To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4375fje; doi: 10.1096/fj.05-4375fje SPECIFIC AIMSAlzheimer's disease (AD) is characterized by excessive deposition of aggregated ␤-amyloid (A␤) peptide of 40-42 residue, which is derived from the amyloid precursor protein (APP) following processing by ␤-secretase and ␥-secretase. We have reported that developmental exposure to Pb up-regulates the expression levels of APP and its amyloidogenic A␤ products late in life. This provided the first evidence for the developmental and environmental link for disturbances in AD-associated proteins. Our aim was to examine whether latent up-regulation in APP expression and A␤ levels are exacerbated by concurrent disturbances in APP processing or A␤ aggregation. PRINCIPAL FINDINGS Nanomolar levels of Pb promote A␤ aggregationIncreased APP expression and/or processing are believed to accelerate AD pathogenesis in humans and animal models of AD due to the increased production of the amyloidogenic peptides A␤ 1-40 and A␤ 1-42 . Environmental agents could exacerbate such a situation by directly promoting A␤ aggregation; therefore, we decided to examine the effects of xenobiotic metals on human A␤ aggregation in vitro.The fluorescent dye 1,1-bis(anilino)napthaline-5,5-disulfonic acid (bis-ANS) was used to probe the conformational properties of A␤ and A␤ aggregates in the absence and presence of metals at various concentrations. Bis-ANS is known to bind aggregated but not monomeric A␤. The non-amyloidogenic A␤ 40-1 was used as a control in these experiments. Among the environmental metals tested, only A␤ solutions containing Pb were observed to bind tightly to bis-ANS, consistent with the presence of A␤ aggregates. The aggrega-

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Following assembly, the anthrax protective antigen (PA) forms an oligomeric translocon that unfolds and translocates either its lethal factor (LF) or edema factor (EF) into the host cell. Here, we report the cryo-EM structures of heptameric PA channels with partially unfolded LF and EF at 4.6 and 3.1-Å resolution, respectively. The first α helix and β strand of LF and EF unfold and dock into a deep amphipathic cleft, called the α clamp, which resides at the interface of two PA monomers. The α-clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins. EF undergoes a largescale conformational rearrangement when forming the complex with the channel. A critical loop in the PA binding interface is displaced for about 4 Å, leading to the weakening of the binding interface prior to translocation. These structures provide key insights into the molecular mechanisms of translocation-coupled protein unfolding and translocation.

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Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin’s translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell’s cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest–host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest–host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest–host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with L-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,L-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation.