Submitted to: Dr.

Mehwish Salman
Topic: Protein misfolding and its therapeutic approaches
Course Title: Proteins and amino acids
Department: Biochemistry (BS HONS)
Semester: 5TH (Morning)
Submitted by: M Jamshed (6101)
Hadia Hanif (6106)
Maryam Akmal (6107)
M Mohsin (6129)
Ameena Talib (6143)
Maryam Eman (6148)

Department of Biochemistry
Government College University Faisalabad
 Proteins misfolding and its therapeutic approaches
Contents:
 Introduction
 History
 Reasons of misfolding
 Amyloid and tangles
 Diseases
 Therapeutic approaches

Introduction
Protein folding:
The physical process by which a protein chain is translated into its native three-
dimensional structure, typically a "folded" conformation, by which the protein becomes
biologically functional is known as protein folding (Balsam, 1856).
Each protein exists first as an unfolded polypeptide or random coil after being translated
from a sequence of mRNA into a linear chain of amino acids. At this stage, the polypeptide lacks
any stable (i.e., long-lasting) three-dimensional structure (see the left side of the first figure). As
the polypeptide chain is synthesized by a ribosome, the linear chain begins to fold into its three-
dimensional structure. The folding of many proteins begins even during the translation of the
polypeptide chain. The amino acids interact with each other to produce a well-defined three-
dimensional structure, the folded protein (see the right side of the figure), known as the native
state. The resulting three-dimensional structure is determined by the amino acid sequence or
primary structure (Blancou, 1995).

The correct three-dimensional structure is essential to function, although some parts of

functional proteins may remain unfolded indicating that protein dynamics are important. Failure
to fold into a native structure generally produces inactive proteins, but in some instances,
misfolded proteins have modified or toxic functionality (Fullerton, 2004).
Protein misfolding:
The process by which a protein does not fold into its correct three-dimensional structure
due to different reasons is known as protein misfolding.
Misfolded proteins (also called toxic conformations) are typically insoluble, and they
tend to form long linear or fibrillar aggregates known as amyloid deposits. It is a common
cellular event that can occur throughout the lifetime of a cell, caused by different events
including genetic mutations, translational errors, abnormal protein modifications, thermal or
oxidative stress, and incomplete complex formations (GJ, 2015).

History :
In 1917 the German chemist Hermann Staudinger proposed that organic molecules such
as proteins were organized in polymers, giant molecules made of small-molecule constituents
linked together by chemical bonds in long chains. This idea contradicted the prevailing
hypothesis, and it took some years for biochemists to accept it. Today researchers know that
proteins are long polymers made out of a set of twenty small constituents called amino acids
(Handbook, 2012).

Reasons for misfolding:

 A mutation that changes an amino acid in the protein chain, makes it difficult for a
particular protein to find its preferred fold or “native” state. This is the case for inherited
mutations, for example, those leading to cystic fibrosis or sickle cell anemia. These
mutations are located in the DNA sequence or “gene” that encodes one particular protein.
Therefore, these types of inherited mutations affect only that particular protein and its
related function.

 When proteins are created, the machine (ribosome) that reads the directions from DNA to
create the long chains of amino acids can make mistakes (Hannah, 2010).
 Protein folding becomes even more difficult if the conditions in the cell, like acidity and
temperature, change from those to which the organism is accustomed.
Mechanism of misfolding:
The mechanism of protein misfolding and aggregation follows the so-called “seeding-
nucleation” model. In this process, the initial steps of misfolding are thermodynamically
unfavorable and progress slowly, until the minimum stable oligomeric unit is formed that then
grows exponentially at a fast speed. There are two kinetic phases in the seeding-nucleation
model of polymerization. Firstly, during the lag phase, a low amount of misfolded and
oligomeric structures are produced in a slow process, generating seeds for the next step. Once
nuclei are formed, the elongation phase takes place and results in the fast growth of the
polymers. The addition of pre-formed seeds can reduce the length of the lag phase, accelerating
the exponential phase. Oligomers are perhaps the best seeds to propagate the misfolding process
exponentially. However, larger structures such as fibrils could be as well important to propagate
this event in vivo, due to their higher resistance to biological clearance than smaller aggregates
(Health, 13 April 2016).

