Dirk Beher

γ-Secretase Complex Assembly within the Early Secretory Pathway

γ-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the β-secretase-processed β-amyloid precursor protein to liberate the neurotoxic amyloid β-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the γ-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that γ-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state γ-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete γ-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid β-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of β-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of γ-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the γ-secretase complex within the ER.

C‐terminal PAL motif of presenilin and presenilin homologues required for normal active site conformation

The Alzheimers disease‐associated β‐amyloid peptide is produced through cleavage of amyloid precursor protein by β‐secretase and γ‐secretase. γ‐Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH‐1) and presenilin enhancer‐2 (PEN‐2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane‐embedded aspartic acid residues for catalysis. Apart from the two aspartate‐containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C‐terminus. Although it has been well documented that this motif is essential for γ‐secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of γ‐secretase resulting in a complete loss of PS binding to a γ‐secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and γ‐secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of γ‐secretase and of SPP.

Protease inhibitors as potential disease-modifying therapeutics for Alzheimer's disease

The current lack of an effective treatment for Alzheimer’s disease (AD) has fuelled an intense search for novel therapies for this neurodegenerative condition. Aberrant production or decreased clearance of amyloid-β peptides is widely accepted to be causative for AD. Amyloid-β peptides are produced by sequential processing of the β-amyloid precursor protein by the two aspartyl-type proteases β-secretase and γ-secretase. Because proteases are generally classified as druggable, these secretases are a centre of attraction for various drug discovery efforts. Although a large number of specific drug-like γ-secretase inhibitors have been discovered, progress towards the clinic has been slowed by the broad substrate specificity of this unusual intramembrane-cleaving enzyme. In particular, the Notch receptor depends on γ-secretase for its signalling function and, thus, γ-secretase inhibition produces distinct phenotypes related to a disturbance of this pathway in preclinical animal models. The main task now is to define the therapeutic window in man between desired central efficacy and Notch-related side effects. In contrast, most studies with knockout animals have indicated that β-secretase inhibition may have minimal adverse effects; however, the properties of the active site of this enzyme make it difficult to find small-molecule inhibitors that bind with high affinity. In most instances, inhibitors are large and peptidic in nature and, therefore, unsuitable as drug candidates. Thus, there are many issues associated with the development of protease inhibitors for AD that must be addressed before they can be used to test the ‘amyloid cascade hypothesis’ in the clinic. The outcomes of such trials will provide new directions to the scientific community and hopefully new treatment options for AD patients.

Functional Overexpression of γ-Secretase Reveals Protease-independent Trafficking Functions and a Critical Role of Lipids for Protease Activity

Presenilins appear to form the active center of γ-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue γ-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, γ-secretase appears to play a role in the trafficking of the β-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized γ-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. γ-Secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the γ-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive γ-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of γ-secretase.

Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and γ‐secretase activity

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and γ‐secretase cleavage of substrates. The structural requirements for PS incorporation into the γ‐secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and γ‐secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and γ‐secretase activities. Swap mutations identified an N‐terminal TM1 domain that is important for γ‐secretase activity only and a C‐terminal TM1 domain that is essential for both presenilinase and γ‐secretase activities. Exchange of residues 95–98 of PS1 (sw95–98) completely abolishes both activities while the familial Alzheimers disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95–98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog γ‐secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with γ‐secretase substrates and γ‐secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. γ‐Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for γ‐secretase that alters the ratio of amyloid‐β peptide species produced, leading to the amyloid‐β peptide aggregation that causes familial Alzheimers disease.

γ-Secretase Inhibitors – from Molecular Probes to New Therapeutics?

Publisher Summary The hallmark lesions in Alzheimers disease (AD) brains described by Alois Alzheimer are extracellular proteinaceous deposits, found either as amyloid plaques in the brain parenchyma or as vascular amyloid surrounding the brain blood vessels, and intracellular neurofibrillary tangles consisting of an abnormally phosphorylated microtubule-associated protein tau. Over the past several years, extraordinary progress has been made in understanding mechanistic details of the amyloid hypothesis of AD. A part of this understanding has arisen as a result of the availability of molecular probes targeted toward γ-secretase. Although the final proof of the identity of γ-secretase by reconstitution of the γ-secretase complex in vitro is a difficult task, substantial evidence generated by affinity labeling of presenilins by transition state analog inhibitors points toward a critical role of polypeptides in the enzyme complex.

P1-359: Identification of genes that regulate the processing of the β-amyloid precursor protein and are candidates for association with late-onset Alzheimer’s disease by a genome-wide siRNA screen

Although dysregulation of cholesterol balance may be related to the onset of neurological disease, the molecular mechanisms that underlie the brainspecific expression of the human CYP46 (hCYP46) have never been described. Objective(s): The human CYP46 5’-flanking region has been cloned in order to identify cis-regulatory elements involved in the brainspecific expression of this gene. Methods: PCR was used to amplify the human CYP46A1 genomic fragment. Several promoter deletion reporter vectors were used in transfection/transactivation studies. Results: Like many neuronal genes, the hCYP46 5’ upstream region is GC-rich and lacks a consensus TATA box. Transfection-transactivation studies using hCYP46 reporter gene constructs have allowed us to identify the presence of a CCAAT/Enhancer Binding Proteins (C/EBPs) responsive region in the proximal promoter. Electrophoretic mobility shift assays, have shown that the C/EBP-responsive elements do not correspond to the canonical C/EBP binding site. Cross-talk with the multiple SP-1 binding sites was assessed. Conclusions: Several recent studies in mammals show that C/EBP mRNA is widely expressed in adult mouse brain and that this protein could be implicated in long-term synaptic plasticity and memory consolidation in rat hippocampus. Interestingly, significant increases in the C/EBP expression levels have been shown in AD compared to non-demented cortex. The identification of C/EBP responsive region involved the CYP46 brainspecific expression, may therefore contribute to novel approaches to the characterization of endogenous regulatory circuits that control pathophysiological situations.