Thekla S. Diehl

Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.

Accumulation of the amyloid-β protein (Aβ) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimers disease. The final step in the generation of Aβ from the β-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive γ-secretase(s). The most common cause of familial Alzheimers disease is mutation of the genes encoding presenilins 1 and 2, which alters γ-secretase activity to increase the production of the highly amyloidogenic Aβ42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces γ-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Aβ production and increases the amounts of the carboxy-terminal fragments of β-amyloid precursor protein that are the substrates of γ-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp → Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 → TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimers disease and which does not require this cleavage, the Asp 385 → Ala mutation still inhibited γ-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and γ-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for γ-secretase or is itself γ-secretase, an autoactivated intramembranous aspartyl protease.

Transition-state analogue inhibitors of γ-secretase bind directly to presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

Activity-dependent isolation of the presenilin– γ-secretase complex reveals nicastrin and a γ substrate

Presenilin heterodimers apparently contain the active site of γ-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-β precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimers disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional γ-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (β-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated γ-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-β precursor protein substrate of γ-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active γ-secretase complex, indicate that β-catenin, calsenilin, and presenilin-associated protein are not required for γ activity, and suggest an unprecedented mechanism of substrate–protease interaction.

Presenilin Proteins Undergo Heterogeneous Endoproteolysis between Thr291and Ala299and Occur as Stable N- and C-Terminal Fragments in Normal and Alzheimer Brain Tissue

Humans inheriting missense mutations in the presenilin (PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid beta-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimers disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T1/2 approximately 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.

Transition-state analogue inhibitors of γ-secretase bind directlyto presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

Active gamma-secretase complexes contain only one of each component.

γ-Secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid β-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional γ-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid β-peptides and amyloid β-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of γ-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.

Inhibition of Amyloid β-Protein Production in Neural Cells by the Serine Protease Inhibitor AEBSF

Cerebral deposition of amyloid beta protein (A beta) is an early and critical feature of Alzheimers disease. A beta production requires the proteolytic release of A beta from the beta-amyloid precursor protein (beta APP). Thus, inhibition of A beta release is a prime therapeutic goal. Here, we show that the broad spectrum, irreversible serine protease inhibitor, AEBSF, inhibits the constitutive production of A beta in five different human cell lines, both neural and nonneural. AEBSF also stabilizes full-length beta APP and enhances alpha-secretion, as shown by an increase in the proteolytic derivative, alpha-APPS. Further, we demonstrate that the inhibitory effect of AEBSF is specific for A beta proteins starting at Aspartate 1, suggesting that AEBSF directly inhibits beta-secretase, the Methionine-Aspartate (Met-Asp)-cleaving enzyme. These results indicate that specific inhibition of this A beta-generating protease is possible in living human neural cells and provide information about the characteristics of this as yet unidentified enzyme.

Toward the Characterization and Identification of γ‐Secretases Using Transition‐state Analogue Inhibitors

Abstract: The amyloid‐β protein (Aβ), strongly implicated in the etiology of Alzheimers disease (AD), is formed from the amyloid‐β precursor protein (APP) through sequential proteolysis by β‐ and γ‐secretases. Cleavage by γ‐secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40‐ and 42‐amino acid Aβ C‐terminal variants, Aβ40 and Aβ42. The latter form of Aβ is highly fibrillogenic, is invariably elevated in autosomal‐dominant forms of AD, and is the major Aβ component found presymptomatically in cerebral deposits. Thus, blocking production of Aβ in general and Aβ42 in particular is considered an important therapeutic goal. We have developed transition‐state analogue inhibitors of γ‐secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of γ‐secretase activity in APP‐transfected cells, strongly suggesting an aspartyl protease mechanism; (2) γ‐secretases that form Aβ40 and Aβ42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for γ‐secretases; (4) Aβ42 production is increased several fold over control by these γ‐secretase inhibitors after replacement with inhibitor‐free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total Aβ and Aβ42 production hours after replacement with compound‐free media and should help identify the target(s) of these protease transition‐state mimics.

Transition-state analogue inhibitors of |[ggr]|-secretase bind directlyto presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.