homologs). They act as molecular scaffolds for signaling pathway components, regulate synaptic structure and function and ion channels by mediating specific interactions and (in drosophila) act as tumor suppressor. In addition, they recruit molecules into localized multi-molecular complexes and can cluster these complexes at the plasma membrane, such as cell junctions or the apicolateral or basolateral surface, and pre- or post-synapses. Our goal is to understand on the molecular level the basis for their specificity as protein-protein binding partners and their method of regulation.

Le Breton, Guy C., Ph.D.
In order to study human blood platelet signal transduction pathways, our research program utilizes a wide range of chemical, biochemical, immunological, pharmacological and molecular biology techniques. Specifically, novel chemical compounds are synthesized and employed as molecular probes to elucidate the sequence of events leading to platelet activation, and to identify the signal transduction components within the thromboxane receptor pathway. In addition, biochemical, immunological and molecular biology techniques are utilized to study selective interactions within this pathway, including ligand-receptor binding, receptor-G-protein coupling, and receptor/G-protein phosphorylation. The final section of this program involves experiments designed to investigate the pharmacological interaction between discrete and separate signal transduction pathways within human platelets. A separate research program currently in progress is aimed at studying the involvement of TXA2 receptors in the central nervous system. Specifically, work from our laboratory has uncovered evidence pointing to a new signaling system in oligodendrocytes, the cells responsible for central nervous system myelination. In this regard, we originally demonstrated that myelinated fiber tracts in both the brain and spinal cord have a high density of the inflammatory TXA2 receptor. As an extension of these findings, we subsequently determined that both neonatal rat oligodendrocytes and human oligodendroglioma cells possess TXA2 receptors; and that these receptors are functionally coupled to modulation of intracellular calcium levels. Most recently, we have obtained evidence that this receptor also exists on Schwann cells. Thus, the two highly specialized cell lines which regulate central and peripheral system myelination possess significant concentrations of functional TXA2 receptors. While the role of TXA2 in modulating oligodendrocyte physiology is presently unclear, our findings, in conjunction with other published evidence raise the possibility that activation of oligodendrocyte TXA2 receptors may influence the proliferation, differentiation and/or survival of these cells, and thereby directly affect the process of myelin homeostasis.

Liang, Jie, Ph.D
Structural Bioinformatics: We are developing computational methods to calculate the shapes of proteins and other biological molecules. Our approach uses cutting edge developments from computational geometry and computational topology. Studies of protein shapes have two focuses: 1) the surface regions, including pockets, binding sites, and their precise cast or mold. The goal is to predict protein-ligand binding, protein-protein interactions and uncover novel biochemical functions based on full characterization of protein whole surfaces of the universe of all known protein structures. 2) the interior packing of proteins, and its relationship with protein stability and folding, We are also developing empirical statistical potential useful for protein fold recognition problem and for protein design.
Cheminformatics and Drug Discovery: We apply an integrated approach for drug discovery. On the small molecule side, novel shape and chemistry based descripters have been developed to provide the metrics for managing chemical diversity of compound database and combinatorial libraries. On the receptor side, pocket surface analysis and precise cast of binding site provide additional rich information for rapid virtual screening of compounds to achieve enhanced enrichment of useful lead compounds. Our approach emphasizes the physicochemical properties of the molecules rather than bond connectivities, and we are developing methodology that allows lead hopping where compounds of related biological activity but different underlying medicinal chemistry can be identified. Existing close collaboration with pharmaceutical industry is an important component of research in this area.
Data Mining: We are applying various statistical pattern recognition techniques and mathematical and statistical methods for classification and prediction problems arising from high dimensional data in drug discovery. These include discriminant analysis, parametric and nonparametric methods, hybrid models, neural nets and analysis tools complementing Principal Component Analysis and other Gaussian-distribution based methods.
Computational Biology: We study the molecular electrostatics and solvation problem using continuum model. We are developing a boundary element method for the Poisson-Boltzman equation, with emphasis on accurate shape representation, as well as the application of fast multilevel method. Of particular interest is the differential treatment of surface and core region of proteins embedded in solution. In addition, we are studying interactions between cosolvent and proteins in terms of both osmotic stress and preferential exclusion related hydration changes. Our approach uses detailed geometric analysis and is applied to the study of enzyme reaction to understand the relationship between water transfer and enzyme mechanism.

