C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-β (Aβ) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by β-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an α-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of α-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Aβ production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by β-and γ-secretase and resulting amyloid-β production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.The human amyloid precursor protein (APP) 1 is a single-span membrane protein that is alternatively processed by either α-or β-secretase to release its large ectodomain from the cell surface, a process referred to as "shedding". β-Secretase (β-site APP cleaving enzyme 1, BACE1) cleaves APP after Met671, leading to production of the C-terminal 99-residue domain of APP, C99, a single-span membrane protein. Subsequent cleavage of C99 at membranedisposed sites by γ-secretase leads to release of both the amyloid-β (Aβ) peptides and the water-

Gamma-Secretase is a promiscuous protease that cleaves bitopic membrane proteins within the lipid bilayer. Elucidating both the mechanistic basis of gamma-secretase proteolysis and the precise factors regulating substrate identification is important because modulation of this biochemical degradative process can have important consequences in a physiological and pathophysiological context. Here, we briefly review such information for all major classes of intramembranously cleaving proteases (I-CLiPs), with an emphasis on gamma-secretase, an I-CLiP closely linked to the etiology of Alzheimer's disease. A large body of emerging data allows us to survey the substrates of gamma-secretase to ascertain the conformational features that predispose a peptide to cleavage by this enigmatic protease. Because substrate specificity in vivo is closely linked to the relative subcellular compartmentalization of gamma-secretase and its substrates, we also survey the voluminous body of literature concerning the traffic of gamma-secretase and its most prominent substrate, the amyloid precursor protein.

SummaryIt is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimer's disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appears to be a pro-amyloidogenic factor. The recent observation that the Cterminal transmembrane domain (C99, also known as the β-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimer's-cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway-amyloid-β and the intracellular domain of the APP (AICD) -may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies in AD.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.