Specific Binding of Cholesterol to C99 Domain of Amyloid Precursor Protein Depends Critically on Charge State of Protein.
ABSTRACT: Recent NMR chemical shift measurements of the 99 residue C-terminal fragment of amyloid precursor protein (APP-C99) in the presence of cholesterol provide evidence of binary complex formation between C99 and cholesterol in membrane mimetic environments. It has also been observed that the production of A? protein is enhanced under conditions of high cholesterol concentration. In this study, we investigated the impact of the charge state of C99 on the structure and stability of the C99-cholesterol complex. We observed that the binding of C99 to cholesterol depends critically on the charge state of Glu 693 (E22) and Asp 694 (D23). Evaluation of the pKa values of the Asp and Glu side chains suggests that these residues may be predominantly neutral in existing experimental observations of a stable C99-cholesterol complex at lower pH (characteristic of the endosomal environment), while binding is destabilized near neutral pH (characteristic of the cytoplasm). These observations suggest that specific binding of cholesterol to C99 is a sensitive function of the pH encountered in vivo, with key E22 and D23 residues serving as a "pH switch" controlling C99-cholesterol binding.
Project description:The 99-residue transmembrane C-terminal domain (C99, also known as ?-CTF) of the amyloid precursor protein (APP) is the product of the ?-secretase cleavage of the full-length APP and is the substrate for ?-secretase cleavage. The latter cleavage releases the amyloid-? polypeptides that are closely associated with Alzheimer's disease. C99 is thought to form homodimers; however, the free energy in favor of dimerization has not previously been quantitated. It was also recently documented that cholesterol forms a 1:1 complex with monomeric C99 in bicelles. Here, the affinities for both homodimerization and cholesterol binding to C99 were measured in bilayered lipid vesicles using both electron paramagnetic resonance (EPR) and Förster resonance energy transfer (FRET) methods. Homodimerization and cholesterol binding were seen to be competitive processes that center on the transmembrane G???XXXG???XXXG??? glycine-zipper motif and adjacent Gly709. On one hand, the observed Kd for cholesterol binding (Kd = 2.7 ± 0.3 mol %) is on the low end of the physiological cholesterol concentration range in mammalian cell membranes. On the other hand, the observed K(d) for homodimerization (K(d) = 0.47 ± 0.15 mol %) likely exceeds the physiological concentration range for C99. These results suggest that the 1:1 cholesterol/C99 complex will be more highly populated than C99 homodimers under most physiological conditions. These observations are of relevance for understanding the ?-secretase cleavage of C99.
Project description:The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol-rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER for cleavage by ?-secretase, acts as a lipid-sensing peptide that forms regulatory DRMs in the ER, called mitochondria-associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.
Project description:NhaB is a bacterial Na(+)/H(+) antiporter with unique topology. The pH dependence of NhaB from Vibrio alginolyticus differs from that of the Escherichia coli NhaB homolog. Replacement of Asp-147 with Glu made high H(+) concentrations a requirement for the NhaB activity. Replacement of Asp-147 with neutral amino acids inactivated NhaB.
Project description:?-Secretase is an intramembrane aspartyl protease that cleaves the C99 fragment of amyloid precursor protein to generate extracellular A? peptides. These peptides can oligomerize and aggregate to form amyloid plaques, processes that are widely believed to be causal for Alzheimer's disease. In spite of this critical function, it remains unknown how ?-secretase recognizes C99 and its other substrates, including Notch. In this study we determined E22-K55 as the minimal C99 fragment that was sufficient and required for initial cleavage. Within this fragment, we identified four determinants: (i) a transferable extracellular determinant that differed between C99 and Notch, and which included negative charge in the case of C99, (ii) the amino acid sequence of the C-terminal half of the transmembrane helix, (iii) an invariant lysine or arginine at the intracellular membrane border, and (iv) a positive charge cluster that included the invariant lysine/arginine. We demonstrated that the charge clusters of C99 and Notch receptors could directly bind phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-binding cluster was required for ?-secretase cleavage, and modulation of membrane PIP2 levels strongly affected ?-secretase cleavage levels and the A?40/A?42 ratio, providing support for the importance of the PIP2 interaction in cells. Together, these studies provide critically needed insight into substrate recognition by ?-secretase.
