ABSTRACT
Amyloidoses are characterised by the deposition of insoluble protein that occurs in the extracellular compartment of various tissues. One form of amyloidosis is caused by transthyretin (TTR) misfolding and deposition in target tissues. It is clear that many amyloidoses share common features of fibrillogenesis and toxicity. This chapter examines the mechanisms of TTR aggregation with a view to understanding the possible therapeutic interventions in amyloid disease.
Subject(s)
Amyloid Neuropathies, Familial/metabolism , Amyloid/metabolism , Prealbumin/metabolism , Protein Folding , Amyloid/chemistry , Amyloid Neuropathies, Familial/pathology , Animals , Humans , Prealbumin/chemistryABSTRACT
BACKGROUND: Alzheimer's disease (AD) is caused by accumulation of Aß, which is produced through sequential cleavage of ß-amyloid precursor protein (APP) by the ß-site APP cleaving enzyme (BACE1) and γ-secretase. Enoxaparin, a low molecular weight form of the glycosaminoglycan (GAG) heparin, has been reported to lower Aß plaque deposition and improve cognitive function in AD transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: We examined whether heparin and enoxaparin influence APP processing and inhibit Aß production in primary cortical cell cultures. Heparin and enoxaparin were incubated with primary cortical cells derived from Tg2576 mice, and the level of APP and proteolytic products of APP (sAPPα, C99, C83 and Aß) was measured by western blotting. Treatment of the cells with heparin or enoxaparin had no significant effect on the level of total APP. However, both GAGs decreased the level of C99 and C83, and inhibited sAPPα and Aß secretion. Heparin also decreased the level of ß-secretase (BACE1) and α-secretase (ADAM10). In contrast, heparin had no effect on the level of ADAM17. CONCLUSIONS/SIGNIFICANCE: The data indicate that heparin and enoxaparin decrease APP processing via both α- and ß-secretase pathways. The possibility that GAGs may be beneficial for the treatment of AD needs further study.
Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/physiology , Enoxaparin/pharmacology , Heparin/pharmacology , Neurons/drug effects , Neurons/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Blotting, Western , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Fibrinolytic Agents/pharmacology , Mice , Mice, Transgenic , Neurons/cytology , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathologyABSTRACT
BACKGROUND: Familial amyloidotic polyneuropathy (FAP) is a peripheral neuropathy caused by the extracellular accumulation and deposition of insoluble transthyretin (TTR) aggregates. However the molecular mechanism that underlies TTR toxicity in peripheral nerves is unclear. Previous studies have suggested that amyloidogenic proteins can aggregate into oligomers which disrupt intracellular calcium homeostasis by increasing the permeability of the plasma membrane to extracellular calcium. The aim of the present study was to examine the effect of TTR on calcium influx in dorsal root ganglion neurons. RESULTS: Levels of intracellular cytosolic calcium were monitored in dorsal root ganglion (DRG) neurons isolated from embryonic rats using the calcium-sensitive fluorescent indicator Fluo4. An amyloidogenic mutant form of TTR, L55P, induced calcium influx into the growth cones of DRG neurons, whereas wild-type TTR had no significant effect. Atomic force microscopy and dynamic light scattering studies confirmed that the L55P TTR contained oligomeric species of TTR. The effect of L55P TTR was decreased by blockers of voltage-gated calcium channels (VGCC), as well as by blockers of Nav1.8 voltage-gated sodium channels and transient receptor potential M8 (TRPM8) channels. siRNA knockdown of TRPM8 channels using three different TRPM8 siRNAs strongly inhibited calcium influx in DRG growth cones. CONCLUSIONS: These data suggest that activation of TRPM8 channels triggers the activation of Nav1.8 channels which leads to calcium influx through VGCC. We suggest that TTR-induced calcium influx into DRG neurons may contribute to the pathophysiology of FAP. Furthermore, we speculate that similar mechanisms may mediate the toxic effects of other amyloidogenic proteins such as the ß-amyloid protein of Alzheimer's disease.