Malik, Asrar, Ph.D.
A major interest of the laboratory is to understand the regulation of the barrier properties of the endothelial and epithelial cells. Dr. Malik studies the events occurring at the level of the receptors and the signaling pathways regulating the barrier function of these monolayers. As thrombin has been shown to increase endothelial permeability, we are studying, using this agonist, how the activation of its proteolytically cleaved receptor leads to the increase in permeability. Studies have localized the domains of the receptor involved in activation and in shutting off endothelial cell signaling. Another approach taken is to clone a dominant negative form of the receptor and to use it to inhibit thrombin receptor activation. These studies are also pursuing the cellular effector pathways increasing permeability to understand how the activation of the signaling pathways mobilizes these effectors (i.e., actin-myosin motor, cadherin-catenin complex and the intermediate cytoskeletal filaments).

Another objective of the laboratory is to develop and to test novel strategies for drug and gene delivery. We are interested in targeting specifically the cells of the vessel wall which are critical in the pathogenesis of variety of inflammatory diseases, atherosclerosis and cancer metastasis. The intent of this strategy is to prevent in a specific manner the expression of endothelial adhesion molecules. Among the approaches being studied include the selective expression using inducible promoters in order to target the expression of anti-adhesive proteins in endothelial cells. This laboratory is also developing non-viral means of gene delivery to transduce endothelial proteins of interest. The approaches taken involve molecular biology as well as physiological monitoring in experimental models.

Finally, this laboratory is studying the expression of the adhesion molecular ICAM-1 at the level of gene transcription. In particular, we are interested in how certain cytokines and oxidants induce the expression of the ICAM-1 gene at the level of its promoter, the intra-cellular signaling pathways regulating ICAM-1 expression, and how gene activation is regulated by the redox state of the cell. This group identified an element on the promoter that is activated by hydrogen peroxide. Activated transcription factors bind in a complex manner to this element and initiate ICAM-1 transcription. The objective in these studies is to understand the genetic basis of ICAM-1 expression, and then to develop strategies for controlling the expression of this adhesion molecule and to regulate leukocyte trafficking across the vascular endothelium

Nakajima, Shigehiro, M.D. Ph. D.
The aim is to clarify the mechanism by which brain neurons are excited by transmitter substances, such as neurotensin and substance P. We have been using cultured neurons from brain nuclei such as: (a) the nucleus basalis (main acetylcholine-containing neurons in the brain; degeneration of these neurons leads to Alzheimer’s disease), (b) the locus coeruleus (the main noradrenergic neurons of the brain), and (c) the substantia nigra and the ventral tegmental area (the main dopaminergic neurons of the brain). Neurons are excited mainly by the action of ion channels. We have been investigating two types of ion channels: one is a family of K+ channels (G protein-coupled inward rectifier K+ channels) and the other is the family of TRPC channels (transient receptor potential canonical). Activity of these two channels is the principal determinant of neuronal excitability. Our goal is to elucidate the signal transduction cascades, by which these two types of ion channels are regulated. The methods we are using are electro-physiological and the molecular biological techniques.

