Group 2 Research Proposal

Background:
Tertiary structure of a protein is vital for its function, as it determines the shape and
properties of the reactive sites that connect with other molecules in the cell[1]. Misfolded
proteins are toxic to cells due to deprivation from normal function, harmful gain of function, and
tendency to undergo non-native interactions, such as forming aggregates[2]. Proteopathic
diseases, such as Huntingtons disease, are caused by mutations that lead to conformational
changes leading to a tendency to aggregate[3]. Huntingtons disease is caused by an autosomal
dominant mutation that increases the length of the poly-glutamine sequence (polyQ) and
increases the tendency of huntingtin to misfold and aggregate. This aggregation leads to a
harmful gain-of-function, interfering with cellular machinery needed for cell viability[4].
However, the cell has an adaptive mechanism by which it combats the detrimental effects of
misfolded proteins to maintain proteostasis, the stable folding, trafficking and degradation of
proteins. The maintenance of proteostasis is accomplished by the attenuation of mRNA
translation, increased transcription of ER chaperones and the degradation of misfolded proteins
along the ER associated degradation pathway[2].
Specific Aims
The aim of the experiment is to observe the effect of drs-1 knockdown in C. elegans.
This will reaffirm what the genes normal function is and demonstrate the role it plays in
maintaining proteostasis. Drs-1 codes for aspartyl-tRNA synthetase, which charges tRNA with
aspartate during translation. Our hypothesis is that drs-1 knockdown will lead to reduced protein
translation and subsequently reduced protein aggregation in our Huntingtons model worms.
In Anderson, et al., the knockdown of arginyl-tRNA synthetase reduced translation rate
in C. elegans and completely blocked induction of biomarkers (P hsp-4::GFP) for misfolded
proteins [5]. With fewer proteins were synthesized, there was a much smaller incidence of
aggregates forming, and the worms were saved. In relation to our experiment, we believe that the
knockdown of Drs-1 will elicit a similar effect on protein translation, aggregate formation and C.
elegans phenotype. However, the knockdown of drs-1 should reduce translation below the
threshold at which aggregates start to form, but not to the point that the cell dies due to lack of
protein synthesis. The potential for suppression of protein translation as an evolutionary safety
mechanism to reduce aggregation further supports our hypothesis [5].
Experimental Design
In these experiments, we will be working with three variants of the L4440 plasmid. This
plasmid contains an origin of replication, a gene for ampicillin resistance (Ampr), a multiple
cloning site (MCS) which is flanked by two T7 promoters. The first variant is the empty vector
(L4440). This control is to show that the plasmid itself has no effect on aggregation. The second
variant has the YFP gene spliced into the MCS (L4440/YFP). Our final experimental plasmid
variant has drs-1 spliced into the MCS (L4440/Drs-1). Our plasmid variants were transformed
into HT115 bacterial cells and grown on an ampicillin plate. Specific colonies will be selected,
grown in an overnight culture and stimulated with IPTG. The IPTG will lead to the production of
T7 RNA Synthetase. The flanking T7 promoters will then lead to the production of dsRNA of
our inserts because of the two flanking T7 promoters. For the L4440/YFP plasmid, the IPTG
plasmid will lead to the production of dsRNA for YFP, leading to a knockdown of YFP
signaling. In our experimental plasmid, L4440/drs-1, IPTG stimulation will lead to knockdown
of drs-1.

Group 2 Research Proposal

We will be using two types of transgenic C. Elegans worms. Our control strain of worms
is transgenic for a construct containing a muscle specific promoter upstream of YFP gene
(mus/YFP). Our experimental strain of worms is transgenic for a construct containing a muscle
specific promoter upstream of Q40 (Huntingtons Model) which in frame with YFP. Due to the
muscle specific promoter, we should see YFP production in only the muscle cells of our C.
Elegans.
The bacterial culture will then be plated, creating an RNAi Bacterial Lawn. For each
plasmid variant, we will create two bacterial lawns. Half of the lawns will be plated with our
control mus/YFP eggs. The remaining plates will be plated with our experimental mus/Q40/YFP
eggs. Over the course of a week, the C. Elegans eggs will mature and consume the HT115
bacterial cells and internalize the dsRNA. One week later, we will collect our data from the
worms. Through the use of WormTracker and Fluorescent Microscopy, we will be able to see
what, if any, effect knocking down Drs-1 had on worm movement, size, speed and aggregation
of our Huntingtons model.
Expected Results
Preliminary results have been encouraging and in-line with our expected results. Worms
fed the empty vector (L4440) will be our control for this experiment. As expected, the mus/YFP
worms show high levels of YFP expression, while our mus/Q40/YFP worms show high levels of
protein aggregation and less movement.
In worms fed L4440/YFP, we expect less YFP expression due to the YFP RNAi
produced by our plasmid. Our results to this point support this, as the YFP expression in both
mus/YFP and mus/Q40/YFP is less than the empty vector control. These results show us that our
YFP gene is working as expected.
Finally, in the worms fed L4440/drs-1, we expect less protein aggregation and more
worm movement, due to the reduction in protein translation. Our preliminary results have not
shown anything conclusive, but other papers give us confidence in this hypothesis.
Alternative Strategies/Future Plans/Conclusions
The results of our experiment have significant implications regarding other proteopathic
conditions. Should the knockdown of drs-1 lead to reduced protein aggregation, then this may
suggest that the knockdown of other translational proteins could also yield similar preventive
containment of protein aggregation. Examples of such illnesses include ALS (Amyotrophic
lateral sclerosis) and CreutzfeldtJakob disease (commonly known as mad cow), both of which
are invariably fatal.
There are many future directions with this project. Pertaining to this experiment
specifically, there are other ways to quantitatively determine the effect of drs-1 knockdown. For
example, we could lyse the C. elegans cells and run a western blot or a quantitative-PCR for
specific marker proteins for the presence of misfolded proteins. We would expect levels of these
marker proteins to go down in mus/Q40/YFP worms with the drs-1 knockdown. Additionally,
we could run a similar experiment in human muscle cells and see the results of a drs-1
knockdown. This experiment will show us how cells maintain proteostasis and molecular
mechanisms behind Huntingtons disease.

Group 2 Research Proposal

REFERENCES

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Reynaud, E., Protein Misfolding and Degenerative Diseases. Nature Education, 2010. 3(9): p. 1.
Cao, S.S. and R.J. Kaufman, Endoplasmic reticulum stress and oxidative stress in cell fate
decision and human disease. Antioxid Redox Signal, 2014. 21(3): p. 396-413.
Carrell, R.W. and D.A. Lomas, Conformational disease. Lancet, 1997. 350(9071): p. 134-8.
Hatters, D.M., Protein misfolding inside cells: the case of huntingtin and Huntington's disease.
IUBMB Life, 2008. 60(11): p. 724-8.
Anderson, L.L., et al., Survival from hypoxia in C. elegans by inactivation of aminoacyl-tRNA
synthetases. Science, 2009. 323(5914): p. 630-3.