Abstract/Details

Characterization of roles for the disintegrin and metalloproteases, kuzbanian and tumor necrosis factor-alpha converting enzyme, in mammalian Notch signaling


2009 2009

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Abstract (summary)

Notch signaling is a highly conserved cell-cell communication system that is essential not only during embryogenesis but also for the maintenance of tissue homeostasis in adults. Upon ligand binding, the Notch receptor undergoes two proteolytic cleavages releasing the Notch intracellular domain, which then translocates to the nucleus to directly activate Notch target gene expression. The first activating cleavage at site 2 (S2) is the rate-limiting step in signal generation and is mediated by A Disintegrin And Metalloproteases (ADAMs). In the absence of ligand, a negative regulatory region (NRR) within the extracellular domain of Notch maintains the Notch receptor in a metalloprotease-resistant state by burying the S2 site in a hydrophobie pocket, thus preventing Notch activation. Importantly, mutations in the NRR lead to aberrant ligand-independent Notch activation that has been associated with more than half of the T-cell lymphoblastic leukemia (T-ALL) cases in humans.

Two highly related ADAMs; ADAM10/Kuzbanian, and ADAM17/Tumor necrosis factor-alpha converting enzyme have been implicated in Notch signaling. Genetic studies have suggested a requirement for ADAM10 in both the signal-receiving and signal-sending cell; however, biochemical studies employing constitutively active forms of Notch1 have suggested that ADAM17 mediates S2 cleavage. Given these inconsistencies and the lack of mechanistic knowledge of ADAM-mediated S2 cleavage in ligand-induced Notch signaling, the studies described in this dissertation characterize the role of ADAM10 and ADAM17 in ligand-dependent and ligand-independent Notch signaling by using a cell culture based system.

My analyses using a loss-of-function approach demonstrate that ligand-induced Notch1 proteolysis and activation are significantly reduced in both ADAM10-deficient (ADAM10-/-) Mouse Embryonic Fibroblasts (MEFs) and in C2C12 cells treated with ADAM10 specific siRNA duplexes. On the other hand, neither MEFs deficient in ADAM17 activity nor C2C12 cells depleted for ADAM17 protein show losses in Notch signaling. In addition, ADAM10 -/- MEFs stably expressing the ligand Delta-like1 (Dll1) can activate Notch signaling, excluding a role for ADAM10 in the ligand cell. Furthermore, consistent with previous findings, my results implicate a major role for ADAM17 in ligand-independent activation of Notch1 induced by either protein overexpression, calcium chelation, or NRR mutations that destabilize the Notch1 heterodimeric structure and deprotect the S2 site. Together these findings define a specific requirement for ADAM10 in ligand-induced S2 processing and activation of the Notch1 receptor, and implicate ADAM17 in ligand-independent Notch signaling.

Indexing (details)


Subject
Biochemistry
Classification
0487: Biochemistry
Identifier / keyword
Pure sciences; Disintegrin; Kuzbanian; Metalloproteases; Notch signaling; TNF-alpha
Title
Characterization of roles for the disintegrin and metalloproteases, kuzbanian and tumor necrosis factor-alpha converting enzyme, in mammalian Notch signaling
Author
Cagavi Bozkulak, Esra
Number of pages
125
Publication year
2009
Degree date
2009
School code
0031
Source
DAI-B 70/07, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781109236538
Advisor
Weinmaster, Gerry
University/institution
University of California, Los Angeles
University location
United States -- California
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3364016
ProQuest document ID
304852491
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304852491
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