Brittany Niece
Writing Assignment
Alpha-synuclein (αS) is an abundant protein found in the neurons of the brain that is
thought to play a key role in synaptic vesicle transmission. In our brains, we have presynaptic
terminals at the end of every neuron; it is here that αS interacts with lipids and proteins to, as it
is suggested, maintain levels of synaptic vesicles these terminals. It is worth noting that αS is
thought to regulate the dopamine levels of the brain. Dopamine is a critical neurotransmitter
responsible for voluntary and involuntary movements of the musculoskeletal system, which,
when misfolded or present in toxic amounts, can impair neuron function or even kill the neuron
completely. The importance of this is to help understand the uncontrolled and twitch-like
movements of someone diagnosed with Parkinson's disease or Multiple System Atrophy. With
aging and certain neurodegenerative diseases, such as those mentioned and dementia with
Lewy bodies, the αS protein can improperly fold and cluster into aggregates which are insoluble
in neurons due to "missense mutations, copy number variants, or up-regulated expression"
(Dettmer). Although it was initially believed to strictly form as an unfolded monomer, it has
been discovered that αS can also form "physiological multimers, principally tetramers, which
have α-helical conformation" (Dettmer). This infers that αS can form multimers which are
soluble and do not cause any mutation or neurodegenerative diseases; the physiological forms
are distinct from the pathological forms (Beta-sheet rich aggregates). Beta-synuclein (βS), the
homolog of alpha-synuclein, exhibit cell lysis sensitivity in chemical cross-linking experiments,
which suggests "that a dynamic intracellular population of metastable αS multimers and
monomers coexist normally" (Dettmer). The importance of alpha-synuclein is proven through
this minimal information: if there is too much present, or if αS is present as aggregated clumps
throughout the brain, there will be serious neurodegenerative complications, whether they be
mental, musculoskeletal, or both.
The structure of KTKEGV, as noted in the image below, is polar, charged, and mostly
hydrophilic, having only two nonpolar amino acids in the polypeptide chain. These finding
indicate that αS will be found on the surface of the protein. Of note: KTKEGV has a net +1
charge.
The secondary structure of normal αS has long been thought to be an unfolded
monomer; however, through recent research, it has come to light that these proteins can also
take on alpha-helical conformations forming multimers and tetramers, which are referred to
simply as multimers. Pathological alpha-synuclein, on the other hand, was found to have a
secondary structure described as beta-sheet rich aggregates which are called oligomers. From
attempts to research further, it is noted that while tertiary and quaternary forms “…could
provide some insight into the pathogenic mechanism associated to point mutations,” there is
still no solid evidence that they exist (Kara et al.). In their article, Kara et al. state that there
have been studies suggesting that a tertiary structure of “…α-synuclein consists of two
antiparallel α-helices linked through a short protein chain that naturally assembles into
tetramers presumably preventing α-synuclein monomer aggregation,” but again, there is not
much evidence supporting this.
Dettmer et al. want to show that a mutation causing the abrogation, or retraction, of αS
tetramers can cause the very insolubility, inclusions, and neurotoxicity of αS that causes
neurodegenerative diseases. Therefore suggesting that the “folded tetrameric form is
indispensable for normal αS homeostasis” (Dettmer). That is to say that this folded tetrameric
form of alpha-synuclein actually lessens the likelihood of mutations leading to the negative
neurodegenerative effects.
Based on the common recurrence of each amino acid at each position, it is clear why the
particular sequence, KTKEGV, is used as the reference sequence. In more than half of the 9
samples, each amino acid is found in the same position. This leads to the idea that it is always
more likely for that particular amino acid to appear in that location pending possible mutations.
For each mutation, the charge, size, or polarity of the amino acid changed. For ease of
understanding, the different mutations used in this experiment are listed below:
a. KTKKGV – this mutation of E to K caused a change in the charge of the amino
acid (negative to positive), thus affecting the net charge of the whole
polypeptide chain (+1 to +3). This mutation deferred the formation of tetramers.
b. KTEEGV – this mutation of K to E also caused a change in the charge of the amino
acid, but it went from positive to negative this time, giving the whole chain a net
charge of -1. This mutation still allowed for the formation of tetramers.
c. KTKEIV – inserting I in place of G caused the polypeptide chain to become larger.
This mutation deferred the formation of tetramers.
d. GTKEGV – the charge of the amino acid G is 0, whereas the charge of K is +1. This
mutation causes the net charge to be 0. This mutation still allowed for the
formation of tetramers.
e. KLKEGV – switching the T for L changes the polarity, with T being a polar
uncharged amino acid, and L being hydrophobic. This mutation deferred the
formation of tetramers.
f. KTKEGR – this switch also affects the polypeptides polarity along with its size and
charge. V is a small hydrophobic amino acid while R is much larger, hydrophilic,
and positively charged (net charge = +2). This mutation still allowed for the
formation of tetramers.
g. KTKEGW – mutating the V to a W changes the size, causes the mutant to be
larger. This mutation deferred the formation of tetramers.
