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The Prolactin Family -- Cytokine Receptor Recognition
        The overall goal of this long-term project is to delineate the specific structural and functional interactions involved in prolactin receptor (PRLr) recognition and signal transduction. Human prolactin (hPRL1) is a 23 kDa protein hormone closely related, both functionally and evolutionarily, to human growth hormone (hGH) and placental lactogen. Together, these three hormones are part of the larger family of hematopoietic cytokines, which contains erythropoietin, granulocyte-colony stimulating factor, interleukin-6, interleukin-4, and others. Proteins in this family share a common structural fold and recognize a conserved family of cell surface receptors. Although best known for its traditional role as a pituitary-derived hormone, recent research has established important autocrine/paracrine functions of hPRL in the growth and development of a diversity of tissues. Multiple breast and prostate cancer cell lines express hPRL and its cell-surface receptor (hPRLr). hPRL has mitogenic and angiogenic functions in these tumors and increases cancer cell motility. The biology of peripheral hPRL synthesis is distinct from the pituitary, including alternative mechanisms for transcriptional regulation, RNA splicing, and hormone storage and secretion. Whereas a majority of pituitary-derived PRL is secreted as the full-length, unmodified protein, glycosylated, phosphorylated and proteolytically-cleaved variants of PRL have been identified. Research has demonstrated functional consequences of these modifications, some of which may act to counter the tumorigenic effects of native hPRL.

        Our primary objective is to identify, at an atomic level, the precise intermolecular interactions responsible for PRL receptor recognition specificity. Towards this goal, we have determined the tertiary structure of human prolactin using NMR spectroscopy (see below). We hypothesize that distinct subsets of PRL residues are responsible for differences in receptor recognition specificity displayed by PRL and its variants towards PRLr isoforms. We are currently using NMR spectroscopy to investigate the structural interactions between prolactin and the extracellular domain of the PRLr and its ΔS1 isoform. Once identified, the structural epitopes on the surface of prolactin for receptor recognition will be compared to the functional epitopes as defined by mutagenesis of the interacting residues, followed by measurements of receptor binding kinetics and cellular assays of prolactin signaling. We will also investigate the consequences of various post-translational modifications, with demonstrated importance to prolactin function, on both the structure of the hormone and its interactions with the PRLr isoforms. This research will aid the development of potential therapeutic agents designed to modulate specific activities of PRL, including PRLr antagonists, which will have significance for the treatment of breast and prostate cancer.

        We originally determined the solution structure of hPRL using NMR spectroscopy and confirmed that the hormone adopts the conserved topology of the hematopoietic cytokines, consisting of four primary a-helices bundled together lengthwise. The helices span 25-32 residues in length and are interrupted by two stretches of residues, one between the 1st and 2nd helices and another between the 3rd and 4th helices, which correspond to the two long loops necessary for the “up-up down-down” topology of the four-helical bundle. These two long loops were demonstrated to be highly dynamic and mostly unstructured in solution according to an analysis of their backbone atom NMR chemical shifts and NMR relaxation rates. Experimentally, structure determination was frustrated by multiple technical limitations including reversible oligomerization of the hormone in solution resulting in low signal-to-noise and broad linewidths of NMR signals. Recently, a greatly improved solution structure of hPRL was reported where the investigators overcame these technical problems through the use of a higher field (800 MHz) NMR spectrometer, incorporation of a new class of NMR structural restraints known as residual dipolar couplings, and altered solution conditions (pH 8.0 and 37 °C) where reversible oligomerization of the hormone is minimized. This newly reported solution structure of hPRL represents an improvement over our original model and clarifies subtle structural features of the hormone. We are currently intepreting all of our results using this updated structural model.

