AMYLOID PROTEINS
What are amyloid proteins?
Amyloid proteins are formed from normal, soluble proteins that are misfolded to produce aggregates of insoluble proteins. These then form stable, elongated fibres with β-sheet configurations that are able to be stained by the dye Congo Red (Arce, 2014; Eisenberg & Jucker, 2012). The fibres become resistant to degradation and deposit extracellularly in the tissues and organs. Some examples of amyloid protein include amyloid-β-protein (Aβ), α-synuclein, islet amyloid polypeptide (IAPP) and prion protein (PrP).
Amyloid proteins are thoroughly being studied since they are associated with certain pathologies known as protein conformational diseases (Pearson & Peers, 2006; Liu & Zhang, 2011). These diseases include neurodegenerative disorders like Alzheimer’s disease which is associated with the pathological overproduction of Aβ proteins leading to the formation of amyloid plaque formation and neurofibrillary tangles which damage the blood brain barrier (BBB) and are linked to memory impairment, confusion, difficult with language and lack of physical control (Morley et al., 2010). There are other pathologies associated with amyloid proteins such as type 2 diabetes and spongiform encephalopathies such as Mad cow disease (Liu & Zhang, 2011).
Types of amyloid proteins
Amyloid protein can be broken down into 6 main types. These are:
Amyloid beta peptide (Aβ): Aβ is synthesised from amyloid precursor proteins (APPs) through proteolytic processing.
α-Synuclein
Islet amyloid polypeptide
Prion Protein
Copper/zinc superoxide dismutase
Huntingtin protein.
Amyloid beta peptide (Aβ) is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by β- and γ-secretases. Aβ accumulation in the brain is proposed to be an early toxic event in the pathogenesis of Alzheimer's disease, which is the most common form of dementia associated with plaques and tangles in the brain. Aβ monomers aggregate into various types of assemblies, including oligomers, protofibrils and amyloid fibrils. Amyloid fibrils are larger and insoluble, and they can further assemble into amyloid plaques, while amyloid oligomers are soluble and may spread throughout the brain. The different forms of Aβ include soluble Aβ, Aβ oligomer and Aβ present in amyloid plaques. In addition, a dynamic compartmentalization of the different types of Aβ may exist between plaques and soluble Aβ196, and the different Aβ forms may contribute to neurodegeneration at different stages of the disease197. Aβ has also been reported to form aggregates in two fundamental types of reactions: non-metal-dependent association and metal-dependent association. Non-metal Aβ aggregates form soluble oligomers and amyloid fibrils, while metal Aβ aggregates form ionically bridged aggregates, covalently crosslinked oligomers, and seeds for non-metal-dependent Aβ fibrillization198. Accumulating Aβ first forms Aβ oligomers and gradually deposits as fibrils and senile plaques.
Amyloid fibrils and disease
Fibrils are insoluble, long, straight, and unbranched protein aggregates composed of subunits called 'protofilaments'. A single fibril consists of 4 protofilaments which wind and twist around a central core. Each protofilament has a cross-β structure and may be formed by 1 to 6 β-sheets stacked on top of each other. In addition, protofilaments can have β-strands contributed from protein molecules. Such strands may form parallel and anti-parallel β-sheets. However, it should be noted that only a small fraction of the polypeptide chains are in a β-strand conformation in the fibrils, with the remainder forming structured or unstructured loops and tails. Interestingly, β-sheets and β-strands can interact with one another to form an interface called a 'steric-zipper interface'. This forms due to neighboring β-sheets which are tightly packed together via a water-free interface along with the opposing β-strands which are arranged in such a way that their side-chains interlock with each other. The resulting dehydrated interface is called the steric-zipper interface.
The formation of fibrils often accompanies diseases, the most well-known of which include Alzheimer's disease, Type II diabetes, and spongiform encephalopathies (for example, Mad Cow Disease and Creutzfeldt-Jakob Disease/CJD).
Amyloid fibrils are formed when normally soluble proteins assemble and aggregate to form insoluble fibres which are resistant to degradation, via a process of polymerisation. These fibrils are deposited extracellularly in the tissues and result in pathogenic effects. Such agglomeration of fibrils is inherently stable, with structural studies revealing that these are mainly composed of β-sheet structures in a cross-β conformation. As such, the fibrils are self-assembled and proteins themselves can fold into alternative conformations which allows for this self-assembly.
There are 3 main proposed models for the self-assembly of amyloid fibrils from their monomers, these being:
The refolding model
The natively disordered model
The gain-of-interaction model
The refolding model is the most well-known model and it describes a situation wherein a protein refolds from its native state to an amyloid-forming state.
The natively disordered model suggests that the protein is natively disordered and folds directly into an amyloid-forming state.
The gain-of-interaction model is where part of one amyloid protein monomer becomes a domain in another amyloid protein monomer which eventually forms the amyloid fibril.
It should also be noted that while, on the whole, amyloid fibrils of the same protein form consistent structures, other fibril features such as morphology and properties can vary according to exposure to different environmental conditions during growth. Hence, minute changes to the growth conditions can have a significant effect on the potential cytotoxicity of the formed amyloid fibril.