Nakajima, Yasuko, M.D., Ph.D.
We are conducting molecular biological, physiological and cell biological investigations of the signal transduction mechanisms of neurotransmitter effects on receptors of brain neurons. For this purpose we have developed a unique method of culturing dissociated neurons from specific brain nuclei. Particular emphasis is placed on the study of the role of G proteins which mediate the effects of neurotransmitters. The following are the current topics. (1) We are investigating neuropharmacologically the cellular mechanisms of arousal and sleep. Hypocretins/orexins, newly discovered peptide neurotransmitters, and their receptors play a role in narcolepsy and sleep disorders. We are investigating the effects of these transmitters on dissociated cultured brain neurons which regulate arousal and sleep. (2) The effects of substance P on cultured brain neurons are being investigated. Particularly we are interested in possible direct interaction between Gqalpha and G protein-coupled inward rectifier K+ channels. 3) We have started to investigate the signal transduction mechanisms of ghrelin (newly discovered neuropeptide transmitter related to obesity) effects on the Kir3 and Trp channels...

Olson, Steven T., Ph.D.
Two main projects are currently being investigated in our laboratory. One concerns elucidating the mechanism by which the serpin, antithrombin, and its effector, heparin, regulate the activity of blood clotting proteinases and determining how this anticoagulant mechanism contributes to hemostasis and the response to vascular injury, i.e., wound healing. This project involves identifying the amino acid residues of antithrombin which are responsible for the protein’s specificity for inhibiting clotting proteinases, those which mediate heparin activation of the serpin and those which confer antiangiogenic activity. The molecular mechanisms of antithrombin antiangiogenic activity are addiitonally being investigated using mutagenesis, gene expression and cell biology approaches. A second project is aimed at understanding the general mechanism by which proteins of the serpin superfamily inhibit serine and cysteine proteinases. This project is focussed on understanding the dynamics and role of the F helix in the mechanism by which serpins trap proteinases in stable complexes and the structural basis for the remarkable stability of these complexes.We additionally are interested in elucidating how factor Xa procoagulant function is regulated by the protein Z-dependent blood serpin, ZPI, on cell membranes using mutagenesis and biophysical approaches.

Prins, Gail, Ph.D.
My research interests concern basic and applied studies in prostate gland growth and carcinogenesis. The primary research focus of my laboratory is the hormonal control of prostatic development, growth and function and how abnormalities in these systems contribute to aging-associated disease. Areas of emphasis include steroid receptor expression, developmental regulatory networks, developmental estrogenization, environmental estrogenic exposures, growth hormone and carcinogenesis, and the role of selenium and selenoproteins in prostate cancer prevention.
A major research effort, which has spanned two decades, is elucidation of the hormonal regulation of steroid receptor expression and the interrelationship between this regulation and the normal and pathological growth of the prostate gland. Although we have applied some of these findings to the human prostate, the majority of these studies have used the rodent as an animal model for tissue heterogeneity and disease. Our work has described the ontogeny and adult expression patterns of prostatic androgen receptor, estrogen receptors (? and ß), progesterone receptor, retinoic acid receptors (RAR and RXR ?,ß, ?) as well as retinoid metabolizing enzymes and binding proteins and have found that each of these receptors has distinct localization in specific cell types which change over the course of development in the three prostate lobes. Importantly, they are differentially regulated by hormones (androgens, estrogens, prolactin) which leads to complexity in response to the hormonal milieu. Recent studies on regulatory mechanisms have determined that autoregulation of the androgen receptor as well as its down-regulation by estrogens is mediated at the posttranscriptional level through targeted proteolysis via ubiquitination and proteosome-mediated protein degradation.

Another major research interest concerns the effects of early developmental exposures to estrogens on the prostate gland, a phenomenon referred to as estrogen imprinting or Developmental Estrogenization. The rat and mouse prostate undergo morphogenesis after birth and this process can be imprinted by brief exposure to endogenous and exogenous hormones. We have demonstrated that neonatal estrogen exposure permanently imprints prostatic development and is associated with an increased incidence of hyperplasia, dysplasia and adenocarcinoma with aging. Thus, neonatal estrogenization of the rat has evolved as a useful model to evaluate the role of exogenous and endogenous estrogens as a predisposing factor for prostatic diseases later in life. The current objective of our research is to elucidate the cellular and molecular mechanisms by which neonatal estrogens initially imprint or transform the prostate gland. Towards this end, our recent studies with ER? knockout mice and ßERKO mice demonstrated that effects of estrogens are initially mediated through ER? which is amplified within prostatic stromal cells. We further determined that key members of the steroid receptor superfamily - AR, ER?, ERß, PR and RAR ? and ß - which are expressed in a temporal and cell-specific manner during prostate development are drastically altered by early estrogenic exposure as shown in the diagram on the right. This effectively shifts the developing prostate from an androgen-regulated gland to one driven by estrogens and retinoids. The net result is that programming and organizational signals which normally dictate and determine prostate development during discrete temporal windows are permanently and irretrievably altered.

