Michael Hodsdon, MD/PhD

Our primary scientific interest is to better understand how the structural and biophysical properties of proteins contribute to their role in pathophysiology of human disease. NMR spectroscopy is a central tool in our research program used to determine the three-dimensional structures of proteins in solution and to monitor a variety of biophysical properties under physiological conditions. At the moment, we are investigating a number of biomedically important protein systems, which are briefly summarized below. A detailed description of some of our ongoing research efforts can be found below.

Molecular basis for pH-dependent regulation of protein hormone function. The overall goal of this project is to define the biophysical basis for the pH dependence of human prolactin (PRL) and growth hormone (GH)-receptor recognition and to characterize its functional importance during intracellular trafficking of endocytosed hormone-receptor complexes. Secreted protein hormones and cytokines experience multiple regulated variations in solution acidity, or pH, during their functional lifecycles. Briefly, after synthesis and transport into the endoplasmic reticulum, secreted proteins are initially exposed to a near neutral pH, which is later acidified to around 6.0 in the trans-Golgi and to 5.0 in the dense cores of secretory granules. Once released into extracellular fluid, the pH varies from 6.5 – 7.8; but, after recognition and activation of cell surface receptors, endocytosed ligand-receptor complexes experience a step-wise lowering of acidity first to pH ~ 6.5 in mature endosomes, to pH ~ 5.5 in late endosomes, and, if not directed back to the cell surface for recycling, to a lysosomal pH ²⁺ and Ca²⁺ that allosterically regulate protein function. In particular, the imidazole side-chain of histidine residues is ideally suited to serve as the primary sensor of variations in acidity across the pH range 5.5 – 7.5.

We have chosen to focus on the protein hormones, hPRL and hGH, and their respective cell surface receptors (hPRLr and hGHr) due to our previous experience investigating this protein family and our discovery of a nearly one thousand-fold loss of binding affinity of hPRL for its receptor over the narrow range of pH from 7.5 to 6.0; whereas, the affinity of hGH binding to the same receptor remains essentially constant. We hypothesize that this difference in pH dependence depends entirely on structural changes in the intermolecular interfaces of these two hormone receptor complexes. We will also test the hypothesis that acid-induced dissociation of endocytosed hormone-receptor complexes (or lack thereof) regulates intracellular trafficking towards either lysosomal degradation or recycling back to the cell surface. Our approach relies on a combination of measurement of site-specific protonation reactions using both NMR spectroscopy and tandem mass spectrometry, modeling of the thermodynamic linkage between receptor-binding and protonation, high resolution structural analysis of hormone-receptor complexes, and complementary cellular studies. Projects are currently available combining any of the above methodolgies.

Molecular Mechanism for Hematopoietic Cytokine Receptor Activation and Transduction of Cell Signaling. The long-term goal of this project is to delineate the specific structural and functional interactions involved in hematopoietic cytokine receptor activation and signal transduction. However, given our previous experience with the hPRL and hGH receptor subfamily, current efforts are exclusively focused on these two cytokines. Although best known for its traditional role as a pituitary-derived hormone, recent research has established important autocrine/paracrine functions of prolactin (PRL) in the growth and development of a diversity of tissues. PRL and its receptor are expressed in cancers of the breast, prostate and female reproductive tract. PRL has mitogenic and angiogenic function in these tumors and increases cancer cell motility. Whereas a vast majority of pituitary-derived PRL is secreted as the full-length, unmodified protein, in peripheral tissue glycosylated and phosphorylated variants of PRL are found. Research has demonstrated functional consequences of these modifications, some of which may act to counter the tumorigenic effects of unmodified PRL.

Recently, we have successfully expressed, purified and refolded the extracellular domain of the hPRL receptor extracellular domain (ECD), along with its separate fibronectin-like III) subdomains (S1 and S2). The isolated S1 and S2 domains can be prepared in milligram quantities and are both highly soluble and stable in aqueous buffer, allowing the application of high-resolution NMR spectroscopy and other biophysical techniques to the analysis of their structural properties. We hypothesize that the hPRL receptor homodimerizes in at least two alternate conformational states, associated with the active and inactive states of the receptor. Projects are immediately available in the laboratory involving the structural and biophysical characterization of these receptor ECDs.

Polymorphic Drug Metabolizing Enzymes. Research over the past 30 years has demonstrated striking genetic variability in the enzymatic pathways used to metabolize xenobiotics (drugs, poisons, pollutants, etc.) in the human body. This metabolic diversity complicates the administration of pharmaceutical agents to combat disease, resulting in variable levels of efficacy and toxicity from the same dosage of medications applied across a population. 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.

One common biological mechanism for generating diversity in metabolic pathways involves inherited polymorphisms in the protein sequence of enzymes, which appear to target the polymorphic proteins for intracellular degradation. We would like to understand the structural and biophysical mechanisms by which genetic polymorphisms within the protein sequences of these enzymes modulate their relative role in drug metabolism and, consequently, on the variable efficacy and toxicity of administered pharmaceuticals. Our work so far has concentrated on the enzyme, thiopurine methyltransferase (TPMT), which metabolizes the class of 6-thiopurine medications, including 6-mercaptopurine, 6-thioguanine and azathioprine. Large variations of TPMT activity exist in humans and a variety of genetic polymorphisms in the TPMT protein sequence have been identified that target the allelic variants for proteasomal degradation.

We have determined the three-dimensional structure of TPMT using NMR spectroscopy and characterized the consequences of ligand-binding on the conformation and molecular dynamics of the polypeptide backbone. We are currently 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.

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 of 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.

1. Molecular basis for pH-dependent regulation of protein hormone function.
2. Molecular mechanism for prolactin receptor activation and transduction of cell signaling.
3. Biophysical characterization of single-site polymorphisms in drug-metabolizing enzymes targeting them for proteasomal degradation.
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