The reasons why amyloid fibrils cause diseases are still unclear. In some cases, the deposits physically disrupt tissue architecture, suggesting disruption of function by some bulk process. An emerging consensus implicates prefibrillar intermediates, rather than mature amyloid fibers, in causing cell death, particularly in neurodegenerative diseases.
MECHANISMS OF TOXICITY:
Calcium dysregulation has been observed to occur early in cells exposed to protein oligomers. These small aggregates can form ion channels through lipid bilayer membranes and activate NMDA and AMPA receptors. Channel formation has been hypothesized to account for calcium dysregulation and mitochondrial dysfunction by allowing indiscriminate leakage of ions across cell membranes. Studies have also shown that amyloid deposition is associated with mitochondrial dysfunction and a resulting generation of reactive oxygen species (ROS), which can initiate a signaling pathway leading to apoptosis.
Beta‐amyloid (Aβ) accumulation is also commonly associated with Alzheimer's disease. Aβ toxicity results in an age‐related increase in cholinergic loss and microglial activation in the brain, along with neuronal loss. Studies have also shown that age plays a crucial role in susceptibility of the brain to certain Alzheimer's pathologies including accumulation of beta-amyloid.
All these mechanisms of toxicity are likely to play a role. In fact, the aggregation of a protein generates a variety of aggregates, all of which are likely to be toxic to some degree. The oligomers have also been reported to interact with a variety of molecular targets. The misfolded nature of protein aggregates causes a multitude of aberrant interactions with a multitude of cellular components, including membranes, protein receptors, soluble proteins, RNAs and small metabolites. Hence, it is unlikely that there is a unique mechanism of toxicity or a unique cascade of cellular events.
The Proteostasis network
The majority of proteins existing within us must be accurately folded into three dimensional structures. This conformation needs to be maintained as long as they remain circulating our bodies in order for them to effectively carry out their biological functions. Additionally, the relative amounts of each individual kind of protein needs to be precisely controlled. This state of consistent balance in the proteomes within our cells is referred to as proteostasis – a complex system which is under the control of numerous molecular chaperones, proteolytic systems, and a multitude of regulators. Research on the organization and regulation of this network of different systems that serve to maintain proteostasis, and how these systems are altered in response to both exogenous and endogenous stressors is of paramount importance, since a dysregulation of these processes has been heavily implicated in ageing and several degenerative diseases.
The proteostasis network works to:
Make sure that accurately-folded proteins are produced at the specific time and cellular location that they are needed.
Make sure that these proteins are produced in the exact quantity necessary for their stoichiometric assembly with other proteins when these are needed in the formation of oligomeric protein complexes.
Preventing proteins from incorrect assembly, from misfolding, and from aggregation with other misfolded proteins.
Essential role in removing any extra, redundant, or misfolded proteins – carried out by several processes such as autophagy or proteosome-mediated degradation.
Altogether, these processes serve to circumvent the accumulation of protein aggregates since these can be toxic to the cell.
Proteostasis is disrupted in several pathologies, the majority of which are diseases that are heavily associated with old age, such a neurodegenerative disorders. This implies that the capacity of the PN decreases with aging. This correlation between increasing and decreasing capacity of cells to maintain a functional proteome is implicated as a central provocation for age-related cellular dysfunction and degenerative diseases. The reason for the decline in functionality of the PN with age is still ambiguous but is most likely due to a lack of evolutionary pressure for proteome upkeep past the time when organisms have already passed on their genome to their progeny.
Cells work to manage the functions of three interrelated aspects of the PN to maintain a balanced proteosome. These include:
protein synthesis and folding,
maintenance of conformational stability, and
protein degradation.
Eukaryotic proteosomes are highly complex and consist of several thousands of different proteins within one cell. To further complicate things, proteome composition varies between cell types and tissues. The amounts of specific proteins occurring within cells varies greatly depending on protein type, for example only around 50 copies of some transcription factors are produced, whereas 10 million or more copies of histone molecules or cytoskeletal proteins are needed. This means that the totals of each protein produced within a cell must be meticulously controlled in order to support cell signaling, metabolic pathways, and to permit an equal ratio of proteins needed for their assembly into macromolecular structures; such as ribosomes and mitochondrial respiratory complexes.
For most proteins to be able to present their biological function, it is necessary for them to be folded into defined 3D structures. This well-defined folding structure is called the protein’s native state – a state which is thermodynamically favourable to ensue. Although all the information needed for the protein to fold into its native structure is held within the amino acid sequence of the polypeptide chain, the proteins must still maneuver through a complex energy landscape as they are folding, during which they may potentially assume a different kinetically-stable structure that is non-native for that protein. This is why the folding process must be so meticulously controlled.
Initially, it was expected that the folding process occurred spontaneously, as defined by the amino acid sequence. However, it has now become clear that complex proteins and those that are composed of multiple domains, actually require molecular chaperones to fold properly. These molecular chaperones are additional factors that are interacting with the protein components during their folding processes, and do not form a part of the final protein structure. These chaperons - which are classified into different protein families such as small heat shock proteins, HSP60, or HSP90 - serve to avert aggregation of proteins and encourage their precise folding through a recognition of specific protein components such as hydrophobic amino acid residues. Moreover, chaperones that are involved in the folding of recently-synthesised proteins are active both during and after translation in order to prevent misfolding and aggregation.
