Characteristics of amyloidogenic proteins and the related peptides
1. Introduction
Oligomerization of amino acids by binding their peptide bonds (-CONH-) forms proteins (or peptides), which are the major components of our bodies. Although the primary sequence (the linear sequence of amino acids) of the protein mainly determines its characteristics, its secondary structures (the conformations) are also critical determinants of their shapes and functions. The conformation (random coil, α-helix, and ß-sheet) is restricted by the circumstances nearby proteins. The hydrogen bond between the amino acids in the peptide chain forms the α-helix structure. Meanwhile, the ß-sheets (ß-plated sheets) consist of ß-strands which are laterally connected peptide bonds with hydrogen bonds.
Recent neurochemical evidence indicates that the oligomerization of proteins and the formation of ß-sheet structures are linked with several neurodegenerative diseases such as Alzheimer’s disease (AD), prion diseases, triplet repeat diseases, dementia with Lewy bodies (DLB). The disease-related proteins, such as ß-amyloid protein (AßP) in AD, prion protein in prion diseases, polyglutamine in triplet repeat disease, α-synuclein in DLB, are identical in each disease (Table 1). However, all of these amyloidogenic proteins share common characteristics in the formation of amyloid with ß-sheet structures, and in the exhibition of cytotoxicity. Therefore, a new concept termed “conformational disease” was proposed, suggesting that protein conformation is an important determinant of its toxicity, and consequently, the development of the related disease [1].
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Alzheimer’s disease | Al, Zn, Cu, Fe | + | + | + | |
Prion disease | Prion protein: MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKP |
Zn, Cu, M, Fe |
+ | + | + |
Dementia with Lewy bodies (DLB) | α-synuclein; MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVTTVAEKTKEQVSNVGGAVVTGVTAVAHKTVEGAGNFAAATGLVKKDQKNESGFGPEGTMENSENMPVNPNNETYEMPPEEEYQDYDPEA |
Cu, Fe, Al |
+ | + | + |
Triplet- repeat disease | MATLEKLMKAFESLKSF |
Fe | + | + | + |
Diabetes mellitus | KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY |
Cu, Al | + | + | + |
These conformational diseases are included in amyloidosis. At 1853, Virchow found the abnormal accumulates in tissues and named “amyloid”, since they exhibited similar characteristics with
In this chapter, we review the implication of protein oligomerization in the pathogenesis of these neurodegenerative diseases. Considering that the amyloidogenic proteins are commonly present in our brain, factors which influence oligomerization play crucial roles in their pathogenesis. As such factors, we focus on trace elements such as Al, Zn, Cu, and Fe. Metals have a property of firmly binding to metal-binding residues of proteins, such as tyrosine (Tyr) or histidine (His) or phosphorylated amino acids, and cause cross-linking of the proteins (Fig. 1). Furthermore, all of these amyloidogenic proteins were reported to have the ability to bind metals as shown in Table 1. Our and other numerous studies reported that oligomers cause neurodegeneration by induction of Ca2+ dyshomeostasis through the formation of amyloid channels on neuronal membranes [3,4]. The beneficial characteristics of carnosine (ß alanyl histidine) as a drug for the treatment for these neurodegenerative diseases are also discussed.
2. Alzheimer’s disease and oligomerization of AßP
2.1. Amyloid cascade hypothesis
Alzheimer’s disease (AD) is a severe type of senile dementia, affecting a large portion of elderly people worldwide. It is characterized by profound memory loss and inability to form new memories. The pathological hallmarks of AD are the presence of numerous extracellular deposits (senile plaques) and intraneuronal neurofibrillary tangles (NFTs). The degeneration of synapses and neurons in the hippocampus or cerebral cortex is also observed. The major components of NFTs are phosphorylated tau proteins, and that of senile plaques are ß-amyloid proteins (AßPs) [5]. Although the precise cause of AD remains elusive, numerous biochemical, cell biological, and genetic studies have supported the idea termed “amyloid cascade hypothesis” that the AßP accumulation and the consequent neurodegeneration play a central role in AD [6]. Moreover, recent studies on the identified AßP species have indicated that the oligomerization of AßP and the conformational changes are critical in the neurodegeneration process [7].
AßP is a small peptide of 39–43 amino acid long. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP). AßP is secreted by the cleavage of its N-terminal by ß-secretase (BACE), followed by the intra-membrane cleavage of its C-terminal by γ-secretase. Genetic studies of early-onset cases of familial AD indicated that APP mutations and AßP metabolism are associated with AD. It was also revealed that mutations in the presenilin genes account for the majority of cases of early-onset familial AD. Presenilins have been revealed to be one of γ -secretases, and their mutations also influence the production of AßP and its neurotoxicity.