Polymorphic Drug Metabolizing Enzymes
        Xenobiotics (drugs, poisons, pollutants, etc.) are metabolized in the human body by a variety of enzyme pathways. The general consequence of these enzymatic reactions is to "detoxify" the xenobiotic and target it for excretion. As organisms are continually exposed to unexpected and diverse chemicals, these enzymatic pathways must be adaptive to assure proper detoxification. Many mechanisms for adaptation exist. For example, expression of some enzyme systems is induced upon exposure to specific molecules. A second mechanism, which operates on the species level, involves genetic diversity in the activities of many competing metabolic pathways. Upon exposure of a population to a novel xenobiotic, variability within the balance of these competing pathways results in heterogeneous metabolism throughout the population. Some individuals will efficiently detoxify and eliminate the xenobiotic, while others may produce metabolites that are more or less toxic than the original compound. This same metabolic diversity complicates the administration of pharmaceutical agents to combat disease, resulting in variable levels of efficacy and toxicity. Ideally, the selection and dosing of individual medications would be specifically tailored to a predicted response within an individual. The scientific study of this genetic diversity and its relation to the administration of pharmaceuticals is the focus of the developing field of pharmacogenetics.
        Our overall goal is to understand the molecular mechanisms by which genetic polymorphisms within the protein sequences of enzymes modulate their relative role in drug metabolism and, consequently, on the variable efficacy and toxicity of administered pharmaceuticals.  Thiopurine S-methyltransferase (TPMT) metabolizes the class of 6-thiopurine medications, including 6-mercaptopurine, 6-thioguanine and azathioprine, and has been extensively investigated over the past few decades. Large variations of TPMT activity exist in humans and a variety of genetic polymorphisms in the TPMT protein sequence have been identified. Approximately 10% of people are heterozygous for one of the polymorphisms, resulting in an intermediate reduction of their cellular TPMT activity. More seriously, 1 in 300 people are homozygous and almost completely enzyme deficient. A clear relationship has been established between these variations in TPMT activity and the risk of life-threatening hematopoietic toxicity as well as the efficacy of 6-thiopurine medications.
fig2
        We have determined the three-dimensional structure of TPMT using NMR spectroscopy (figure above) and found it to adopt the topology of the conserved family of SAM-dependent methyltransferases. We have also characterized the consequences of SAM-binding on the conformation and molecular dynamics of the TPMT polypeptide backbone (figure below). Previous research has established that decreased tissue TPMT activities are the result of increased susceptibility of the polymorphic enzymes to proteasomal degradation. We are interested in analyzing the consequences of the polymorphic mutations on the structural and functional properties of TPMT in order to characterize the molecular basis for increased susceptibility to intracellular degradation. We hypothesize that the polymorphisms destabilize the tertiary structure of TPMT and have only minimal effects on their enzymatic activity. Although the structural basis for destabilization has many possibilities, we will specifically distinguish whether the polymorphs adopt an alternative static conformation or, more likely, produce a dynamic and fluctuating structural state of the proteins. Our current results suggest a simple model where an early intermediate in the equilibrium unfolding pathway for TPMT could represent a single destabilized state independently accessed, to varying degrees, by multiple polymorphs under native conditions. In the future, similar studies will be extended to other drug metabolizing enzymes.
fig34

        Note: A poster is available for download summarizing all of our work on psTPMT to date: psTPMT poster (2.7 MB).

The GLUT4-tethering protein, TUG.
        This project focuses on the interactions between the insulin-regulated glucose transporter, GLUT4, found in muscle and adipose cells and a recently discovered protein, TUG, that regulates GLUT4 trafficking. Discovered by our collaborator, Dr. Jonathan Bogan, TUG binds directly to GLUT4-containing vesicles and tethers them intracellularly. In response to insulin, TUG releases GLUT4 allowing translocation to the plasma membrane. Like many other proteins, TUG is composed of a modular array of independent protein domains. Our long term goal is to determine the tertiary structures of these TUG domains, to structurally characterize their interactions with each other and with a number of associated proteins, and ultimately to develop a detailed molecular model for TUG-regulated GLUT4 trafficking. A combination of sequence analysis and experimental studies has identified a number ubiquitin-like (UBL) domains in TUG. We have chosen these UBL domains as the initial focus of our structural studies because of (1) their demonstrated functional importance in TUG-mediated GLUT4 tethering and release, (2) the clear delineation of their structural domain boundaries based on sequence alignment, and (3) a pre-existing knowledge base of their potential interactions partners based on the conserved functions of homologous UBL domains in other proteins. The results of these studies will  benefit diabetes research both by contributing to a better understanding of the cellular mechanism for insulin-regulated GLUT4 trafficking, and also by structurally characterizing novel targets for the rational design of pharmaceutical agents with the potential to modulate cellular glucose uptake.

        As part of our ongoing effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10 – 83 of full length TUG and confirms a b-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and note that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the overall activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal di-glycine (“GG”) motif required for conjugation to an acceptor lysine and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the “Ile-44 hydrophobic face” typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site.

The figure to the right displays various structural properties of the N-terminal ubiquitin-like domain in TUG. Starting in the upper left and progressing clockwise:

Superposed Ca traces for the ensemble of 20 NMR structures, rainbow colored from red at the N-terminus to blue at the C-terminus, prepared using MOLSCRIPT (Kraulis 1991).

Backbone ribbon diagram for a representative member of the NMR ensemble demonstrating the b-grasp topology conserved within this protein family, prepared using MOLMOL (Koradi et al. 1996).

Electrostatic potential mapped onto the molecular surface of the TUG-UBL1 tertiary structure, with coloring in shades of red representing negative potential (q/d, charge per distance) from -5.5 to -1.5 and shades of blue representing positive potential from 1.5 to 5.5, as calculated using MOLMOL (Koradi et al. 1996).

NMR-derived order parameters (S2) are mapped onto a backbone ribbon diagram with coloring from red to light-blue for S2 values from 0.7 to 1.0, respectively. Prolines and degenerate residues are colored gray. Side chains are shown for the four residues requiring conformational exchange (Rex) terms during the Model-free analysis, with coloring according to S2 values.



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