Research / Clinical
Summary
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Diseases/Research Topics
Folding, Interleukin-1b, NMR, Src kinase, Structural Dynamics
While it is critical for a protein's structure to be unique and stable, changes in this structure are imperative for binding and catalysis. It is now evident that many protein recognition processes incorporate conformational changes as a requisite event for function. In this manner, protein structure and dynamics are intimately linked with biological activity.
Conformational changes in proteins may occur upon binding of small molecules, formation of protein-protein interactions, or changes in solution conditions. These conformational changes may result from subtle movements such as the rotation of a peptide bond and/or the stabilization of secondary structure elements. It is also possible that large-scale fluctuations such as the folding of the protein around a prosthetic group, or from changes in the relative orientation of domains (proteins) with respect to one another in a multidomain (multiprotein) complex are essential prerequisites for functional activity.
The Jennings laboratory is interested in characterizing the structural fluctuations in proteins that are essential for regulating subcellular localization and catalysis. In particular, they are investigating the regulatory processes in the Src tyrosine kinase family and in a separate project, the biological function of the cytokine, interleukin-1 beta. Src.
The Src family kinases (SFKs) serve as molecular switches that regulate numerous biological events, including cell growth, division, and differentiation. Tight regulation of these enzymes is very important because of their potential to be cell transforming. Unrestricted SFK activity can lead to cancers, including human colon cancer, breast cancer, lymphoma, and leukemia. While the Src enzymes comprise a large sub-family of non-receptor protein tyrosine kinases (nrPTKs), all are regulated through a single nrPTK, Csk (C-terminal Src kinse). Csk down-regulates kinase activity by phosphorylating a single tyrosine residue in the C-terminus of SFKs.
SFK activity is in equilibrium between active and inactive states due to balance regulation by Csk and its counteracting tyrosine phosphatase. Disturbance of this equilibrium can be induced by up/down regulation of Csk leading to cancer progression.
Owing to this premier regulatory function, Csk has direct effects on many biological functions, including T cell activation, neuronal development, cytoskeletal organization, and cell cycle control. Like the Src enzymes, Csk is a complex protein composed of noncatalytic SH2 and SH3 domains linked to a C-terminal catalytic core. While X-ray diffraction methods have provided a three-dimensional framework, the molecular mechanisms for activity regulation through these noncatalytic sequences is still intensely debated.
Dr. Jennings and her team apply hydrogen-deuterium [H-D] exchange techniques, coupled with electrospray mass spectrometry, fast mixing kinetic techniques, and TROSY NMR methods to define molecular motions and interaction surfaces important for Csk function. They have demonstrated using H-D exchange and mass spectrometric methods that nucleotides induce long-range conformational changes in a simple protein kinase, cAMP-dependent protein kinase.
In an effort to determine long-range motions are important for function, they have initiated an H-D study on Csk. Such findings will provide the first solution-derived link between distal regulatory domains and the active site of Csk. Interleukin-1beta, the pro-inflammatory cytokine interleukin-1beta (IL-1b ), plays many physiological roles and affects nearly all cell types, often in concert with other cytokines and small molecules.
While IL-1b is central in many physiological processes, its roles in inflammation and cell growth make it a contributing factor in several cancers including the progression and severity of lung, hepatic, gastric,and colorectal cancers. The adhesion of circulating cancer cells to capillary walls is a major step in the invasion of cancers throughout the body. IL-1b acts as an upstream activator of VCAM-1 and promoter of metastases. In the case of noncardia gastric adenocarcinoma, tumors arise after acute infection (atrophic gastritis) by Helicobacter pylori, during which IL-1b production is up-regulated in the gastric mucosa.
Several genetic polymorphisms in the IL-1 gene cluster have been correlated with higher incidences of gastric carcinomas following H. pylori infection. Upon stimulation by microbial products, IL-1b is synthesized as a 31 kDa precursor protein which becomes biologically active upon cleavage to the 17 kDa mature form and export out of the cell. IL-1b, however, does not contain a classic leader sequence, and the translocation across the cell membrane occurs through a novel mechanism not presently understood. It is synthesized as a larger precursor protein of 269 residues which is biologically inactive and referred to as ProIL-1ß.
The entire precursor protein is protease sensitive and chemical modification experiments indicate that residues buried in the IL-1ß are exposed in ProIL-1ß. Processing to the mature form and secretion are thought to be closely coupled events, with processing preceding secretion. Based on the available data, it has been suggested that IL-1b must be exported in a partially/fully unfolded form and fold to the biologically active conformation in the extra-cellular space. Thus, Dr. Jennings and her laboratory colleagues are interested in describing the mechanism of export and folding in the extra-cellular space. It is hoped that a detailed understanding of folding of IL-1b may lead to a greater understanding both of the important determinants in the folding of this class of proteins as well as of the structural factors involved in secretion and biological function.
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