Yankner
Furthermore, the longer peptide variant, AßP(1–42), has the characteristics of immediate polymerization compared to AßP(1–40). AßP(1–42) enhances the aggregation of AßP(1–40) and becomes a seed of the amyloid fibrils. AßP (1–42) is more abundant in the brains of AD patients as compared to those of age matched controls. The mutations of APP and those of presenilin genes induce the increased production of AßP (1–42) in the transfected cell lines.
Recent approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy have demonstrated that there are several stable types of soluble oligomers: naturally occurring soluble oligomers (dimers or trimers), ADDLs (AßP-derived diffusible ligands), AßP globulomers, or protofibrils. Hartley separated aggregated AßP(1–40) into low–molecular-weight (mainly monomer), protofibrillar, and fibril fractions by size-exclusion chromatography, and found that the protofibrillar fraction caused marked changes in the electrical activity of cultured neurons and neurotoxicity. Walsh
2.2. Metal-induced oligomerization of AßP
Considering that AßP is secreted from APP into the brain of young people or of normal subjects, factors which influence (accelerate or delay) the oligomerization may become important determinants of the pathogenesis of AD. Various factors, such as the concentration of peptides, the oxidations, mutations, and racemization of AßP, pH, composition of solvents, temperature, and trace elements, can influence the oligomerization processes. A considerable amount of asparagines (Asp) or serine (Ser) residues of AßP accumulated in senile plaques are racemized. Tomiyama
Among these factors, trace elements such as aluminum (Al), zinc (Zn), copper (Cu), iron (Fe) are of particular interest. The accumulation of AßP is rarely observed in the brains of rodents (rats or mice) as compared to humans or monkeys. As shown in Fig.2, the amino acid sequence of human and rodent AßP are similar, yet they differ by three amino acids. However, rodent AßP exhibits less tendency to oligomerization compared to human AßP [10]. Considering that these three amino acids (Arg5, Tyr10, and His13) have the ability to bind metals and that trace metals have cross-linking ability, trace elements might play important roles in the accumulation of AßP in the human brain.
Exley
Bush
Considering the implications of metals in AD pathogenesis, chelation therapy for AD treatment is of great interest. Clioquinol (quinoform), a chelator of Cu2+ or Zn2+, inhibits oligomerization of AßP and attenuates the accumulation of amyloid in the brains of experimental animals. Clinical trials using its analogue PBT2 are under investigation. DFO, a chelator of Al and Fe, attenuates the decline of daily living skills in AD patients. Silicates, which couple with Al and reduce its toxicity, are also candidates for chelation therapy in AD [17].
2.2. Oligomerization-induced neurotoxicity of AßP
There is a considerable interest regarding the mechanisms by which AßP oligomers cause neurotoxicity. Exposure to AßP causes various adverse effects on neuronal survivals such as the production of reactive oxygen species, the induction of cytokines, the induction of endoplasmic reticulum (ER) stresses, and the abnormal increase of intracellular calcium levels ([Ca2+]i),
There is considerable interest regarding the mechanism by which AßPs interact with neurons and disrupt Ca2+ homeostasis. In 1993, Arispe
To determine whether AßPs form channels on neuronal cell membranes as well as artificial lipid bilayers, we employed membrane patches from a neuroblastoma cell line (GT1-7 cells), which exhibit several neuronal characteristics such as the extension of neuritis and the expression of neuron-specific proteins or receptors [24]. After exposing the excised membrane patches of GT1-7 cells in the bath solution to AßP(1–40), the current derived from the amyloid channels appeared. The amyloid channels formed on the GT1-7 cell membranes were cation-selective, multilevel, voltage-independent, long-lasting ones; the channel activity was inhibited by the addition of Zn2+, and recovered by a zinc chelator,
Considering the results of our study together with those of the other studies, we propose the following hypothetical scheme of neurodegeneration induced by oligomerization of AßP (Fig. 4).