Amyloid plaques:
Amyloid plaques are the gradual buildup and accumulation of protein fragments between
neurons; these form when Alzheimer's disrupts the brain's normal disposal process for the
proteins, eventually impacting cognitive function.
Several drugs, known as monoclonal antibodies, may prevent beta-amyloid from
clumping into plaques or remove beta-amyloid plaques that have formed and help the body clear
the beta-amyloid from the brain (Herman, Feb 2017).

Tangles:
Neurofibrillary tangles are formed by hyperphosphorylation of a microtubule-associated
protein known as tau, causing it to aggregate, or group, in an insoluble form. (These aggregations
of hyperphosphorylated tau protein are also referred to as PHF, or "paired helical filaments")
(Lawrence, 1950).

Diseases due to misfolding:

Several diseases are caused by the misfolding of proteins. These include:
 Alzheimer's disease
 Huntington disease
 Cystic fibrosis
 Parkinsons disease
 Prion disease
Alzheimer disease:
Alzheimer's disease has been identified as a protein misfolding disease, or proteopathy,
due to the accumulation of abnormally folded Amyloid-beta proteins in the brains of AD patients
(Lester W, Kaye S, Robertson, 2017).
Misfolding of protein in AD
AD is a progressive neurological disease of the brain characterized by the presence of
protein aggregates and irreversible loss of intellectual abilities, like reasoning and memory;
thereby detrimentally affecting the normal social and occupational lifestyle of the individual. The
aggregates, known as neuritic plaques, chiefly comprise amyloid-β peptide (Aβ), which is
formed by the proteolytic cleavage of amyloid precursor protein (APP), a large type I integral
membrane protein of 695–770 aa that is expressed in many tissues but particularly concentrated
within the synapse of neurons (Loveday, et al., January 2014).

Cleavage of APP by two different enzymatic activities, sequentially by BACE-1 (rather

than normal processing by α-secretase) followed by γ-secretase, releases 40 or 42 aa long Aβ
peptide fragments. These fragments can then combine into oligomers, which can further
aggregate to ultimately form insoluble Aβ clumps surrounding neurons, and these oligomers and
insoluble Aβ forms have been reported to possess detrimental toxic effects. The Aβ containing
aggregates have a β-sheet structure that is a characteristic of amyloid. AD is therefore
distinguished by the accumulation of insoluble Aβ plaques within key regions of the brain
(Manual, 2020).
Symptoms:

 Memory loss disrupts daily life.

 Poor judgment leads to bad decisions.
 Loss of spontaneity and sense of initiative.
 Losing track of dates or knowing the current location.
 Taking longer to complete normal daily tasks.
 Repeating questions or forgetting recently learned information. (Moorer, dec 2021)
Treatment:
Despite the best efforts of medical science, Alzheimer’s remains incurable. However,
some progress has already been made in the fight against this condition. Drugs can help stabilize
mental performance and slow the progression of symptoms. As well as a medicinal treatment,
many concomitant therapies are in use today aimed at promoting sufferers’ mental faculties or
preserving these capabilities for as long as possible, improving the overall quality of life
(Rabenau, October 2005).
Huntington disease:
Huntington's disease, a lethal neurodegenerative condition, is believed to be caused by
misfolding of mutated versions of huntingtin protein in which a glutamine-containing sequence
is repeated too many times.
Typically when a protein misfolds, the cell destroys it, but as a person ages, this quality-
control mechanism starts to fail and the rogue proteins build up. In Huntington's, for
example, huntingtin protein—used for many cell functions—misfolds and accumulates.

Huntington's disease (HD) is caused by an expansion of cytosine-adenine-guanine (CAG)

repeats in the huntingtin gene, which leads to neuronal loss in the striatum and cortex and the
appearance of neuronal intranuclear inclusions of mutant huntingtin. Huntingtin plays a role in
protein trafficking, vesicle transport, postsynaptic signaling, transcriptional regulation, and
apoptosis. Thus, a loss of function of the normal protein and a toxic gain of function of the
mutant huntingtin contributes to the disruption of multiple intracellular pathways. Furthermore,
excitotoxicity, dopamine toxicity, metabolic impairment, mitochondrial dysfunction, oxidative
stress, apoptosis, and autophagy have been implicated in the progressive degeneration observed
in HD (Roberts, 2017).
Symptoms:

 difficulty concentrating and memory lapses.

 depression.
 stumbling and clumsiness.
 involuntary jerking or fidgety movements of the limbs and body.
 mood swings and personality changes.
 problems swallowing, speaking, and breathing.
 difficulty moving.
Treatment:
Currently, no cure or treatment exists for Huntington’s disease. However, your doctor can
treat your symptoms and improve your quality of life.

