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Protease Sensitivity Map of the Highly Structured Heterodimerization Domain (HD) of the Human Notch 2 Receptor in the Presence and Absence of the Furin Cleavage Loop
Author(s) -
Cheng Catherine Yanan,
Cabral Kimberly S,
Khan Aliya,
VardarUlu Didem
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.790.10
Subject(s) - furin , notch signaling pathway , protease , microbiology and biotechnology , cleavage (geology) , receptor , proteolysis , biology , chemistry , trypsin , biochemistry , signal transduction , enzyme , paleontology , fracture (geology)
Notch receptors of the highly conserved signaling pathway regulate cell fate decisions in metazoans. Normal activation of the receptor depends on a tightly regulated, ligand dependent, proteolytic cleavage within the extracellular Heterodimerization Domain (HD). Aberrant Notch signaling is associated with a multitude of diseases. The HD consists of an intertwined α/β sandwich around a conserved hydrophobic core, a hotspot for many disease‐inducing point mutations. HD harbors two of the three Notch specific proteolytic cleavage sites: S1, cleaved by a furin‐like protease during receptor maturation, and S2, by an ADAM‐type metalloprotease rendering the receptor susceptible to the activating γ‐secretase cleavage in the membrane. Here, we present our study on the accessibility of specific protease sites within the human Notch2 HD in the presence and absence of the unstructured loop around the S1 site. We used limited proteolysis with trypsin and chymotrypsin followed by Matrix‐assisted Laser Desorption Ionization‐Time of Flight mass fingerprinting and C18 reverse phase Liquid Chromatography to create a relative protease susceptibility map for HD and compared the experimental results to the structure based predictions. Together with other stability data on specific HD mutants, the presented information provides further insight into the mechanism of Notch activation both in the normal and diseased states.