Our current scientific interests focus primarily on investigating the structural basis of signaling by the cytokines that interact with class II receptors (i.e., IL-10, IFN-γ, etc.), aiming at structural characterization of their molecular components (cytokine, receptor, Janus kinase) and interactions. The ultimate goals of this research are to understand the details of signal transduction, specific to a particular cytokine-receptor-JAK system, and to correlate the structural properties of this system with its biological activity, paving a foundation for novel or improved therapeutic interventions targeting these signaling pathways. Although we primarily use x-ray crystallography in these studies, our research techniques also extend into biochemistry and molecular biology. Additional molecules that we are currently studying include defensins and secretoglobins/uteroglobins.

Structural Basis of Signaling by Cytokines. Over 100 cytokines described to date represent a class of extracellular soluble molecules with primary roles in conveying various intercellular messages or initiating a wide range of transmembrane signals. These signals (i.e., messages) initiate and regulate a very broad range of critical physiological and immunological functions that include growth, differentiation, cell division, apoptosis, inflammation, immunity, repair, fibrosis, etc. Not surprisingly, cytokines and their downstream molecules play critical roles in both the functioning of healthy biological systems and in the response of these systems to pathologies. The sequence conservation across the whole class of cytokines is rather low. These proteins are classified in various ways, based on either their molecular properties or their biological effects. Cytokines are produced by most cell types, primarily in response to molecular stimuli, and act through interaction with receptors. The latter are primarily integral membrane proteins that belong to several classes and are usually coupled with specific intracellular proteins. Characteristic to all cytokines is their high receptor affinity, often in the nanomolar range. Despite this high potency, both cytokines and receptors are usually promiscuous. The initial signal (or message) leads to conformational changes of the cytokine receptor molecule and is detected by the receptor-coupled intracellular partner. In the case of chemotactic cytokines (i.e., chemokines) these molecules are heterotrimeric G-proteins. For most of the other cytokines these molecules are Janus kinases. Our interests are focused on structural aspects of the component molecules from this system: the cytokine, the receptor, and the coupled partner, as well as on the interactions among these components at the atomic level. The specific systems we are investigating include those associated with the IL-10 family of cytokines, class II cytokine receptors, and Janus kinases.

Defensins. Defensins are small basic proteins that, until recently, were mainly known for their potent antimicrobial properties. We now know that human beta-defensins are also potent chemoattractants, and that they specifically interact with CCR6, the receptor for the chemokine MIP-3alpha. The mechanisms underlying the antimicrobial and chemotactic activity of defensins are not well understood, although both of these properties are of practical interest. The crystal structures of human beta-defensin-2, solved by my laboratory, were the first published for human beta-defensins. These structures revealed a topology and an oligomerization mode for defensins that had not been reported previously. Our findings shed new light on a possible mechanism for the antimicrobial properties of these proteins, and we recently extended our research by solving the x-ray structure of another human beta-defensin, hBD1. One of our primary goals in studying defensins is to establish the molecular basis of their chemotactic properties, which greatly complements our interest in chemokines. Additionally, we will focus on determining the mechanism of the antimicrobial activity of these proteins. Future studies will involve numerous mutant proteins and will be complemented by extensive biological assays.

Secretoglobins. The family of secretoglobins (SCGBs) consists of a group of small (~10 kDa) secreted proteins with a cytokine-like properties. The most extensively studied member of this family is the Clara cell secretory protein (CSSP), also called secretoglobin 1A1 (SCGB1A1 or CC10), which is primarily found in bronchial epithelial cells. It was suggested that the main physiological role of SCGB1A1 may be as an inhibitor of inflammation. The main reason for this suggestion is the inhibition of phospholipase A2 by SCGB1A1. While SCGB1A1 is present in normal epithelial cells, prostate cancer cells lose SCGB1A1, suggesting a role for the protein in inhibiting malignant growth, a proposition partly supported by in vitro studies. Other properties (i.e., activities) of SCGB1A1, including immunomodulation, progesterone binding, substrate of transglutaminase, protease inhibition, calcium binding, etc., have also been reported in the literature. Despite a number of quite extensive studies of SCGB1A1, the primary physiologic function of this protein remains unknown. In 2001, Dr. Shioko Kimura (Laboratory of Metabolism, CCR) and her collaborators identified another member of the secretoglobin family, the secretoglobin 2A2 (SCGB3A2), also called the uteroglobin-related protein 1 (UGRP1). Even less information is currently available on the biological role and properties of this cytokine-like small (~10kDa) protein compared to SCGB1A1. UGRP1 is predominantly expressed in the pulmonary airways epithelium. It has anti-inflammatory and growth factor activities. Also, it has been reported that UGRP1 is expressed in human carcinomas and it was suggested that this protein may potentially be used as a marker for diagnosis of pulmonary tumors. In collaboration with Dr. Kimura, we are focused on further characterization of mouse and human UGRPs, including structural studies.