Amyloids and Amyloidosis: An overview on mis-folded proteins and their diseases

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Proteins undergo intra-molecular self-assembly processes to form a stable conformation in order to perform diverse and most essential processes in
living organisms. This regular process can be disturbed with mis-folding phenomenon which leads to the formation of insoluble amyloid fibrils resulting
production of a wide range of amyloidogenic diseases. These diseases are both inherited as well as acquired. They are localized and also systemic. Most
of these diseases are chronic and degenerative. Currently diagnosis is carried out by both invasive and non-invasive methods, whereas treatment is
given in the form of chemotherapy, organ transplantation and surgical treatments. However diagnosis and treatment of amyloidosis is more challenging
and hence more knowledge and research is needed in this criteria with molecular and medicinal approach to reveal the mystery of folding and mis-folding
events and also to find a more suitable and lifesaving therapy for different kinds of amyloidosis to save the mankind

Amyloids, Amyloidosis, mis-folding, fibrils, amyloid pathogenesis


Proteins are one of the most important macromolecules that take part in diverse essential processes in all living organisms and hence they form the
fundamental components of all living cells. These protein exhibit an intra-molecular self-assembly process in which the molecules of protein
spontaneously organize themselves to form stable three dimensional conformations, with more specific biological functions, through a number of
different non-covalent interactions such as hydrogen bonds, ionic bonds and van der Waals forces [1]. This self-assembly process is commonly called as
protein folding. At times this regular folding process undergoes malfunction due to various reasons and results in the emergence of inappropriately
folded proteins. Many of such mis-folded proteins often forms insoluble aggregates through deposition and leads to various abnormal diseases. Such
aggregates are often termed as amyloids and their diseased condition as amyloidosis. This review attempts to discuss briefly about the amyloids and its
associated diseases.


Amyloids are insoluble proteins formed as a result of errors of intra molecular self-assembly and it forms the contributory component of many of
degenerative diseases. The word amyloid was coined by Matthias Schleiden, a German botanist in 1838 to describe a normal amylaceous constituent in
plants [2]. However, Virchow used the word first in the medical field to describe some small round deposits in the nervous system in 1854. So far
twenty different unrelated proteins were identified to be the amyloid precursors and are capable of forming stable amyloid fibrils [3].

Formation of amyloids

Amyloids are formed from the rapid aggregation of mis-folded amyloidogenic precursor proteins. These oligomeric aggregations of precursor proteins are
believed to form as a result of various disease specific events. It includes non-physiological and defective physiological proteolysis and various
kinds of mutations producing thermodynamical changes. The mutations in the proteins are the most substantial event which changes the native
conformation of the proteins and supports accumulation of mis-folded proteins. Mutations in non-protein coding genes also produce amyloids (E.g.
Presenilins in AD). These mutations also favor the alteration of proteolytic pathways and forms longer and aggregated proteins [4]. Covalent
modifications of proteins (E.g. Glycation, oxidation etc.) may also endorse mis-folding in addition to ROS production [5]

These aggregates are assembly of higher order structures which are usually insoluble under physiological conditions and are commonly termed as amyloid
fibrils [6]. These amyloid fibrils are formed by inter molecular hydrogen bonding of polypeptide strands produced initially by protein mis-folding.
Initially as a result of mis-folding or denaturation processes, the amyloid precursor proteins acquire the ability to aggregate in an infinite
propagating manner. The aggregates thus formed are of soluble in nature and represents the intermediate in amyloid fibril formation. These soluble
aggregates are spherical, ranges from dimers to polymers and are approximately 3 to 10 nm in size. After a longer period of aggregation, these
aggregates form curvilinear fibres which are bead like structures. These are called as protofibrils which further undergo conformational change or
anneal to produce β fibrils. Mature β fibrils are of 6 to 10 nm in size and have either smooth or helical morphology [7]. This amyloid fibril formation
pathway (Fig 1) is almost found to be similar in all type of amyloid related diseases.

