gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.

Presenilin and nicastrin are essential components of the ␥-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-␤ precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin aminoand carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of ␥-secretase substrates (APP carboxyl-terminal fragments), and reduction of ␥-secretase products (amyloid-␤ peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the ␥-secretase complex required for the intramembrane proteolysis of APP and Notch.Regulated intramembrane proteolysis of the amyloid-␤ precursor protein (APP), 1 Notch, and other selected membrane proteins is a conserved control mechanism essential for cell signaling (1). The cleavage of Notch within its transmembrane domain is preceded by a ligand-dependent "site-2" cleavage step in the extracytosolic domain. The Notch intracellular domain (NICD) liberated by the subsequent "site-3" intramembrane cleavage translocates into the nucleus to regulate transcription of specific target genes important in cell-fate decision. Intramembrane proteolysis of APP is also preceded by a cleavage step at the ␣-or ␤-site in the extracellular domain. Like NICD, the APP intracellular domain released by intramembrane cleavage at the ␥-site undergoes nuclear translocation to regulate gene expression (2). The amyloid-␤ (A␤) peptides released by the ␤-and ␥-site cleavages are believed to be essential for the pathogenesis of Alzheimer's disease.Proteolytic cleavage within the hydrophobic transmembrane domains of Notch and APP are intimately associated with a multimeric ␥-secretase complex that contains the presenilin-1 or presenilin-2 and the membrane glycoprotein nicastrin. First, presenilin and nicastrin interact stoichiometrically in a high molecular weight complex where the heavily glycosylated nicastrin and the amino-and carboxyl-terminal fragments (NTF and CTF) of the presenilin heterodimers are tightly regulated in a mutually dependent way (3-9). Second, ␥-secretase inhibitors can capture both presenilin and nicastrin, and semipurified presenilin-nicastrin complex has ␥-secretase activity (10 -12). Third, when mutated in cells or animals, either presenilin or nicastrin affects APP and Notch processing (5,(13)(14)(15)(16)(17)(18)(19)(20).Genetic studies in Caenorhabditis elegans have demonstrated that the worm presenilin homologs, sel-12 and hop-1, along with two "anterior-pharynx-defective" genes, aph-1 and aph-2 (the nicastrin homolog), are essential for notch/glp-1 signaling (5, 21-23). Rece...

Single-stranded nucleic acids, which carry multiple negative charges in an aqueous medium at near neutral pH, are found to induce the aggregation and self-assembly of the positively charged alkynylplatinum(II) terpyridyl complexes via electrostatic binding of the platinum complexes to the single-stranded nucleic acids, as revealed by the appearance of new UV-vis absorption and emission bands upon addition of single-stranded nucleic acids to a buffer solution of the complex. Changes in the intensity and pattern of circular dichroism (CD) spectroscopy are also observed, many of which are consistent with the assembly of the platinum complexes into helical structures, via metal⅐⅐⅐metal and ⅐⅐⅐ stacking interactions. The induced spectroscopic property changes are found to depend on the structural properties of the nucleic acids.helicity ͉ noncovalent interactions ͉ platinum complex N ucleic acids are, in a sense, the most fundamental and important class of biomolecules in a living cell. Detection of nucleic acids, analysis of their sequence, structure, and the corresponding properties, as well as their self-replication, interactions with regulation factors, and the details of transcription and translation are among the major foci of numerous biochemical and biophysical research works (1, 2). Single-stranded nucleic acid sensing and characterization are therefore of great importance, which not only can help us to understand how the cell functions and to assist biological research, but also facilitate the development of new tools for disease diagnosis and treatment and new drug developments.It has been known for many years that many of the square planar metal complexes arrange themselves into highly ordered extended linear chain or oligomeric structures in the solid state. Depending on the extent of the metal⅐⅐⅐metal and the ligand ⅐⅐⅐ stacking interactions, different colors could be observed (3, 4). Square planar platinum(II) complexes belong to a particularly interesting class of this type of complexes, as a result of their rich spectroscopic and luminescence properties revealed during the past few decades (3-16).Recently, we have synthesized a number of platinum(II) polypyridyl alkynyl complexes that show interesting spectroscopic and luminescence properties (17-21). One particularly interesting observation is that solvents of different polarity and also polyacrylate could induce the aggregation of the positively charged platinum complexes in organic solvent mixtures (19, 21). As a result, remarkable spectroscopic property changes were observed. Because single-stranded nucleic acids carry multiple negatively charged phosphate functional groups, they are polyanions. It is therefore reasonable to envisage that single-stranded nucleic acids may have the possibility to induce the self-assembly of the square planar platinum(II) complexes in an aqueous environment. Here, we describe the synthesis and characterization of a water-soluble platinum(II) terpyridyl alkynyl complex, [Pt(tpy)C'CC'CCH 2 OH]OTf (1) (Fig. 1), and it...

The presenilins and nicastrin, a type 1 transmembrane glycoprotein, form high molecular weight complexes that are involved in cleaving the beta-amyloid precursor protein (betaAPP) and Notch in their transmembrane domains. The former process (termed gamma-secretase cleavage) generates amyloid beta-peptide (Abeta), which is involved in the pathogenesis of Alzheimer's disease. The latter process (termed S3-site cleavage) generates Notch intracellular domain (NICD), which is involved in intercellular signalling. Nicastrin binds both full-length betaAPP and the substrates of gamma-secretase (C99- and C83-betaAPP fragments), and modulates the activity of gamma-secretase. Although absence of the Caenorhabditis elegans nicastrin homologue (aph-2) is known to cause an embryonic-lethal glp-1 phenotype, the role of nicastrin in this process has not been explored. Here we report that nicastrin binds to membrane-tethered forms of Notch (substrates for S3-site cleavage of Notch), and that, although mutations in the conserved 312-369 domain of nicastrin strongly modulate gamma-secretase, they only weakly modulate the S3-site cleavage of Notch. Thus, nicastrin has a similar role in processing Notch and betaAPP, but the 312-369 domain may have differential effects on these activities. In addition, we report that the Notch and betaAPP pathways do not significantly compete with each other.