Project description:Hysteresis in equilibrium protein folding titrations is an experimental barrier that must be overcome to extract meaningful thermodynamic quantities. Traditional approaches to solving this problem involve testing a spectrum of solution conditions to find ones that achieve path independence. Through this procedure, a specific pH of 3.8 was required to achieve path independence for the water-to-bilayer equilibrium folding of outer membrane protein OmpLA. We hypothesized that the neutralization of negatively charged side chains (Asp and Glu) at pH 3.8 could be the physical basis for path-independent folding at this pH. To test this idea, we engineered variants of OmpLA with Asp ? Asn and Glu ? Gln mutations to neutralize the negative charges within various regions of the protein and tested for reversible folding at neutral pH. Although not fully resolved, our results show that these mutations in the periplasmic turns and extracellular loops are responsible for 60% of the hysteresis in wild-type folding. Overall, our study suggests that negative charges impact the folding hysteresis in outer membrane proteins and their neutralization may aid in protein engineering applications.
Project description:Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH's (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.
Project description:At neutral pH, oxidation of CH(3)OH --> CH(2)O by an o-quinone requires general-base catalysis and the reaction is endothermic. The active-site -CO(2)(-) groups of Glu-171 and Asp-297 (Glu-171-CO(2)(-) and Asp-297-CO(2)(-)) have been considered as the required general base catalysts in the bacterial o-quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o-PQQ when MeOH is hydrogen bonded to Glu-171-CO(2)(-) and the crystal water (Wat1) is hydrogen bonded to Asp-297-CO(2)(-) is DeltaG++ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated DeltaG++ when MeOH is hydrogen bonded to Asp-297-CO(2)(-) is >50 kcal/mol. The Asp-297-CO(2)(-)...Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDH.PQQ.Wat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297-CO(2)(-) bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297-CO(2)(-), we find that MeOH returns to be hydrogen bonded to Glu-171-CO(2)(-) and Asp-297-CO(2)(-) coordinates to Ca(2+) during 3 ns simulation. The Asp-297-CO(2)(-)...Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation DeltaG++ = 1.1 kcal/mol for Asp-297-CO(2)(-) general-base catalysis of Wat1 hydration of the immediate CH(2)==O product --> CH(2)(OH)(2). By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH(2)(OH)(2) is exothermic.
Project description:Endosomal-autophagic-lysosomal (EAL) dysfunction is an early and prominent neuropathological feature of Alzheimers's disease, yet the exact molecular mechanisms contributing to this pathology remain undefined. By combined biochemical, immunohistochemical and ultrastructural approaches, we demonstrate a link between EAL pathology and the intraneuronal accumulation of the ?-secretase-derived ?APP fragment (C99) in two in vivo models, 3xTgAD mice and adeno-associated viral-mediated C99-infected mice. We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomal-autophagic function. The deleterious effect of C99 was found to be linked to its aggregation within EAL-vesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment. This effect was A? independent and was even exacerbated when ?-secretase was pharmacologically inhibited. No effect was observed in inhibitor-treated wild-type animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation. In some brain areas, strong C99 expression also led to inflammatory responses and synaptic dysfunction. Taken together, this work demonstrates a toxic effect of C99 which could underlie some of the early-stage anatomical hallmarks of Alzheimer's disease pathology. Our work also proposes molecular mechanisms likely explaining some of the unfavorable side-effects associated with ?-secretase inhibitor-directed therapies.
Project description:C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by ?-secretase to release the amyloid-? 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 ? helix, making it well suited for processive cleavage by ?-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.
Project description:The class IV adenylyl cyclase from Yersinia pestis has been engineered by site-specific mutagenesis to facilitate crystallization at neutral pH. The wild-type enzyme crystallized only below pH 5, consistent with the observation of a carboxyl-carboxylate H bond in a crystal contact in the refined structure 2FJT. Based on that unliganded structure at 1.9 A resolution, two different approaches were tested with the goal of producing a higher-pH crystal needed for inhibitor complexation and mechanistic studies. In one approach, Asp 19, which forms the growth-limiting dicarboxyl contact in wild-type triclinic crystals, was modified to Ala and Asn in hopes of relieving the acid-dependence of that crystal form. In the other approach, wild-type residues Met 18, Glu 25, and Asp 55 were (individually) changed to lysine to reduce the protein's excess negative charge in hopes of enabling growth of new, higher-pH forms. These 3 sites were selected based on their high solvent exposure and lack of intraprotein interactions. The D19A and D19N mutants had reduced solubility and did not crystallize. The other 3 mutants all crystallized, producing several new forms at neutral pH. One of these forms, with the D55K mutant, enabled a product complex at 1.6 A resolution, structure 3GHX. This structure shows why the new crystal form required the mutation in order to grow at neutral pH. This approach could be useful in other cases where excess negative charge inhibits the crystallization of low-pI proteins.