Personnel
Igor Prudovsky
http://www.mmcri.org/cmm/prudovsky.html
Center for Molecular Medicine, Maine Medical Center Research Institute.
Non-classical protein secretion - a flexible mechanism of cytokine delivery
A large group of exported proteins do not exhibit a signal peptide in their primary structure. Among them are interleukin (IL) 1? and 1?, fibroblast growth factor (FGF) 1 and 2, secretory transglutaminase, sphingosine kinase 1(SK1), galectins, Annexin (Anx) 1 and 2, and a number of viral proteins. The ability of these proteins to undergo non-classical release attracted considerable attention since they play roles in important physiological processes such as cell growth and differentiation, inflammation and angiogenesis. Our laboratory has been involved in studies of non-classical release of FGF1 since the 1990s. This protein and FGF2 are prototype members of the FGF family. While most of the other FGF family members display a signal peptide in their primary structure and exhibit tissue-specific patterns of expression, FGF1 and FGF2 are signal peptide-less proteins expressed in practically all tissues of the mammalian organism. Unlike mammalian FGF1 and FGF2, FGFs of C. elegans and Dr. melanogaster have signal peptides. We hypothesized that the loss of signal peptides in FGF1 and FGF2 represents a protective evolutionary device. Indeed, while strictly programmed mosaic development of round worms and insects may rely on the regulation of FGF availability solely at the expression level, a less hierarchical and more complex development of chordates might require the evolution of FGFs, whose secretion is not automatic and thus may be finely regulated at the post-translational level.
Although FGF1 is not released at normal conditions, its export is stimulated by a number of cell stresses. Similar stress situations occur in vivo in the course of inflammation and tumor development. FGF1 released from heat shocked cells is represented by a cysteine-dependent homodimer. The FGF1 dimer is a part of a copper-dependent multiprotein release complex which includes the small calcium-binding protein S100A13 and a 40 kDa isoform of the p65 docking protein Synaptotagmin 1. More recently we found that another potent signal peptide-less angiogenic cytokine, IL1?, is released through a similar stress-activated pathway. IL1? is translated as a large precursor (p), and the calpain-mediated cleavage of its N-terminal prodomain results in the production of the mature (m) IL1? form that is released at stress. Interestingly, FGF1 and mIL1?, which are proteins of similar size exhibit strikingly similar 3D structures despite a virtual absence of homology between their amino acid sequences. We demonstrated that S100A13 forms a Cu2+-dependent release complex with IL1?, and heat shock-induced release of IL1? depends both on Cu2+ ions and S100A13. Interestingly, pIL1? is neither cleaved in nor released from NIH 3T3 cells, and it blocks the stress-induced release of FGF1. This observation further indicated the convergence of two export pathways. We found that both IL1? and FGF1 undergo stress-induced transport to the cell membrane. Both proteins and other components of the release complexes are able to permeabilize liposomes consisting of acidic phospholipids.
In the course of the studies of FGF1-mediated angiogenesis, our laboratory found that FGF1 upregulates the expression of Jagged 1, a transmembrane ligand of Notch receptors in endothelial cells. Notch signaling induced by the Jagged and Delta families of Notch ligands is crucial for cell fate determination during most morphogenetic processes including angiogenesis. Further, we demonstrated that the expression of a soluble extracellular form of Jagged 1 (sJg1) in NIH 3T3 cells inhibits Notch signaling and induces a phenotype reminiscent of angiogenic endothelial cells and is indicative of FGF signaling activation. More recently, we found that NIH 3T3 cells with Notch signaling downregulated due to the expression of sJg1 or soluble Delta 1 release FGF1 constitutively. When such cells are cotransfected with constitutively active Notch, FGF1 secretion is blocked. Conversely, Notch activation does not block the stress-induced release of FGF1. Additionally, we recently demonstrated that thrombin, the major protease of the coagulation cascade known to also exhibit mitogenic activity induces FGF1 release in the absence of cellular stress. Significantly, thrombin specifically cleaves Jagged 1 producing an extracellular Jagged 1 fragment, and thrombin-induced FGF1 release is blocked by Notch signaling activation. This body of data demonstrates the existence of an alternative pathway of FGF1 release, which is stress-independent and requires the repression of Notch signaling.
The understanding of molecular mechanisms underlying non-classical FGF1 and IL1? release is of considerable importance because these proteins are involved in the regulation of a plethora of physiological and pathological processes.
The major aims of our laboratory are:
1. To define the mechanisms of transport of FGF1 and IL1? release complexes to the cell membrane.
2. To understand how non-classically exported proteins pass through the cell membrane.
3. To elucidate molecular mechanisms underlying the cross-talk between Notch signaling and FGF1 release.
We are looking forward to cooperation with the IMB especially in the studies of the intracellular transport and membrane penetration of FGF1 and IL1?, where microscopic and biophysical methods of high resolution are required.
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