Competition Between Homodimerization and Cholesterol Binding to the C99 Domain of the Amyloid Precursor Protein

Song, Yuanli; Hustedt, Eric J.; Brandon, Suzanne; Sanders, Charles R.

doi:10.1021/bi400735x

Cited by 112 publications

(191 citation statements)

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“…Yet, the Km is significantly more favorable for the reaction in bicelles, which contains CHAPSO, a cholesterol mimic. Cholesterol is a modulator of both membranes (51,52) and C100 catalysis (53)(54)(55)(56)(57). Indeed, relevant regions of C100 are partitioned well inside the lipid bilayer and specifically bind cholesterol (53).…”

Section: Discussionmentioning

confidence: 99%

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Naing

Kalyoncu

Smalley³

et al. 2018

Journal of Biological Chemistry

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Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen, and retain the predominant cleavage site (His-Thr). The aromatic ring, but not its hydroxyl substituent, in Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala-Thr) and the other the nonpathogenic Aβ48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen indicate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions. INTRODUCTIONIntramembrane proteases (IPs) cleave within a transmembrane (TM) helix of membranebound substrates to release cytoplasmic or extracellular proteins/peptides, which in turn translocate to different regions of the cell where they elicit their corresponding biological response related to, for example, cell differentiation, development, and metabolism (1). Despite their broad biomedical reach, basic questions surrounding the structure of the active enzyme, how substrates are presented, and how hydrolysis chemistry occurs in an active site sequestered within the hydrophobic lipid membrane, remain active areas of research.The least biochemically understood IP is the intramembrane aspartyl protease (IAP) enzyme class, one of just three bona fide IP types that hydrolyze substrates within the hydrophobic lipid environment by using different catalytic nucleophiles (2). IAPs employ membrane-

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Section: Discussionmentioning

confidence: 99%

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Naing

Kalyoncu

Smalley³

et al. 2018

Journal of Biological Chemistry

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“…The formation of homodimers of C99 has been proposed to be critical to the mechanism by which C99 is cleaved by γ-secretase, a process that is known to depend on a number of factors including peptide sequence (9-13) and lipid composition of the membrane environment (14,15). However, recent studies have questioned the role (16) and importance (17,18) of homodimerization in the processing of full-length C99 by γ-secretase. Regardless of whether C99 homodimer is a natural substrate for γ-secretase, its ready formation both in vitro and in vivo raises the question, what is the functional role of the dimer?…”

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confidence: 99%

Impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein

Domı́nguez

Foster

Straub

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

116

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Cleavage of the amyloid precursor protein (APP) by γ-secretase is a crucial first step in the evolution of Alzheimer's disease. To discover the cleavage mechanism, it is urgent to predict the structures of APP monomers and dimers in varying membrane environments. We determined the structures of the C99 23−55 monomer and homodimer as a function of membrane lipid composition using a multiscale simulation approach that blends atomistic and coarsegrained models. We demonstrate that the C99 23−55 homodimer structures form a heterogeneous ensemble with multiple conformational states, each stabilized by characteristic interpeptide interactions. The relative probabilities of each conformational state are sensitive to the membrane environment, leading to substantial variation in homodimer peptide structure as a function of membrane lipid composition or the presence of an anionic lipid environment. In contrast, the helicity of the transmembrane domain of monomeric C99 1−55 is relatively insensitive to the membrane lipid composition, in agreement with experimental observations. The dimer structures of human EphA2 receptor depend on the lipid environment, which we show is linked to the location of the structural motifs in the dimer interface, thereby establishing that both sequence and membrane composition modulate the complete energy landscape of membrane-bound proteins. As a by-product of our work, we explain the discrepancy in structures predicted for C99 congener homodimers in membrane and micelle environments. Our study provides insight into the observed dependence of C99 protein cleavage by γ-secretase, critical to the formation of amyloid-β protein, on membrane thickness and lipid composition.nderstanding the structural and thermodynamic properties of transmembrane (TM) helical dimers is of fundamental importance to molecular biology. It is known that the association of "bitopic" proteins, having single pass TM helical domains, is essential to immunoreceptors and protein kinases that play critical roles in cellular function. Computational and experimental studies have provided insight into the role of sequence-specific interactions stabilizing helix dimerization (1, 2). Examples include the heptad repeat motif responsible for the stability of coiled-coils in GCN4 phospholamban (3) and the M2 proton channel (4), the role of the GxxxG motif in stabilizing TM helix-helix association in systems including the glycophorin A (GpA) homodimer (5, 6), found in the human erythrocyte membrane, and GxxxG and heptad repeat motifs, which play a role in stabilizing homodimers of APP-C99 (C99), the 99-aa C-terminal fragment of the amyloid precursor protein (APP) (7,8).The amyloid β (Aβ) peptide aggregation pathway, known to be crucial to the evolution of Alzheimer's disease (AD), begins with the cleavage of C99 by γ-secretase leading to the formation of a number of isoforms of Aβ. The formation of homodimers of C99 has been proposed to be critical to the mechanism by which C99 is cleaved by γ-secretase, a process that is known to dep...

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“…A possible mechanism is preferable interaction between GXXXG and cholesterol to suppress helix-helix contact, as reported for the C99 domain of the amyloid precursor protein. [19,20] However, the degree of FRET from a fluorescent sterol, dehydroergosterol, to NBD-labeled helices suggested no association between them (Supporting Information, Figure S2). …”

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confidence: 99%

GXXXG‐Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single‐Pair FRET and 2D IR Studies

et al. 2017

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Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single-pair FRET technique to examine the effect of a GXXXG motif on the association of denovo designed (AALALAA) 3 helices in liposomes. Dimerization occurred with sub-second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time-resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface. Taken together, the data illustrate that the GXXXG motif controls helix packing but still allows for a dynamic and lipid-regulated oligomeric state. HHS Public Access AbstractUnique interaction inside membrane: Model transmembrane helices containing a GXXXG motif were synthesized to explore interplay of the motif and lipid composition on the intramembrane helix associations. Single-pair FRET with controlled association topology and isotope-edited 2D-IR experiments revealed the dynamic nature, distinct interfaces, and high cholesterol sensitivity of the GXXXG-mediated interactions, which can actively regulate the functions of membrane proteins.

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Competition Between Homodimerization and Cholesterol Binding to the C99 Domain of the Amyloid Precursor Protein

Cited by 112 publications

References 100 publications

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein

GXXXG‐Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single‐Pair FRET and 2D IR Studies

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