ABSTRACT
Alzheimer's disease (AD) is characterized by the extracellular deposition of the beta-amyloid protein (Abeta). Abeta is a fragment of a much larger precursor protein, the amyloid precursor protein (APP). Sequential proteolytic cleavage of APP by beta-secretase and gamma-secretase liberates Abeta from APP. The aspartyl protease BACE1 (beta-site APP-cleaving enzyme 1) catalyses the rate-limiting step in the production of Abeta, and as such it is considered to be a major target for drug development in Alzheimer's disease. However, the development of a BACE1 inhibitor therapy is problematic for two reasons. First, BACE1 has been found to have important physiological roles. Therefore, inhibition of the enzyme could have toxic consequences. Second, the active site of BACE1 is relatively large, and many of the bulky compounds that are needed to inhibit BACE1 activity are unlikely to cross the blood-brain barrier. This review focuses on the structure BACE1, current therapeutic strategies based on developing active-site inhibitors, and new approaches to therapy involving targeting the expression or post-translational regulation of BACE1.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/physiology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/physiology , Drug Design , Protease Inhibitors , Amyloid Precursor Protein Secretases/chemistry , Animals , Aspartic Acid Endopeptidases/chemistry , Catalytic Domain , Gene Expression Regulation, Enzymologic , Humans , Protease Inhibitors/adverse effects , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Substrate SpecificityABSTRACT
The presenilins form part of a complex of membrane proteins that are involved in the proteolytic cleavage of cell-surface molecules. This article reviews the history of the discovery of the presenilins, their role in the pathogenesis of Alzheimer's disease and in the metabolism of the amyloid-beta precursor protein. Unanswered questions about their biochemical mechanism of action and their effects on Ca2+ homeostasis are examined.
Subject(s)
Alzheimer Disease/pathology , Alzheimer Disease/physiopathology , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Presenilins/metabolism , Alzheimer Disease/etiology , Alzheimer Disease/genetics , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Protein Precursor/genetics , Calcium/metabolism , Cell Membrane/metabolism , Humans , Mutation , Presenilins/geneticsABSTRACT
The beta-site APP cleaving enzyme (BACE1) is responsible for the first step in the production of the beta-amyloid protein of Alzheimer's disease. BACE1 is synthesized as a partially active zymogen (proBACE1). We previously showed that the glycosaminoglycan (GAG) heparin can increase the enzyme activity of proBACE1. In this study, the structural requirements and the mechanism for the GAG-induced activation were examined. The effect of heparin on proBACE1 was influenced by the degree of sulfation and carboxylation of the GAG, as well as by the length of the sugar. Although low molecular weight heparin fragments did not strongly stimulate proBACE1, they inhibited heparin-induced activation of the enzyme. The structure of the zymogen was modeled using the known X-ray structures of the BACE1 catalytic domain and the homologous prodomain of porcine pepsinogen. The modeled structure suggested that a heparin-binding domain may reside close to the prodomain, and that movement of a loop region between residues 46-65, lying adjacent to the prodomain, may be needed to accommodate heparin binding. The presence of the loop domain adjacent to the active site may account for the lower activity of the zymogen relative to the mature enzyme. Movement of the loop region upon heparin binding could expose the active site region to allow for increased substrate binding. The results suggest a model in which conformational changes close to the prodomain may be involved in the mechanism of heparin-induced activation of proBACE1.
Subject(s)
Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Fibrinolytic Agents/pharmacology , Heparin/pharmacology , Amyloid Precursor Protein Secretases/chemistry , Animals , Aspartic Acid Endopeptidases/chemistry , Computer Simulation , Enzyme Activation/radiation effects , Enzyme Precursors/metabolism , Humans , Models, Molecular , Molecular Structure , Polyelectrolytes , Polymers/pharmacology , Protein Carbonylation/drug effects , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity/drug effectsABSTRACT
The beta-secretase (BACE1) is initially synthesized as a partially active zymogen containing a prodomain which can be further activated through proteolytic cleavage of the prodomain by a furin-like protease. The active site of BACE1 is large and although a number of high-affinity active-site inhibitors of BACE1 have been described, most of these compounds are large, polar and do not cross the blood-brain barrier. However, it may be possible to target other regions of the protein which regulate BACE1 allosterically. We have found that proBACE1 can be stimulated by relatively low concentrations (e.g. 1 microg/ml) of heparin. Heparin initially increases proBACE1 activity, probably by binding to the prodomain, which decreases steric inhibition at the active site. However, the heparin-activated zymogen also undergoes autocatalysis, which ultimately leads to a loss of enzyme activity. We speculate that proBACE1 can be regulated by endogenous heparan sulfate proteoglycans and that drugs which target this interaction may have value in the treatment of Alzheimer's disease.