AßPs are normally secreted from APP into the cerebrospinal fluid and are usually degraded proteolytically by neprilysin within a short period. However, upregulation of the AßP secretion from APP, or an increased ratio of AßP(1–42) to AßP(1–40) may render AßPs liable to be retained in the brain. It has been demonstrated that APP or presenilin gene mutations promote this process. AßP possesses positive charges at neutral pH. Therefore, the net charge of the outer membrane surface may be a determinant when secreted AßPs bind to cellular membranes (Fig.4 (A)). The distribution of phospholipids on cellular membranes is usually asymmetrical and negatively charged phospholipids such as PS exist on the inner membrane surfaces. Disruption of the assymetrical distribution is the first hallmark of apoptotic cell death [30]. Therefore, the binding of AßP to neuronal membranes seldom occur in normal and young brains. This idea may explain why AD occurs in aged subjects meanwhile AßPs are secreted in the brains of young subjects. After incorporation into the membrane, the conformation of AßPs change and the accumulated AßPs aggregate on the membranes (Fig. 4(B)). The ratio of cholesterol to phospholipids in the membrane may alter membrane fluidity, thereby affecting the process from step (A) to (B). AßP oligomerization
Once AßP channels are formed on neuronal membranes, homeostasis of Ca2+ and other-ion will be disrupted. Disruption of Ca2+ homeostasis triggers several apoptotic pathways such as the activation of calpain, the induction of caspase, and promote numerous degenerative processes, including the production of reactive oxygen species (ROS) and the phosphorylation of tau, thereby accelerating neuronal death. Mutations of presenilins cause disturbances in the capacitive Ca2+ entry and may influence these pathways. Free radicals also induce membrane disruption, by which unregulated Ca2+ influx is further amplified. The disruption of Ca2+ homeostasis also influences the production and processing of APP. Thus, a vicious cycle of neurodegeneration is initiated. This hypothesis explains the long delay in AD development; AD occurs only in senile subjects despite the fact that Aßs are normally secreted also in younger or in normal subjects. Various environmental factors, such as foods or trace metals, as well as genetic factors will influence these processes and contribute to AD pathogenesis [31].
3. Prion diseases and other amyloidosis
The disease-related amyloidogenic proteins exhibit similarities in the formation of ß-pleated sheet structures, abnormal deposition as amyloid fibrils in the tissues, and introduction of apoptotic degeneration. Prion diseases, including human kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (BSE), are associated with the conversion of a normal prion protein (PrPC) to an abnormal scrapie isoform (PrPSC) [32]. The ß-sheet region of PrPSC is suggested to play a crucial role in its transmissible degenerative processes. A peptide fragment of PrP corresponding to residues 106–126 (PrP106–126) has been reported to cause death in cultured hippocampal neurons. We investigated the oligomerization of PrP106-126 and its neurotoxicity on primary cultured rat hippocampal neurons [33]. As AßP, PrP106-126 formed amyloid-like fibrils with ß–sheet structures by observation with atomic force microspope and by thioflavin T staining during the aging process. The oligomerization and formation ß-sheet structure enhanced the neurotoxicity of PrP106-126. The co-existence of Zn or Cu inhibited ß-sheet formation of PrP106-126 and attenuated its neurotoxicity. Furthermore, the thickness of PrP106-126 fibrils was decreased in the presence of Zn or Cu.
Electrophysiological and morphological studies have revealed that PrP106-126 exhibits similarities in the formation of amyloid channels as well as AßP [34]. Lin
The oligomerization and fibrillation of α-synuclein has been implicated in the formation of abnormal inclusions, termed Lewy bodies, and the etiology of dementia with Lewy bodies (DLB). Non-amyloid component (NAC), a fragment peptide of α-synuclein, accumulates in Alzheimer’s senile plaques and causes apoptotic neuronal death. Lashuel
The elongation of a polyglutamine-coding CAG triplet repeat in the responsible genes is based on the pathogenesis of triplet-repeat disease such as Huntington’s disease or Machado-Joseph disease. Hirakura
Lal
4. Conclusion
This hypothesis about the pathogenesis of conformational diseases may help in the development of drugs for these diseases. We focus carnosine (ß-alanyl histidine) as such a protective drug. Carnosine is a naturally occurring dipeptide and is commonly present in vertebrate tissues, particularly within the skeletal muscles and nervous tissues [37]. It is found at high concentrations in the muscles of animals or fish which exhibit high levels of exercise, such as horses, chickens, and whales. Thus, it is believed that carnosine plays important roles in the buffering capacities of muscle tissue and the administration of carnosine has been reported to induce hyperactivity in animals.
Secretion from synapses of AßP, and its direct incorporation into membranes and formation of oligomeric amyloid channels are depicted. Details are discussed in the text.
In the brain, a considerable amount of carnosine is localized in the neurons of the olfactory bulb. It is secreted into synaptic clefts along with the excitatory neurotransmitter glutamate during neuronal excitation. Carnosine reportedly has several beneficial effects including the antioxidant activity, the chelating ability to metal ions, the inhibition of the Maillard reaction. Furthermore, carnosine is reported to have anti-crosslinking properties. Attanasio
In conclusion, further research into the role of protein oligomerization and Ca homeostasis via amyloid channels might lead to the development of new treatments for neurodegenerative diseases.
Acknowledgments
The authors would like to thank Mr. M. Yanagita, Ms. A. Komuro, and Ms. N. Kato for their technical assistance. This work was partially supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant from Cooperation for Innovative Technology and Advanced Research in Evolutional Area (CITY AREA) from the Miyazaki Prefectural Industrial Support Foundation.
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