 Medications to lessen the mental and physical effects of the disease

 Therapy for depression and anxiety that often accompany Huntington’s disease
 Exercise therapies to help movement problems
 Access to support services in your community (Rutala, 2008).

Prion disease:
Transmissible spongiform encephalopathies (TSEs or prion diseases) are a rare group of
invariably fatal neurodegenerative disorders that affect humans and other mammals. TSEs are
protein misfolding diseases that involve the accumulation of an abnormally aggregated form of
the normal host prion protein (PrP)
The misfolding and aggregation of the human prion protein (PrP) are associated
with transmissible spongiform encephalopathies (TSEs). Intermediate conformations forming
during the conversion of the cellular form of PrP into its pathological scrapie conformation are
key drivers of the misfolding process (SM, May 2006).

Symptoms:
 Rapidly developing dementia.
 Difficulty walking and changes in gait.
 Hallucinations.
 Muscle stiffness.
 Confusion.
 Fatigue.
 Difficulty speaking.
Treatment:
 Unlike bacteria and viruses, prion diseases are irreversible and can not be overcome by
the body's immune system. As of yet, there are no medical drugs or other types of advances for
the treatment of prion diseases. There are no known ways to cure prion diseases but RML
scientists are working to develop treatments. Using both cell-free and cell-based assays, NIAID
researchers have tested thousands of compounds and identified hundreds of molecules that
inhibit the formation of the abnormal form of the prion protein (Semmelweis, September 15,
1983).

Therapeutic approaches to protein misfolding:

For AA amyloidosis
In 1928, Henning Waldenström reported the rapid resolution of hepatomegaly and liver
amyloid deposits after successful surgical treatment of “lymphoid TBC fistulae” in a child
presumably affected by AA amyloidosis. Similar successful regression of AA amyloid deposits
has been described after surgical excision of localized Castleman’s disease leading to the
resolution of the associated chronic inflammation.
Studies of experimental murine AA amyloidosis show that AA amyloid can be indeed
spontaneously cleared upon cessation of inflammation through redundant innate immune
mechanisms). They also demonstrate that, after the apparent resolution of AA amyloid deposits,
a relapse of inflammation can lead to a dramatic, rapid exacerbation of amyloid deposits,
presumably due to residual AA amyloid fibrils resisting clearing mechanisms and serving as
nucleating seeds, further stressing the importance of maintaining reduced SAA levels throughout
the disease (http://www.fornariindustria.com.br/en/uncategorized/how-the-disinfection-process-
works/, 2017)
For ATTR Amyloids:
Tetramer dissociation is currently regarded as the rate-limiting factor for ATTR
amyloidogenesis, as it releases TTR monomers which can then misfold and aggregate. A parallel
mechano-enzymatic amyloidogenic mechanism has also been proposed. The first drug to obtain
approval for the treatment of ATTRv amyloidosis, tafamidis, was the result of a structure-based
drug design. This molecule was indeed designed to bind to one of the two T4-binding pockets of
the TTR tetramer and to slow tetramer dissociation. (lenntech.com, 12 December 2019.)
GAIM technology:
The protein coat of bacteriophage M13 binds that disaggregates and prevents the
formation of multiple amyloid structures. GAIM selectively targets misfolded protein
aggregation in diseases such as prion and Alzheimer's disease. (sedman, 2003)
Neurophage pharmaceuticals:
Preclinical development and clinical approach that NPT088 recognizes and disrupts the
shape of misfolded proteins and targets them for degradation through the body's natural
mechanism. Misfolded proteins can, however, be rescued via the use of proteostasis regulators
and/or pharmacological chaperones, suggesting that treatments with small molecules might be
developed for a range of genetic diseases. (toxnet.nlm.nih.gov., 2016)

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