The Aβ42 peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low temperature and low salt can stabilize disc-shaped oligomers (pentamers) that are significantly more toxic to murine cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the β-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C-terminus is protected from solvent exchange and which have a turn conformation placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Aβ42 oligomers to fibrils involves the association of these loosely aggregated strands into β-sheets whose individual β-strands polymerize in a parallel, in-register orientation and are staggered at an inter-monomer contact between Gln15 and Gly37.

The time-dependent complete-active-space self-consistent-field (TD-CASSCF) method for the description of multielectron dynamics in intense laser fields is presented, and a comprehensive description of the method is given. It introduces the concept of frozen-core (to model tightly bound electrons with no response to the field), dynamical-core (to model electrons tightly bound but responding to the field), and active (fully correlated to describe ionizing electrons) orbital subspaces, allowing compact yet accurate representation of ionization dynamics in many-electron systems. The classification into the subspaces can be done flexibly, according to simulated physical situations and desired accuracy, and the multiconfiguration time-dependent Hartree-Fock (MCTDHF) approach is included as a special case. To assess its performance, we apply the TD-CASSCF method to the ionization dynamics of one-dimensional lithium hydride (LiH) and LiH dimer models, and confirm that the present method closely reproduces rigorous MCTDHF results if active orbital space is chosen large enough to include appreciably ionizing electrons. The TD-CASSCF method will open a way to the first-principle theoretical study of intense-field induced ultrafast phenomena in realistic atoms and molecules.PACS numbers: 32.80. Rm, 42.65.Ky

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.

The ␤-amyloid peptide (A␤) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer disease. A␤ is produced by the sequential cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases. Cleavage of APP by ␥-secretase also generates the APP intracellular C-terminal domain (AICD) peptide, which might be involved in regulation of gene transcription. APP contains three Gly-XXX-Gly (GXXXG) motifs in its juxtamembrane and transmembrane (TM) regions. Such motifs are known to promote dimerization via close apposition of TM sequences. We demonstrate that pairwise replacement of glycines by leucines or isoleucines, but not alanines, in a GXXXG motif led to a drastic reduction of A␤40 and A␤42 secretion. ␤-Cleavage of mutant APP was not inhibited, and reduction of A␤ secretion resulted from inhibition of ␥-cleavage. It was anticipated that decreased ␥-cleavage of mutant APP would result from inhibition of its dimerization. Surprisingly, mutations of the GXXXG motif actually enhanced dimerization of the APP C-terminal fragments, possibly via a different TM ␣-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production.The progressive deposition of ␤-amyloid peptide (A␤) 3 leading to the formation of senile plaques is an invariant feature of Alzheimer disease. A␤ is a 39 -43-amino acid peptide, with two major isoforms of 40 and 42 amino acids (1, 2). A␤ is produced by the amyloidogenic cleavage of its precursor, the amyloid precursor protein or APP (3).The amyloidogenic processing of APP is initiated by ␤-cleavage within the lumenal/extracellular domain of the protein.The ␤-cleavage of APP is performed by the BACE proteins (BACE1 and -2) that are integral membrane proteins belonging to the aspartyl protease family (4 -8). ␤-Cleavage produces a 99-amino acid, membrane-anchored APP C-terminal fragment (␤CTF), which is further cleaved by the ␥-secretase activity to generate A␤. The ␥-secretase activity is contained in a high molecular weight multiprotein complex formed at least by the following proteins: a presenilin (PS1 or PS2), nicastrin (Nct), Pen-2, and Aph-1 (9). The activity of the ␥-secretase complex is also required for the generation of the intracellular fragment named (APP intracellular C-terminal domain (AICD). AICD was shown to translocate to the nucleus (10, 11), and there is growing experimental evidence suggesting a role for AICD in the regulation of gene transcription (12-17) even if the identity of APP target genes remains a matter of debate (18). The ␥-secretase complex, therefore, plays a central role in the onset and progression of Alzheimer disease not only because proteolysis of ␤CTF controls the production of A␤, but it also controls the intracellular signaling associated with APP, which in turn might regulate the expression of genes involved in the disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "ad...

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A new method to calculate the atom-atom dispersion coefficients in a molecule is proposed for the use in density functional theory with dispersion (DFT-D) correction. The method is based on the local response approximation due to Dobson and Dinte [Phys. Rev. Lett. 76, 1780 (1996)], with modified dielectric model recently proposed by Vydrov and van Voorhis [J. Chem. Phys. 130, 104105 (2009)]. The local response model is used to calculate the distributed multipole polarizabilities of atoms in a molecule, from which the dispersion coefficients are obtained by an explicit frequency integral of the Casimir-Polder type. Thus obtained atomic polarizabilities are also used in the damping function for the short-range singularity. Unlike empirical DFT-D methods, the local response dispersion (LRD) method is able to calculate the dispersion energy from the ground-state electron density only. It is applicable to any geometry, free from physical constants such as van der Waals radii or atomic polarizabilities, and computationally very efficient. The LRD method combined with the long-range corrected DFT functional (LC-BOP) is applied to calculations of S22 weakly bound complex set [Phys. Chem. Chem. Phys. 8, 1985 (2006)]. Binding energies obtained by the LC-BOP+LRD agree remarkably well with ab initio references.