Ongoing studies in my laboratory are focused on identifying the developmental genes downstream of androgen and estrogen in the developing prostate. Following estrogenic exposures during development, we have demonstrated decreased expression of the epithelial homeobox genes Hoxb-13 and Nkx3.1, which are critical for normal epithelial cell differentiation which, in part, explains the epithelial differentiation defects observed as the animal ages. In addition, paracrine signaling pathways show lobe-specific responses to estrogenic exposures which result in branching defects in the dorsolateral prostate lobes. Specific pathways delineated to date include sonic hedgehog-patched-gli, Fgf10/FgfR2iiib and Bmp4/Bmp7/Smad and the current data suggest that the estrogenization is initiated through alterations in Fgf10 signaling which results in a cascade of alterations in critical developmental genes. In summary, these alterations in morphoregulatory gene expression lead to growth retardation, differentiation aberrations and predisposure to adult-onset PIN.

Two related projects in my laboratory on the developmental estrogenization pathway concentrate on 1) gene methylation patterns that are imprinted by early exposures to both natural and environmental that may help to explain the molecular basis for permanent alterations in gene expression, and 2) oxidative stress pathways that are initiated in response to estrogenic imprints which in turn lead to prostatic dysplasia with aging. This work serves as a model for what might be expected to occur in the prostate gland's of sons of DES-exposed mothers as well as toxicologic exposure to environmental and dietary estrogens which is a major problem now confronting the scientific and medical community.

Rao, Mrinalini C., Ph.D.
A specific focus of my research is examining the role of post-translational modifications, such as protein phosphorylation and dephosphorylation, involved in the regulation of epithelial ion transport. An important corollary is investigating the nature of the cross-talk existing between different signalling systems. I am particularly interested in elucidating, at a molecular level, the processes that modulate ion transport across epithelia. Epithelia are multi-faceted and complex often containing multiple isoforms of the same ion transporter which are compartmentalized to different membranes and perform different functions. My current research projects examine ion transport in gastrointestinal and mammary epithelia. Intestinal epithelial cells are difficult to establish in primary culture, but we have been able to establish primary cultures of human and rabbit colonic epithelial cells which exhibit hormone-sensitive ion transport. We are using these preparations to study the molecular regulation of Cl- transport in human colonocytes as well as the ontogenic regulation of Cl transport in the rabbit colon. We had earlier demonstrated that the rectum in cystic fibrosis (CF) subjects exhibits impaired Cl transport in response to Ca-, cAMP- and cGMP-stimulation. We are delineating the second messenger-specific protein kinase cascades, their cross-talk and targets, including CFTR in normal human colonocytes. Our studies reveal important differences between these cells and colon carcinoma cell lines. We have found age- and segment-specific differences in the cGMP and Ca2+ signalling cascades regulating Cl- transport in rabbit colonocytes. In recent studies we have identified that critical proximal step(s) in Ca2+ signalling, viz., between hormone and increase in intracellular Ca2+, is lacking in the weanling and neonatal animal but is present in the adult. This may prove to be an important defense mechanism by which the developing animal deals with its changing nutrient milieu.