Only several of the observed mutations were cytotoxic; KTKKGV, KLKEGV, KTKEIV, and
KTKEGW. It appears that increasing the size, inducing a positive charge, and altering the polarity
of the sequence plays a key role in ceasing the formation of tetramers.
First looking at living cells, it is noted that there are no inclusions of the αS protein
present. Comparing this finding to several of the mutants, (KLK, KGV, EIV, EGR, and EGW) shows
significant differences in all mutants except EGR, which also has no significant finding for αS
inclusions. For KLK, KGV, EIV, and EGW however, it shows a similar percentage (roughly 100%)
of inclusions in the living cells, with KGV only slightly lesser than the others. Of note: these four
mutants are also the only four tetramer abolishing mutants in the study. The importance of
these inclusions is that they are signs of neurodegenerative diseases.
Based on review of the experiments, there appears to be a pattern displayed within the
various mutations. When amino acid size was increased, it caused the αS protein to become
larger, thus eliminated tetramer formation - causing the protein to be cytotoxic. The same is
seen by increasing the net charge, altering the position of hydrophobic versus hydrophilic
amino acids within the polypeptide chain. When the size, net charge, and polarity of αS was
decreased, it still allowed the mutants to form tetramers. The only exception to the pattern is in
the EGR mutant: as previously stated, V is a smaller, hydrophobic amino acid that is uncharged,
while R is larger, hydrophilic, and has a charge of +1. This finding would lead to the assumption
that this mutant would abolish tetramer formation, however, it displays no such behavior.
The authors’ research does support their hypothesis. It is clearly proven that the
abrogation of tetramers leads to cytotoxicity in cells, inevitably leading to neurodegenerative
diseases. Through this research, scientists can now work on methods to identify and alleviate
αS tetramers and multimers, and “place multimerization of this abundant neuronal protein into
a functional context” (Dettmer). In other words, it has now been discovered how this protein
mutates, what can come from these mutations, and how these mutations appear in cells;
therefore, further research and understanding of these proteins could likely lead to treatment
or a cure. The discovery of what regulates the αS monomeric and tetrameric/related multimeric
homeostasis is the missing piece keeping a treatment or cure out of reach. Knowing that this
protein is a prion leads to the concern for transmission through deep-brain stimulation
equipment used to treat Parkinson’s disease (PD). The concern stems from the fact that other
neurodegenerative diseases, such as multiple system atrophy (MSA), can be initially diagnosed
as PD. In an article, the University of California quotes a published researcher stating, “"You
can't kill a protein…And it can stick tightly to stainless steel, even when the surgical instrument
is cleaned."” Therefore, if a patient with undiagnosed MSA is treated for PD with the same
equipment used for other patients, the prions could be transferred from patient to patient.
Another step into future research concerning αS proteins is in the possibility of pharmacological
treatments. In Reynaud’s article, Protein Misfolding and Degenerative Diseases, he mentions
that “therapeutic inhibition of precursor protein synthesis is within reach…drugs that induce
chaperone expression are also being tested, as well as inhibitors that prevent protein
hyperphosphorylation.” Reynaud also notes that “…scientists have more options to find
common structures for the design of specific chemical inhibitors of aggregation…” with
research providing knowledge of progressively more amyloid beta sheet structures. Lastly,
Reynaud mentions that a vaccine against aggregates are actually in the works. With all of this
tow, Dettmer et al.’s findings are sure to make a big impact in the way future research.
References
Dettmer, U., Newman, A., Von Saucken, V., Bartels, T., & Selkoe, D. (2015). KTKEG repeat motifs
are key mediators of normal α-synuclein tetramerization: Their mutation causes excess
monomers and neurotoxicity. PNAS, 112(31), 9595-9601. doi:10.1073/pnas.1505953112
Kara E, Lewis PA, Ling H, Proukakis C, Houlden H, Hardy J. α-Synuclein mutations cluster around
a putative protein loop. Neuroscience Letters. 2013; 546:67-70.
doi:10.1016/j.neulet.2013.04.058. Available from:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694303/
National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The
Library; 2015 Sep 28. SNCA Gene; [reviewed 2012 May; cited 2015 Sep 29]. Available
from: http://ghr.nlm.nih.gov/gene/SNCA
Reynaud, E. (2010) Protein Misfolding and Degenerative Diseases. Nature Education 3(9):28.
Available from: http://www.nature.com/scitable/topicpage/protein-misfolding-anddegenerative-diseases-14434929
University of California - San Francisco. (2015, August 31). New type of prion may cause,
transmit neurodegeneration: Multiple System Atrophy is described as first new human
prion disease identified in 50 years. ScienceDaily. Retrieved September 29, 2015 from
www.sciencedaily.com/releases/2015/08/150831163735.htm