Processing of amyloid precursor protein (APP) by ␥-secretase is the last step in the formation of the A␤ peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618 -660), we show that the TM domain forms an ␣-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the -cleavage site. This helix-to-coil transition is required for ␥-secretase processing; mutations that extend the TM ␣-helix inhibit cleavage, leading to a low production of A␤ peptides and an accumulation of the ␣-and ␤-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM ␣-helix. The most unusual feature of APP proteolysis is the intramembraneous cleavage by the ␥-secretase complex. The mechanism of proteolysis is of considerable interest because of its role in (i) generating the A␤ peptides associated with Alzheimer's disease and (ii) releasing the AICD involved in APP-dependent gene transcription. Several cleavage sites have been identified that generate different length A␤ peptides. The ␥-cleavage site cuts the APP sequence in the middle of the TM domain to predominantly produce the A␤40 peptide, and to a lesser extent the A␤42 peptide. However, A␤42 has a higher propensity to form aggregates than the shorter isoforms and is the most toxic peptide generated by ␥ cleavage (3). There is another cleavage site (4), referred to as the -cleavage site, a few residues downstream between Leu-645 and Val-646 that has been identified by N-terminal sequencing of the AICD peptide (4). An open question has been whether the same enzyme activity is responsible for both the ␥-and -cleavage sites.The ␥-secretase complex has a diverse set of type I membrane protein substrates. Notch, Cd44, ErbB4, and E-cadherin are cleaved by the ␥-secretase in vivo. For each of these substrates, truncation of the extracellular domain to just a few amino acids is required to bind to the ␥-secretase complex. These substrates are all cleaved near the intracellular TM-JM boundary. However, like APP, Notch, and Cd44 are also cleaved in the middle of the TM domain, although their sequences are not conserved (see SI).To address the mechanism of intramembrane proteolysis, we focus on the structure of the TM domain of APP in membrane bilayers. Proteolysis requires local unraveling of the helical secondary structure of the TM domain to expose backbone carbonyl carbons for nucleophilic attack by polarized water in the enzyme active site. This requirement raises the question of whether there are sequence motifs in the TM domain of APP that destabilize the helical structure in cell membranes in a fashion similar to that proposed for the conserved Asn-Pro sequenc...

Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (⌬5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, ⌬5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. ⌬5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the ⌬5TpoR mutant appears to enhance erythroid differentiation when compared with the Tpo-activated wild-type TpoR. Strikingly, individual substitution of K507 or W508 to alanine also induces constitutive TpoR activation, indicating that the K and W residues within the amphipathic KWQFP motif are crucial for maintaining the unliganded receptor inactive. These residues may be targets for activating mutations in humans. Such a motif may exist in other receptors to prevent ligand-independent activation and to allow signaling via multiple flexible interfaces. IntroductionThe thrombopoietin receptor (c-mpl or TpoR) and its ligand thrombopoietin (Tpo) regulate the proliferation of megakaryocytic progenitors, their differentiation into mature megakaryocytes, and formation of platelets. 1,2 Tpo or TpoR knockout mice exhibit a significant reduction of megakaryocytes and circulating platelets, and they also show reduced number of hematopoietic stem cells (HSCs) in the bone marrow, indicating a role for the TpoR and its ligand in HSC self-renewal. 3 Like the erythropoietin receptor (EpoR), the TpoR is thought to function as a homodimer after binding Tpo. Downstream signaling pathways activated by the TpoR are triggered by 2 cytoplasmic tyrosine kinases, the Janus kinase (Jak) 2, and Tyk2. [4][5][6] Upon receptor activation, phosphorylated tyrosine residues in the cytoplasmic domain of the TpoR and in Jaks provide docking sites for the SH2 domains of many signaling proteins, such as STAT3 and STAT5 (the signal transducers and activators of transcription 3 and 5), shc, SHIP, Grb2, and PI3K. 4,5,[7][8][9] Several activating mutations in the TpoR have been identified. In one, the envelope protein of the myeloproliferative virus replaces the extracellular domain of the receptor and activates it by oligomerization. 10 Introduction of a cysteine residue in the extracellular domain of the TpoR at a position equivalent to that of the constitutively active R129C EpoR mutant 11,12 leads to active disulfide-bonded TpoR dimers. 13 Replacement of S498 (or S505) in the transmembrane (TM) domain by asparagine results in constitutively active receptors, 9,14 most likely by ligand-independent dimerization. 9,10,15 Deletion of the membrane distal extracellular cytokine receptor module of the TpoR results...