Functional and biochemical characterization of the negative regulatory region of mammalian Notch

Notch receptors constitute a family of highly conserved transmembrane receptors that transduce signals between neighboring cells during the development of multicellular organisms. This thesis focuses primarily on human Notch1, one of four mammalian Notch receptors. Though Notch1 signals are normally required for proper T-cell development, gain-of-function mutations in Notch1 lead to T-ALL both in humans and in mouse models.; During maturation, mammalian Notch receptors are cleaved by a furin-like convertase, yielding a heterodimer between extracellular (NEC) and transmembrane (NTM) subunits. Signaling is typically activated when ligands bind to NEC, and trigger a series of proteolytic cleavages in NTM, releasing the intracellular portion of NTM (ICN) into the nucleus to upregulate transcription of target genes.; A N&barbelow;egative R&barbelow;egulatory R&barbelow;egion (NRR) is responsible for maintaining Notch heterodimers in a resting conformation in the absence of ligand. The NRR resides entirely external to the membrane, encompassing the LNR repeats of NEC and a novel domain divided by furin cleavage. This thesis describes functional, biochemical, and biophysical studies of the NRR focusing on the following questions: (i) how do the LNR and the novel domain prevent premature activation of Notch receptors, and (ii) how do leukemia-derived mutations within the Notch1 NRR lead to increased levels of signaling?; Chapter Two shows that the LNR and the novel region have different roles in regulating the activity of Notch receptors. The LNR is necessary for preventing premature proteolytic activation, whereas the novel domain is responsible for holding the furin-cleaved subunits together as a heterodimer (hereafter HD, for heterodimerization domain). These findings facilitated the discovery by collaborators in the Aster group that missense mutations in HD occur frequently in primary human T-ALLs. Chapter Three reports biochemical and functional analysis of fifteen leukemia-associated mutations scattered throughout the HD, showing that HD mutations of human Notch1 induce ligand-independent increases in signaling by either promoting subunit dissociation, or by directly exposing the metalloprotease cleavage site. Chapter Four reports preliminary biophysical analysis of mammalian HDs, showing that furin cleavage does not promote major conformational changes. Together, these studies provide new insight into the biochemistry underlying regulation of Notch receptor activation.