Presenilin-1 (PS1) and presenilin 2 (PS2) are proposed to be transmembrane aspartyl proteases that cleave amyloid precursor protein and Notch. PS1-and PS2-mediated activities were individually characterized using blastocyst-derived (BD) cells and membranes from PS1 ؉/؊ -PS2 ؊/؊ and PS1 PS2؉/؉ mice, respectively. The relative amounts of PS1 and PS2 in the various BD cells were determined from the intensities of the anti-PS1 and anti-PS2 immunoblot signals by comparison with standard curves using radiolabeled PS1 and PS2 standards produced by in vitro transcription and translation. Cellular membranes from wild type, PS1, and PS1 PS2؊/؊ BD cells generated the A␤40 and A␤42 products from the C100FLAG substrate. PS1-associated ␥-secretase displays considerably higher specific activity than PS2-associated ␥-secretase. Moreover, the PS1 PS2؊/؊ BD cells and corresponding membranes exhibited much higher ␥-secretase activity as compared with other BD cells and membranes. The PS1-mediated ␥-secretase activity correlated better with the amount of PS1 that is modifiable by a photoactivated active sitedirected ␥-secretase inhibitor rather than total PS1; hence, only a small portion (<14%) of the PS1 in wild-type membranes appears to be engaged in an active ␥-secretase complex. This finding suggests that PS1 may serve other biological functions in addition to that associated with its ␥-secretase activity. Furthermore, the PS1 ␥-secretase complex and the PS2 ␥-secretase complex activities can be discriminated on the basis of their susceptibility to inhibition by a potent ␥-secretase inhibitor. The distinct yet overlapping enzymatic properties of the PS1 ␥-secretase complex and the PS2 ␥-secretase complex imply that these two putative aspartyl class proteases may contribute to different biological processes.

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron-electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.

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The method of site-directed spin labeling (SDSL) utilizes a stable nitroxide radical to obtain structural and dynamic information on biomolecules. Measuring dipolar interactions between pairs of nitroxides yields internitroxide distances, from which quantitative structural information can be derived. This study evaluates SDSL distance measurements in RNA using a nitroxide probe, designated as R5, which is attached in an efficient and cost-effective manner to backbone phosphorothioate sites that are chemically substituted in arbitrary sequences. It is shown that R5 does not perturb the global structure of the A-form RNA helix. Six sets of internitroxide distances, ranging from 20 to 50 A, were measured on an RNA duplex with a known X-ray crystal structure. The measured distances strongly correlate (R(2) = 0.97) with those predicted using an efficient algorithm for determining the expected internitroxide distances from the parent RNA structure. The results enable future studies of global RNA structures for which high-resolution structural data are absent.

␤-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid ␤ peptide A␤42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC 50 ϳ 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose-and timedependent lowering of brain A␤40 and A␤42. BACE1 inhibition also led to a robust brain secreted (s)APP␤ lowering that was accompanied by an increase in brain sAPP␣ levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (Ϫ/Ϫ) mice, in agreement with previous studies, this effect was also observed in 15-dayold heterozygous (ϩ/Ϫ) mice, but it was not evident in 30-dayold and 2-year-old BACE1 KO (Ϫ/Ϫ) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant A␤ lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major ␤-site cleavage enzyme for APP and that its inhibition can lower brain A␤ and redirect APP processing via the potentially nonamyloidogenic ␣-secretase pathway, without significantly altering NRG-1 processing.