Amyloid formation pathway

Fig 1: Amyloid formation pathway

Structure of amyloid proteins

Amyloid proteins possess many common structural features. They mostly exist in cross β sheet conformation. Amyloid fibrils represent generic,
intermolecular hydrogen bonded structural motif and this hydrogen bonding backbone lies parallel to their fibril axis. These polypeptide amyloids are
commonly arranged in the form of parallel β strands in a sheet in which the amino acid sequence is registered exactly [8]. Each and every amyloidogenic
proteins have the tendency to undergo conformational change to β sheet which forms insoluble fibres. All these protein have similar region with the
ability to bind to glycosaminoglycan portion of proteoglycans and is referred to the GAG binding site. The fibre forming ability and the property of
binding to other elements such as proteoglycan, SAP etc., of these amyloidogenic proteins are same irrespective of their amino acid sequence and origin

Kinds of amyloids

Fibrillar amyloids

These are insoluble fractions produced from the aggregation of amyloid precursors. So far 20 different precursors were identified to form fibrils.
However, all the fibrils exhibit similar structure and properties irrespective of their origin [3].

Nonfibrillar amyloid

Amyloid precursors were also found to produce some nonfibrillar constituents. These nonfibrillar deposits include glycosaminoglycans, proteoglycans and
serum amyloid P component (SAP). These constituents were found associated with all amyloid deposits. SAP bids to fibrils and protect it against
proteolysis and helps in development of amyloidosis [3]. The following table: 1 lists different types of amyloid precursor proteins, amyloid proteins
and their type of deposition.

Amyloid precursors and Amyloids

Table: 1 Amyloid precursors and Amyloids

Amyloid pathogenesis

Amyloid oligomers found in different diseased condition are identified to exhibit common structure and therefore was predicted to have same mechanism
of toxicity. Amyloids arise from both cytosolic as well as from extracellular or secretory proteins and hence their primary target must be accessible
to both the cytosolic and extracellular compartments. The plasma membrane which forms the interface between these two compartments is the most obvious
target of these oligomers. The primary mechanism of pathogenesis of amyloid proteins was believed to be the permeabilization of the plasma membrane,
which is followed by increased intracellular calcium ion concentration [10]. Amyloids such as Aβ, polyglutamine etc., has been identified to produce
discrete pore or single channels in the plasma membranes [11] and these amyloid oligomers specifically increase the conductance of the lipid bilayer
[12]. These oligomers also found to increase the cytosolic free calcium ion concentration and interestingly these ions are found to be liberated from
intracellular stores [13]. It was believed that the oligomers which penetrated the cells disrupts intracellular membranes and paves the way for ion
leakage. But it was also suspected to be due to altered intracellular signaling. This membrane permeabilization and increased ion concentration may
initiate a series of downstream pathological events such as variety of transmembrane signaling processes, production of reactive oxygen species (ROS),
disruption of normal mitochondrial function, induction of oxidative stress etc., [14][15]. Amyloid oligomers can directly penetrate mitochondrial
membrane and along with the stress of increase in energy demand (required to maintain ion homeostasis and membrane polarization) they induce
mitochondrial stress which leads to the mitochondrial dysfunction and increased production of ROS and cytochrome C which results in apoptosis [16]. All
these sequential events ultimately results in the cellular dysfunction and produces up regulation of autophagy and cell death. Besides these,
mis-folded protein fibrils also travel in blood stream and cease osmotic and other filtering processes in the liver, kidneys and heart and produce
other organ based problems.


Deposition of amyloid proteins as extracellular accumulations in various tissues and organs usually results in the disruption of structure and function
of that region and ultimately leads to the diseased condition called “amyloidosis”. In other words amyloidosis is a protein aggregation disease. They
can be acquired or of hereditary type. The deposition of these amyloid fibrils may be of local or systemic. But more commonly amyloidosis is manifested
through systemic involvement rather than local depositions which are uncommon [17]. Amyloidosis involves different parenchymal (E.g. kidney, pancreas,
and liver) and mesenchymal organs (E.g. Cardiac muscles and tendons) and accounts for various degenerative disorders such as systemic amyloidosis,
Alzheimer’s disease, type 2 diabetes, and prion diseases [3]. Amyloidosis is classified based on the types of precursor amyloid proteins. Some of the
different types of amyloidosis are

1. Primary amyloidosis

2. Secondary amyloidosis

3. Familial amyloidosis

4. Senile systemic amyloidosis and

5. Dialysis amyloidosis

The following Table: 2 represent some of the Amyloid proteins and their related diseases

Amyloid proteins and their related diseases

Table 2: Amyloid proteins and their related diseases


Diagnosis of amyloidosis can be done based on traumatic and non-traumatic methodologies. In former, amyloid proteins present in the tissues or organs
can be identified using staining procedures. In later, various non-invasive techniques are being utilized for identifying the amyloids such as serum
and stool examinations and scintigraphy.

