γ-Secretase-regulated Proteolysis of the Notch Receptor by Mitochondrial Intermediate Peptidase

Abstract

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent ␥-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following ␥-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. The Notch signaling pathway is evolutionarily conserved and plays a fundamental role in communication between adjacent cells to influence differentiation, proliferation, survival, and migration (1-3). During its maturation and function as a signal transducer, the Notch receptor undergoes proteolysis by three different enzymes. Notch is processed initially by a furin-like convertase in the trans-Golgi at the S1 site to generate an extracellular domain and a membrane-bound domain, which remain associated during their transport to the plasma membrane.Binding of a ligand to the extracellular domain of Notch stimulates proteolysis of Notch at the S2 site by the metalloprotease tumor necrosis factor ␣-converting enzyme (TACE) and produces a transmembrane fragment referred to as Notch extracellular truncation (N⌬E) 3 (4, 5). The membrane-tethered N⌬E is recruited to the ␥-secretase complex for S3 site cleavage to generate the Notch intracellular domain (NICD) (6 -8). The resultant NICD translocates into the nucleus, where it associates with the transcription factor CBF-1 and with coactivators, such as Mastermind, to initiate gene transcription (9 -12). Notch has mostly been characterized by its transcriptional role in the nucleus, but several studies suggest that it may have functional roles in the cytoplasm (13, 14) as well as in the mitochondria (15)(16)(17)(18)(19)(20) where, in the latter, Notch acts as an interpreter of cellular cues. In this study, we provide evidence that following cleavage by ␥-secretase, NICD is further proteolyzed at a novel S5 site. We identify the protease responsible for this cleavage as the mitochondrial intermediate peptidase (MIPEP), which generates ⌬NICD ( 1752 RQHGQ-…). We showed that Notch function can be modulated by MIPEP-mediated cleavage. Our findings raise the possibility that S5 site proteolysis represents a novel regulatory component of Notch signaling. EXPERIMENTAL PROCEDURES Materials-Chemicals used included ␥-secretase inhibitor N-[N-(3,5-Difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-bu-tyl ester (DAPT) (Calbiochem/EMD) and the EDTA-free complete protease inhibitor mixture tabl...

Alteration of Mitochondrial Proteome Due to Activation of Notch1 Signaling Pathway

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NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation

“…Statistic significance was analyzed with 2-tailed distribution and 2-sample unequal variance Student's t test, 1-way ANOVA followed by Newman-Keuls multiple comparison test, or 2-way ANOVA followed by Bonferroni post tests, as indicated in the figure legends. For survival studies, statistical analysis was determined by log-rank test using http://psort.hgc.jp) and as suggested by a previous study (67). Mitochondrial intermediate peptidase may cleave the N-terminal MTS of NICD1 and release a truncated NICD1 in mitochondria in vitro (67).…”

“…For survival studies, statistical analysis was determined by log-rank test using http://psort.hgc.jp) and as suggested by a previous study (67). Mitochondrial intermediate peptidase may cleave the N-terminal MTS of NICD1 and release a truncated NICD1 in mitochondria in vitro (67). Although ChIP-seq and ChIP-qPCR showed NICD1 recruitment to the mtDNA D-loop, we could not identify a consensus CSL site [G(t)TGGGAAA(c); lowercase letters in parentheses represent letters that can be substituted for the preceding capital letters] in this region.…”

sCD44 Internalization in Human Trabecular Meshwork Cells

“…42 Nuclear CD44 regulates STAT3 and binds to cyclin D1 promoter, leading to cyclin D1 expression and cell proliferation. 43 This suggests that CD44 nuclear trafficking results in an autocrine mechanism.…”