A considerable fraction of proteins exist in partially unfolded states since the native protein structures are only moderately stable. Formation of these partially unfolded states may be further promoted by the presence of stressful or destabilising elements such as mutations, high temperatures, heavy metals or reactive oxygen species. Polypeptides that exist in non-native conformations – comprising nascent chains, folding intermediates and misfolded states – have a tendency to aggregate due to exposed hydrophobic amino acid components or unpaired β-strands. It is essential for this protein aggregation to be impeded as much as possible, since it results in fewer functional proteins, in turn decreasing the functional action of that respective protein type. Additionally, protein aggregates are cytotoxic in several ways.
The proteostasis network works to promote the functional and conformational stability of all protein types via the workings of the salient molecular chaperones. In fact, the transcription of molecular chaperones is significantly upregulated during stressful conditions. When under stress, the primal cytosolic response is known as the heat shock response, whereby several heat shock factors (transcription factors) bind to specific heat shock elements in promoter regions as a means of controlling the production or activation of chaperones and even other factors of the proteostasis network.
These heat shock factors may isolate heat shock proteins in an inactive complex during normal cellular conditions. However, in the presence of non-native protein structures, the molecular chaperones are pulled out of this inactivating complex. Now, the heat shock factors may elicit transcription of heat shock protein genes, further inducing components of the Proteostasis network (PN). Additionally, during stress conditions there is an attenuation of protein synthesis so that there aren’t extra proteins that certainly require the attention of chaperones.
It is not only the production of proteins that must be tightly controlled in order to maintain the correct proportions of different protein types within a cell. Another important mechanism includes the targeted and regulated degradation of proteins, which is necessary to adjust protein levels following environmental alterations and also serves to regulate numerous important cellular processes, such as mitosis. Furthermore, protein degradation is a vital mechanism needed to destroy misfolded proteins, mutant proteins, and unassembled subunits of multimer proteins. Two principal mechanisms are employed for protein degradation; the ubiquitin-proteasome system (UPS) and the autophagosomal-lysosomal pathway. Degradation via the UPS involves ATP-dependent unfolding of single substrate proteins whereas the autophagy pathway is responsible for removal of protein aggregates and organelles. Molecular chaperones are needed in both degradation pathways and serve to identify misfolded proteins and to retain them in a degradation-competent state.
Figure 1
Graphical demonstration of the Proteostasis Network. This diagram was based on a diagram obtained from: Hipp, M. S., Kasturi, P., & Hartl, F. U. (2019). The proteostasis network and its decline in ageing. Nature Reviews Molecular Cell Biology, 20(7), 421-435. doi:10.1038/s41580-019-0101-y
Treatments
Treatments that have been shown to have a potential in treating amyloidosis such as Alzheimer's diseases are cholinesterase inhibitors, NMDA uncompetitive inhibitors, active immunization and passive immunization.
1. Cholinesterase inhibitors:
During the course of Alzheimer's disease (AD), cholinergic neurons that project from the nucleus basalis of Meynert and other septal nuclei to the cortex are lost. As a result, loss of cholinergic input occurs which leads to a reduction in attention and memory. The mode of action of cholinesterase inhibitors (ChEI) is by inhibiting the enzyme acetylcholine esterase and thus, acetylcholine is not degraded which leads to more acetylcholine being present in the synapses. Examples of ChEIs that were approved to treat mild to moderate Alzheimer's diseases are donepezil, rivastigmine and galantamine. A disadvantage of ChEIs treatment is that it does not reduce the rate at which AD progresses.
uncompetitive NMDA inhibitors:
The mode of action of uncompetitive NMDA inhibitors such as memantine is by binding and inhibiting the NMDA receptor of glutaminergic neurons. As a result, the neurons are protected from glutamate mediated neurotoxicity during AD progression. These drugs have been shown to treat moderate to severe AD.
2. Active immunization:
Vaccinations composed of the AB42 amyloid protein with adjuvant have been shown in an AD mouse model to decrease amyloid protein deposits and almost completed prevented the development of amyloid deposits at an old age. Nowadays, vaccination strategies to treat AD are being researched in humans. In fact, CAD106 is currently at stage 3 clinical trials and ABVac40 is in phase 2 clinical trials.
Passive immunization:
Treatment by passive immunization occurs by the infusion of monoclonal antibodies that bind to amyloid proteins in the brain. One such advantage of using passive immunization as a treatment for AD is that the amount of antibodies infused can be controlled and thus minimized any adverse effects. Six different monoclonal antibodies that target the AB proteins have advanced to phase three clinical trials. However, they have been shown that they are not effective in reducing amyloid deposits in the brain and to improve cognitive function. Although the results are discouraging, nowadays these monoclonal antibodies are being tested in phase 2/3 clinical trials to investigate if the monoclonal antibodies are capable to prevent amyloid deposition in the brain of individuals carrying an autosomal dominant AD mutation and thus prevent AD.
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