The roles of prolactin in osmoregulation in non-mammalian species and as a lactogenic hormone in mammals have been well studied. However, its role in regulating fluid transport in mammary epithelia is poorly understood. We recently demonstrated that prolactin can stimulate Na-K-2Cl cotransporter phosphorylation and activity and therefore Cl secretion in mouse mammary epithelial cells. Future studies will examine the molecular basis of prolactin action on fluid transport in epithelia of the mammary gland and amnion. These studies are important in defining fundamental physiological processes of fluid absorption and secretion, which pertain not only to a range of epithelial cells from the cornea to the uterus and colon but also define volume and fluid regulation in non-epithelial cells ranging from oocytes to endothelial cells and fibroblasts.

Rasenick, Mark M., Ph. D.
G protein signaling in neurons was originally described as a linear sequence in which agonist bound to a specific receptor, which in turn activated a single species of G protein. That G protein then went on to activate or inhibit intracellular effectors such as adenylyl cyclase, phospholipase or various ion channels. Over the past several years, it has become clear that G protein signaling is a complex process. Some of this complexity involves direct regulation of receptors and G proteins by a variety of molecules that bind to or phosphorylate receptors (arrestins and receptor kinases) or increase GTPase activity of G proteins (RGS proteins). A more subtle form of regulation appears to be from a series of proteins and lipids that alter the positioning of the molecules of the G protein cascade on the membrane. Some of these are passive positioning molecules, but others, such as microtubules, offer dynamic interactions that both position and activate G proteins. The interface between G proteins and microtubules is bidirectional, as certain bg subunits stabilize microtubules, while a subunits promote rapid microtubule depolymerization. G proteins are highly concentrated at the post-synaptic density and it is hypothesized that their activation leads to cytoskeletal rearrangement and rapid synaptic shape change. It is within this framework, that research in my laboratory is being carried out. Three primary projects diverge from this central theme. The first is designed to explore the relationship of structure and function within the context of G protein signaling. Specifically, we are studying how tubulin transactivated G proteins and how this element of the cytoskeleton can orchestrate G protein signaling. At the same time, we have noted that G proteins can modify the microtubule cytoskeleton. Thus, we are seeking to learn how neurotransmitters might result in rapid changes in synaptic shape and how changes in cell shape (even tiny changes as seen in dendritic spines) could modify neurotransmitter response or responsiveness. Understanding this process could have significant implications for neuronal plasticity as well as for neuropharmacology. We have recently developed a Gsa-GFP fusion protein (modified in the internal sequence) and tool is likely to help us to more clearly understand the relationship between G proteins and the cytoskeleton.

Raychaudhuri, Pradip Ph.D.
Currently, my lab is focused on three distinct projects: (a) Regulators of the mammalian replication genes; (b) Analysis of the role of cullin 4A in tumor development; and (c) Mechanisms of the tumor suppression function of p19ARF.

Regulators of the replication genes: We have identified a new chromatin remodeling protein (yet to be named) that binds to the transcription factor E2F3, which is involved in the regulated expression of the replication genes. This chromatin remodeling protein is related to BRG1, which is mutated in a variety of tumors. Like BRG1, it has a helicase motif and a retinoblastoma-binding motif. Unfortunately, a full-length cDNA clone for this protein has not been isolated. We are in the process of isolating a full-length cDNA clone that will allow us to characterize its function and role in the regulated expression of the mammalian replication genes.

Role of cullin 4A: We study cullin 4A because the gene encoding cullin 4A was shown to be amplified in breast cancers. We recently showed that cullin 4A specifically targets the DNA repair protein DDB2 for proteolysis through the ubiquitin-proteasome pathway. We are planning to test the hypothesis that an enhanced proteolysis of the DNA repair protein DDB2 might be related to the mechanism by which the overexpression of cullin 4A contributes to the development of breast cancer.