Disease suspicion based on the clinical grounds leads to the diagnosis stage, where tissue biopsy is performed followed by staining. For example,
Staining of abdominal fat pad is frequently used for the diagnosis of AL amyloidosis. The dye Congo red is used for staining all types of amyloids.
Congo red is believed to bind with the secondary β-pleated structures of amyloid fibrils through non covalent interactions [18] and it produces
characteristic apple-green birefringence under polarizing microscope. Amyloids can also be identified by fluorescence microscopy using thioflavin
stains which emit green fluorescence when bounded to amyloid proteins [19].

Other non-invasive methods like Serum Paper Electrophoresis, which in combination with urine electrophoresis helps to identify para proteins,
peripheral blood examination show Howell-Jolly bodies in the case of splenic amyloidosis [20], serum free light chain assay measure the production of
abnormal free light chains. In addition to these genetic testing and Scintigraphy is also being performed for different conditions of degenerative
diseases. Genetic testing is used for the detection of transthyretin in case of family (hereditary) amyloidosis. Whereas in scintigraphy radio labeled
macro molecules have been used to quantify protein and it is found to be inexpensive and non-invasive method for diagnosing Protein-Losing Enteropathy


Amyloids can be analyzed to study its fibrilar morphology through electron microscopy by staining with heavy metals such as lead, uranium or tungsten
[22]. The typical secondary structure of the amyloids can be studied by X-ray diffraction analysis of amyloid fibrils which can be isolated from the
tissues and organs by sucrose gradient centrifugation [23] or water extraction methodologies [24].

Current Treatment for amyloidosis

Currently amyloidosis causes death about one per thousand in developed countries and this is mainly because of the fact that there is no treatment to
precisely promote regression of amyloid deposits. Amyloidosis is leading to many complicated conditions such as renal failure in case of long term
haemodialysis, neurodegeneration in case of Alzheimer’s disease, failure of islets in case of type II diabetes etc. Therefore it became a desperate
need for the medical world to identify new therapies to this challenging disease condition [25].

There is no common treatment for different forms of amyloidosis. The treatment given in the case of amyloidosis is purely based on its type and
clinical status of the patient [26]. Chemotherapy is being practised for primary amyloidosis. Here the therapy is focussed to reduce the deposition of
monoclonal immunoglobulins by treating the plasmacyte with radicals. This therapy is now being analysed to replace with autologous peripheral stem cell
or bone marrow grafting. Chemotherapy with the help of melphalan, colchicine and prednisone has been tried to limit the amyloid deposition. Of these,
high dosages of intravenous delivery of melphalan accompanied by autologous stem cell transplantation have shown high degree of success in eliminating
plasma cell dyscrasia. It also showed improvement in functioning of the organs [26].

In many cases of the amyloidosis, due to the lack of the proper therapeutics, the treatment is directed to maintain the proper functioning of the
diseased organs. In the case of congestive heart failure diuretics is being performed, in the case of renal failure kidney transplantation or
hemodialysis is being performed, whereas in the case of familial amyloid neuropathy, liver transplantation is more focussed to inhibit toxic protein
synthesis [26]. Surgical removal of the amyloid deposition is the other widely used treatment under many cases of amyloidosis. These surgical methods
include dermabration, laser vaporization and excision of lesions. Further many treatment procedures which targeting the disruption or inhibition of
deposition of amyloid fibrils, targeting non fibrilar amyloids to increase proteolysis, targeting the membrane permeabilization are still under
investigation and clinical trials.

Future aspects

In all these cases of mis-folding diseases very large numbers of cells are manifested and hence any good therapy targeting the amyloids should diffuse
throughout the diseased tissues to reach most of the cellular compartments. This can be actively done only by small molecules and hence the future of
amyloid treatment lies in the hands of small molecular therapy. However working or designing with small molecules are not that easy comparatively and
therefore a high competency is prevailing among the scientific researchers and the pharmaceutical companies to bring out the remedies. Hence even a
small invention revealing the protein mis-folding or the way to cure or control it will be maximised in the scientific dais and therefore any further
research and development in regarding to amyloids and amyloidosis will be more fruitful.


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About Authors:

Selvakumar K*

Department of Applied Biology,

Global BioLim,

Chennai – 600023

Tamil nadu, India

Madhan R

Department of Applied Biology,

Global BioLim,

Chennai – 600023

Tamil nadu, India

Srinivasan G

Department of Applied Biology,

Global BioLim,

Chennai – 600023

Tamil nadu, India

Baskar V

Department of Informatic Biology,

Global BioLim,

Chennai – 600023

Tamil nadu, India

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