The metastatic process involves a coordinated series of tumor cell behaviors, including adhesion process, migration on extracellular matrix proteins and invasion of the basement membrane. During the invasion process, several tumor cell integrins interact with basement membrane (type IV) collagen. Cell binding to specific sites within collagen and subsequent individual signaling pathways has not been correlated. The triple-helical conformation of collagen has recently been documented as an important cellular recognition element. The investigators have developed methodology of constructing """"""""mini collagens,"""""""" and have applied these synthetic proteins for the identification of individual melanoma cell integrin binding sites within collagen. Our initial work led to the """"""""collagen structural modulation"""""""" model for metastatic melanoma cell progression, involving (1) melanoma cell adhesion to triple-helical type IV collagen via a1b1 and a2b1 integrins, (2) recruitmen of the a3b1 integrin to the triple-helical a1(IV)531-543 site, (3) induction of signal transduction involving pp125FAK, (4) protease production (including early induction of a unique about 50 kDa metalloproteinase), (5) dissolution of the basement membrane, resulting in the denaturation of the type IV collagen triple- helix, (6) reduced levels of p125FAK phosphorylation due to integrin binding to denatured collagen, and (7) increased tumor cell motility and invasion. Overall, the progression of melanoma cell events is dictated by the structural state of type IV collagen. The investigators propose to further examine the collagen structural modulation model by using linear, enantiomeric, glycosylated, triple-helical (homo- and heterotrimeric), and clustered a1(IV)531-543 sequence variants to correlate melanoma cell binding and spreading via the a3b1 integrin with (1) induction of pp125FAK phosphorylation and binding and induction of paxillin and/or Grb2 and (2) induction of metalloproteinases, including the novel about 50 kDa metalloenzyme that have been discovered and members of the MMP family. The investigators will examine time courses and levels of induction, and correlate the results to protease production. Intracellular protein interactions will be analyzed by immunoprecipitation analysis and """"""""peptide insertion"""""""" technology. The novel 50 kDa metalloenzyme will be purified and characterized. Overall, our studies could provide new insights into the mechanism of melanoma cell behaviors as well as identifying a novel metalloproteinase produced melanoma cells. The manner in which the protein three-dimensional structure impacts protein-protein interactions, such as receptor binding to ligand, and subsequent signaling activities will also be better understood.
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| Cudic, Mare; Mari, Frank; Fields, Gregg B (2007) Synthesis and solid-phase application of suitably protected gamma-hydroxyvaline building blocks. J Org Chem 72:5581-6 |
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