Mechanisms of action of p19ARF: The genetic locus encoding the ARF protein is mutated in about 40% of all tumors. ARF acts by increasing the stability of the tumor suppressor p53. Recent studies in my lab provided biochemical evidence for a p53-independent function of the ARF protein. We observed that ARF specifically inhibits E2F-activated transcription, and the inhibition correlates with relocalization of the E2Fs to the nucleolus. Moreover, ARF bypasses a variety of oncogenic pathways to regulate E2Fs. Interestingly, DP1, the DNA binding partner of E2Fs, overcomes the E2F-inhibitory function of ARF. We will be studying the molecular details of the pathway by which ARF inhibits E2Fs and how DP1 blocks the E2F-inhibitory function of ARF. The long-term goal is to investigate the role of E2F-inhibition in the tumor suppression function of ARF.

Skidgel, Randal A., Ph.D.
Sepsis, a leading cause of acute lung injury, causes pulmonary inflammation and increased capillary endothelial permeability and is a potent stimulus for inducible nitric oxide synthase (iNOS) expression. Nitric oxide (NO) plays an important role in regulating lung vascular permeability, and high levels produced during inflammation, or combined with superoxide to form peroxynitrite, are thought to injure the endothelial barrier. Although iNOS is known to be primarily transcriptionally regulated, our evidence shows that iNOS activity and NO production in cytokine-stimulated human lung microvascular endothelial cells (HLMVECs) is highly regulated via G-protein coupled receptor signaling pathways. We have identified a novel phosphorylation site in iNOS that results in the generation of prolonged (90 minutes) “super-high output” NO from endothelial cells. We are currently identifying the novel signaling pathways by which the kinin B1 receptor stimulates iNOS activity. So far, we have identified the heterotrimeric G protein, Gai, and Src kinase as important components of a pathway that leads to activation of MAP kinases and eventual iNOS phosphorylation. Very recently, we have found that B1 receptor activation and super-high output NO result actually promotes recovery of the endothelial barrier, as measured by increases in transendothelial electrical resistance in HLMVEC.

In another set of studies, we are seeking to understand the important roles of regulatory carboxypeptidases in physiological and pathological processes. Known functions include peptide hormone processing, regulation of peptide hormone activity, regulation of fibrinolysis, binding hepatitis B-virus and providing the precursor Arg for nitric oxide synthase. Our studies revealed that carboxypeptidase M (CPM) on the surface of stimulated endothelial cells efficiently converts bradykinin (a B2 receptor agonist) into des-Arg9-bradykinin, a B1 agonist, generating a significant and prolonged output of nitric oxide. In addition, we recently determined the 3 dimensional structure of human CPM and the catalytic subunit of human plasma carboxypeptidase N. These studies have revealed structural features that could affect the orientation of CPM on the cell surface or the interaction of the 50 kDa catalytic subunit of CPN with the 83 kDa non-catalytic subunit that is a member of the leucine-rich repeat family of proteins. We are investigating the structure and function of CPM on the plasma membrane by investigating its role as an important regulator of the B1 kinin system via its ability to generate the B1 receptor agonist and efficiently deliver it to the receptor on the membrane by forming a receptor-enzyme complex.

Solaro, R. John, Ph. D.
Dr. Solaro’s laboratory employs molecular, structural, and integrative approaches to understand the most fundamental mechanisms of control of cardiac function by extrinsic (neuro-humoral) and intrinsic (Starling’s Law) mechanisms. The studies have elucidated molecular mechanisms of cardiac regulation and how they are altered in genetic and intrinsic pathologies, and how drug targeting may modify them. Studies in the Solaro lab demonstrate the feasibility of using transgenesis as an approach for specific alterations in the myofilaments. Results from this approach demonstrate unambiguously that specific changes in the kinetics of myofilament processes are rate limiting and may greatly affect cardiac dynamics. Dr. Solaro and his colleagues have also been able to correlate altered structure and function of the myofilaments to the pathology of diseases genetically linked to the sarcomere and to the injury of ischemia/reperfusion. Major recent accomplishments include the following: (1) Elucidation of the molecular mechanisms of the relaxant effect of b-adrenergic stimulation on the myocardium. These studies first published in a series of 3 papers in Nature identified the major sites (phospholamban and troponin I) of phosphorylation by adrenergic mechanisms and how they change in the beating heart. (2) The first unambiguous demonstration that specific changes in myofilament response to Ca2+ associated with thin filament isoform switching alters cardiac dynamics and make the heart resistant to acidosis and ischemia/reperfusion injury. (3) Identification of C-terminal proteolysis of cardiac troponin I in the molecular pathology of ischemia/reperfusion injury. The American Heart Association listed the elucidation of the role of TnI degradation in cardiac stunning as one of the top ten research accomplishments in the area of heart research. (4) Elucidation of the role of thin filament structure and function as determinants of length dependent activation and Starling’s Law of the Heart. (5) Development of the concept of myofilament Ca-sensitization as pharmacological approach to heart failure. Publication of the first papers demonstrating that inotropic agents, including pimobendan (Acardi) have as their mechanism of action an ability to alter Ca-binding and activity of the myofilaments by direct, unique and stereo-selective actions.

