Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.

Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules. Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1. Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family. Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes Cterminal derivatives of Nedd8. Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720 CUL1 -Nedd8 linkage. At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1. These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720 CUL1 -Nedd8 conjugate, but lacks Nedd8 Cterminal hydrolytic activity and poorly processes hyperneddylated CUL1. These results suggest a unique role for DEN1 in regulating the modification of cullins by Nedd8.Nedd8 is a small ubiquitin (Ub) 1 -like protein that plays a critical regulatory role in cell proliferation and development. In fission yeast, Nedd8 is essential for cell viability (1). In animals, Nedd8 is required for development as inactivation of the Nedd8 pathway in either mouse (2) or Drosophila (3) results in embryonic lethality. The critical biological function of Nedd8 is conferred by its biochemical activity as a protein modifier, being covalently attached to nearly all members of the cullin family (4). This modification, neddylation, is reminiscent of the ubiquitination reaction. Neddylation occurs by the formation of an isopeptide-bond linking the ⑀-amino group of a conserved lysine residue typically within the C terminus of a cullin to the carboxyl-end of Nedd8 Gly-76 (5). The enzyme components of the neddylation reaction include a Nedd8-specific E1 activating enzyme comprised of the APP-BP1/Uba3 heterodimer, an E2 conjugating enzyme known as Ubc12 (6), and the ROC1/Rbx1 RING finger protein (7).Using in vitro systems, several studies have shown that Nedd8 activates the ubiquitination of IB␣ (8) or p27 (9), through its conjugation to cullin 1 (CUL1). These reactions are mediated by SCF E3 Ub ligases, in which CUL1 functions as a molecular scaffold (10 -12). Subsequently, it was observed that degradation of HIF-␣ by von Hippel-Lindau tumor suppressor required Nedd8 (13). In this case, Nedd8 was conjugated to CUL2 that assembles the von Hippel-Lindau protein E3 Ub ligase (reviewed in Ref. 14). These studies thus suggest a role for Nedd8 in the assembly of an active cullin-based E3 Ub ligase.We initially reported that conjugation of Nedd8 to CUL1 increases the ability of ROC1-CUL1, a sub-complex within the SCF E3 Ub ligase, to assemble polyubiquitin chains in a reaction catalyzed by the Cdc34 E2 conjugating enzyme (15). S...

The genetic lesion underlying familial British dementia (FBD), an autosomal dominant neurodegenerative disorder, is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We now report that both BRI-L and its wild-type counterpart, BRI, were constitutively processed by the proprotein convertase, furin, resulting in the secretion of carboxyl-terminal peptides that encompass all or part of ABri. Elevated levels of peptides were generated from the mutant BRI precursor. Electron microscopic studies revealed that synthetic ABri peptides assembled into irregular, short fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates fibrillogenic peptides that initiate the pathogenesis of FBD.

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The lipooligosaccharide from Neisseria gonorrhoeae (GC), consists of lipid A, an oligosaccharide core and three branches, ␣, ␤, and ␥. We report the cloning of the gene (lgtG, lipooligosaccharide glycosyl transferase G) encoding the glucosyl transferase of GC that initiates the ␤ chain which consists of a lactosyl moiety. This gene contains a homopolymeric tract of cytidine [poly(C)] and we demonstrate that changes in the number of Cs in poly(C) account for the variation of ␤ chain expression in different GC strains. Biochemical analyses and mass spectrometry clearly attribute the reactivity of mAb 2C7 to the presence of the lactosyl ␤ chain. In addition, we demonstrate that in the absence of the lactosyl group, a phosphoethanolamine is added to generate a new antigenic epitope as evidenced by the gain of reactivity to mAb 2-L1-8. These results show that, like the ␣ chain, the ␤ chain of lipooligosaccharide is subject to antigenic variation.

The lineage selection in human embryonic stem cell (hESC) differentiation relies on both the growth factors and small molecules in the media and the physical characteristics of the micro-environment. In this work, we utilized various materials, including the collagen-carbon nanotube (collagen/CNT) composite material, as cell culture matrices to examine the impact of matrix properties on hESC differentiation. Our AFM analysis indicated that the collagen/CNT formed rigid fibril bundles, which polarized the growth and differentiation of hESCs, resulting in more than 90% of the cells to the ectodermal lineage in Day 3 in the media commonly used for spontaneous differentiation. We also observed the differentiated cells followed the coarse alignment of the collagen/CNT matrix. The research not only revealed the responsiveness of hESCs to matrix properties, but also provided a simple yet efficient way to direct the hESC differentiation, and imposed the potential of forming neural-cell based bio-devices for further applications.

Abstract. The weighted essentially nonoscillatory (WENO) methods are popular spatial discretization methods for hyperbolic partial differential equations. In this paper we show that the combination of the widely used fifth-order WENO spatial discretization (WENO5) and the forward Euler time integration method is linearly unstable when numerically integrating hyperbolic conservation laws. Consequently it is not convergent. Furthermore we show that all two-stage, second-order explicit Runge-Kutta (ERK) methods are linearly unstable (and hence do not converge) when coupled with WENO5. We also show that all optimal first-and second-order strong-stability-preserving (SSP) ERK methods are linearly unstable when coupled with WENO5. Moreover the popular threestage, third-order SSP(3,3) ERK method offers no linear stability advantage over non-SSP ERK methods, including ones with negative coefficients, when coupled with WENO5. We give new linear stability criteria for combinations of WENO5 with general ERK methods of any order. We find that a sufficient condition for the combination of an ERK method and WENO5 to be linearly stable is that the linear stability region of the ERK method should include the part of the imaginary axis of the form [−ιµ, ιµ] for some µ > 0. The linear stability analysis also provides insight into the behavior of ERK methods applied to nonlinear problems and problems with discontinuous solutions. We confirm the assertions of our analysis by means of numerical tests.Key words. stability analysis, Runge-Kutta methods, WENO method, strong-stabilitypreserving AMS subject classifications. 65L06, 65M20 DOI. 10.1137/0506378681. Introduction. The method of lines (MOL) is a general approach for the treatment of time-dependent partial differential equations (PDEs) [24]. The standard MOL involves two steps. The first step is to discretize the spatial variables of the PDE to obtain a large set of initial-value ordinary differential equations (ODEs). The second step is to integrate the ODEs using a time integration method such as a linear multistep or Runge-Kutta (RK) method [4,5].The essentially nonoscillatory (ENO) methods [6,7] and the weighted essentially nonoscillatory (WENO) methods [15,11] are popular and effective nonlinear spatial discretizations for hyperbolic PDEs. These methods are adept at handling the nonsmooth features that arise in the solutions to hyperbolic PDEs. For example, although these methods are formally first-order accurate once a shock is present, they still have uniform high-order accuracy right up to the location of the shock [11]. Specifically, the fifth-order WENO spatial discretization (WENO5) [11], which uses a convex combination of three third-order ENO stencils, is a widely used and robust spatial discretization for numerical solution of hyperbolic conservation laws.The three-stage, third-order strong-stability-preserving (SSP) explicit RK (ERK) method, which has most recently been referred to as SSP(3,3) [21], is generally viewed as the time integration method of choice to c...

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