Tyner, Angela, Ph.D.
The Tyner laboratory focuses on identifying molecular mechanisms responsible for proper regulation of epithelial cell renewal. The intestinal epithelium provides an excellent system for studying cell differentiation and cell turnover. Efforts to identify novel regulatory genes expressed in the mouse intestine resulted in the cloning of a novel protein tyrosine kinase named PTK6 (also called Brk for Breast tumor kinase, or Sik for Src-related intestinal kinase). PTK6 is expressed in differentiated cells in the linings of the gastrointestinal tract and skin, and it is activated during differentiation. Although it is not expressed in the normal mammary gland, PTK6 is expressed at high levels in human breast tumors and breast tumor cell lines. PTK6 is expressed in nuclei of normal prostate epithelial cells, but relocalized to the cytoplasm in prostate tumors. Using transgenic and knockout mice, the Tyner lab is trying to determine the role that PTK6 plays during normal epithelial cell differentiation and in cancer, with a focus on intestine and prostate. Biochemical approaches are being used to identify PTK6 substrates, interacting proteins, and signaling pathways. The laboratory is also examining the expression localization and activity of PTK6 in human tumors to determine whether it can be used as a prognostic or diagnostic cancer marker.

In other studies, the laboratory is investigating contributions of cyclin dependent kinase inhibitors p21 and p27 to growth and differentiation in the gastrointestinal tract. The lab found that expression of the p21 gene is induced in a p53-independent manner in differentiating cells of the gastrointestinal tract and during liver regeneration. They are identifying cis-acting elements and trans-acting factors responsible for regulating p21 expression in these tissues. They found that p21 and p27 are differentially regulated in the liver following carbon tetrachloride induced injury, and that p21 status has an impact on the extent of toxin induced liver injury. They are determining the functions of p21 and p27 during liver regeneration in mice with disrupted p21 and p27 genes. Decreased expression of p21 and p27 has been detected in human cancers, and lack of both of these critical cell cycle checkpoint proteins in the mouse may lead to increased susceptibility to developing tumors. The laboratory is examining the roles of p21 and p27 in tumorigenesis in the liver and intestinal epithelium in knockout mice.

Unterman, Terry G., M.D.
Studies in this laboratory have focused on the regulation and function of the IGF system for the last 15 years, progressing bioassay studies of IGF inhibitors in the circulation in poorly controlled diabetes and fasting, to the identification of these inhibitors as IGF binding proteins (IGFBP-1), and then to identifying specific signaling pathways and transcription factors mediating effects of insulin on IGFBP-1 gene expression. Subsequent studies have shown that IGFBP-1 is the major short-term modulator of IGF bioactivity, and that it plays an important role in modulating anabolic functions of growth factors in response to metabolic stress. In addition, studies in this laboratory have identified the IGFBP-1 promoter as a key model system for understanding specific mechanisms mediating effects of insulin on hepatic gene expression. Specifically, we have shown that the ability of insulin to suppress IGFBP-1 promoter activity is mediated through a conserved insulin response sequence, that this effect of insulin is mediated through the protein kinase B, that FOXO forkhead proteins interact specifically with the IRS, and that phosphorylation by PKB suppresses transactivation by FOXO proteins. Ongoing studies in the lab now focus on the role of FOXO proteins in mediating effects of insulin and growth factors on gene expression utilizing a variety of approaches, including in vitro studies of recombinant proteins, DNA/protein interaction studies, cell culture reporter gene assays, and transgenic approaches in mouse models. Current studies are examining specific mechanisms mediating effects of phosphorylation on the cellular trafficking of FOXO proteins, and transactivation. In collaboration with Philip Cohen, University of Dundee, we are characterizing novel kinase sites in these transcription factors. Studies also are in progress to identify novel nuclear partners which interact with FOXO proteins and may contribute to gene regulation in the liver and in reproductive tissues.

Voyno-Yasenetskaya, Tatyana, M.D., Ph.D.
My laboratory is interested in heterotrimeric G proteins, a family of signaling proteins that transduce messages from receptors for extracellular stimuli into cellular responses mediated by effector enzymes or ion channels. Several genome sequencing projects have been completed; however, the functions of most gene products predicted from such endeavors remain uncharacterized. Therefore, characterizing and understanding the function of these gene products is the next step to be taken before the genome sequence information can be exploited. Identification of protein-protein interactions and the changes in such interactions that result from ligand binding or covalent modification are clearly central to understanding the mechanisms of signal transduction and the establishment of intracellular signaling networks. To understand G protein signaling and its biological functions, it is necessary to identify effectors that are activated through physical interaction with a G? or G?? subunits. The yeast two hybrid system is a powerful technique to identify those proteins that physically interact with each other and is a method that is amenable to high throughput analysis. Previous studies have investigated protein interactions in organs using yeast two-hybrid analysis, but so far this type of analysis has not been applied to specific cell types, such as, endothelial cells. Additionally, as endothelial cells are an important component of vascular tissue, studying protein interactions in this cell type will not only provide insights into endothelial cell biochemistry, but may also provide insights into disease conditions, such as, vascular disease. We have initiated the program for systemic identification and characterization of the proteins that interact with alpha subunits of heterotrimeric G12 and G13 proteins in human vascular endothelial cells.

Trainees of this grant will have an opportunity to work in the laboratory participating either in on-going projects or in the novel projects that are related to the G protein biology and pharmacology.

Ye, Richard, M.D., Ph.D.
My laboratory is interested in how phagocytic leukocytes respond to environmental stimuli with activation of special cellular functions. These include the abilities of phagocytes to move towards site of infection (chemotaxis), to engulf bacteria (phagocytosis), to produce superoxide (NADPH oxidase activation) and to release enzymes (degranulation) that help to eliminate invading microbes.

Genetic and clinical evidence has shown that failure of phagocytes to produce superoxide is a cause of compromised innate immunity. On the other hand, inappropriate activation of phagocytes can lead to tissue injury and inflammatory disorders. A number of our graduate students and postdoctoral fellows are investigating the mechanisms that are used by phagocytes to regulate the assembly of NADPH oxidase complex, thereby preventing oxidant production and tissue injury under normal conditions while promoting superoxide production when challenged by pathogenic microbes.

Using immuno-pharmacological approaches, we are investigating how cell surface receptors transduce signals for the activation of a multitude of cellular activities. G protein-coupled receptors (GPCRs) constitute a large group of cell surface receptors that are targets of many therapeutic agents. Our laboratory is interested in GPCRs of the immune system, namely those that mediate phagocyte activation. We have developed methods to screen for small, synthetic ligands for a group of GPCRs, and hope to explore the therapeutic value of these novel agents. Our long-term goal is to gain an understanding of the cellular mechanisms of phagocyte activation and use the knowledge to develop clinically useful agents for therapeutic intervention.