Investigating the Effect of Point Mutations in the PRKRA Gene to Understand Molecular Pathogenesis of Dystonia

16 (DYT16)
Autumn Terrelonge, Biological Sciences; Dr. Rekha Patel, Department of Biological Sciences

Background
Dystonia is a neuro-muscular movement disorder characterized by involuntary muscle contractions in the form of
twitching or twisting movements (Geyer and Bressman, 2006). Primary dystonia is caused by mutations in 26 different
genes that have no discernible common biological pathway or theme. Thus, understanding the molecular
pathomechanisms leading to this debilitating disease have remained largely uncharacterized, thus limiting effective
therapeutic strategies. Of the many different types of dystonia, one is DYT16, which has childhood onset and is caused by
mutations in PRKRA gene (Masnada et al., 2021; Carmargos et al. 2008). PRKRA gene encodes for PACT protein, which
activates the protein kinase R (PKR) when cells are exposed to biological stress signals (Figure 1). PKR is involved in
regulating the integrated stress response (ISR), characterized by phosphorylation
serine246
Stress
PACT
serine246 of the translation initiation factor eIF2α (Burnett et al. 2020). The
PACT
serine287
phosphorylation of eIF2a decreases general protein synthesis while also
triggering enhanced translation of specific transcripts leading to homeostasis and
PKR PACT
recovery or cellular apoptosis depending on how intense or persistent the stress
PKR signals are (Burnett et al. 2020). In the absence of cellular stress, PACT and PKR
threonine446
Active PKR
threonine451 heterodimerize (form a complex) with the RNA-binding-protein (TRBP), which
eIF2α phosphorylation
Inhibition of protein synthesis
keeps PKR in an inactive conformation (Burnett et al. 2020). Once stress signal
Apoptosis
induces PACT phosphorylation, there is decreased interaction between PACT and
Fig. 1. PACT activates PKR in response TRBP and increased interaction between PACT monomers thus forming PACT-
to stress.
PACT homodimers, which interact with PKR at a high affinity to bring about
PKR activation (Singh and Patel, 2012). Transient PKR activation and eIF2a phosphorylation is protective to cells but
PKR activation at higher levels or for longer duration is detrimental as the cells undergo apoptosis under these conditions.
We have studied several different PACT mutations that are reported in DYT16 patients (Figure 2), and all of them lead to
increased PACT-PACT and PACT-PKR interactions causing PKR hyperactivation leading to enhanced sensitivity to
stress signals. DY16 patient cells show heightened sensitivity to endoplasmic reticulum stress and higher rate of
apoptosis, thereby contributing to the pathogenesis in DYT16
(Burnett et al. 2020; Vaughn et al. 2015).
Recently two more mutations (G43S and V72F) were
reported in PRKRA that cause DYT16. How these mutations alter
the properties of PACT protein to affect the stress signaling is
unknown and thus, the topic of this proposal is to understand if
the two new point mutations reported to cause DYT16 also cause
Figure 2. DYT16 causing mutations in PACT: the dominant increased PACT-PKR interactions to sensitize the cells to ER
mutations are in red and the recessive mutations are in green.
We have studies all of these mutations before except for G43S stress and cause enhanced apoptosis. This will help to understand
and V72F, which I will generate and study. the disease pathomechanisms, which is an important step for
development of effective therapeutics.
Research Question: What impact do the two newly reported mutations G43S and V72F in PACT protein have on PACT-
PKR interaction?
Project Goals and Objectives: The goal of this research project is to induce a G43S and V72F mutation in PACT’s open
reading frame (ORF) then use it to transfect mammalian cells, express the mutant PACT protein, and determine if the
mutations affect PACT-PKR heterodimerization under cellular stress.
Project Significance: Previous studies have shown that a neurodegeneration mechanism has been detected in DYT16
patients due to increased neuronal apoptosis as the result of abnormal increased activation of PKR under cellular stress
(Masnada et al. 2021). The results of our studies will confirm the molecular pathomechanisms involved in DYT16 and to
study if the two new mutations in PACT also follow the same pattern of PKR hyperactivation. These results can further be
used to find treatment for DYT16, and possibly Alzheimer’s disease (AD), Parkinson's disease (PD), and Amyotrophic
Lateral Sclerosis (ALS) in which PKR hyperactivation has been reported in patient brains.
Methodology:
To begin generating G43S and V72F mutations in PACT we will use site specific mutagenesis with the use of
semi-overlapping primers for whole plasmid PCR. An upstream primer and a downstream primer will be designed to
contain a 19 base pair overlapping sequence region within both primers and non-overlapping sequence regions specific to
each primer. The non-overlapping region of the upstream primer will be 28 base pairs long making it a total of 47 base
pairs long and the downstream primer will have a non-overlapping sequence region that is 40 base pairs long, making it a
total of 59 base pairs. The length of the sequence regions allows optimization of primer melting (Tm) values. We will
induce the G43S and V72F gene at 11 base pairs along the 5’ end of the upstream primer and 9 base pairs along the 3’ end
of the downstream primer all within the overlapping sequence regions. Using this method, Patel lab had previously
generated more than 20 mutations in PACT and PKR genes and a senior graduate student who has mastered this technique
will supervise my primer design and methodology (Burnett et al. 2020; Vaughn et al. 2015).
The plasmid template for our mutations will be tagged with flag wild-type PACT/pCDNA3.1 before conducting a
polymerase chain reaction (PCR) using BioLabs’ high fidelity Q5 DNA polymerase with annealing temperatures of 50° to
72° C. To maximize the mutated plasmid recovery, restriction enzyme Dpn1 will be utilized to digest the methylated
plasmid template of flag-wtPACT/pCDNA3.1. Competent DH5α cells will be treated with 1 µl of the PCR plasmid
product that contains the G43S and V72F mutation for transformation and then will be placed in selective media. The
DH5α cell colonies with then be analyzed for mutation presence using plasmid sequence analysis extracted from 4-6
colonies. The induced mutations will continually be obtained in 100% of the prepared extracted plasmids.
Co-immunoprecipitation (Co-IP) assays will be used to compare dimerization (protein-protein interaction) of the
G43S and V72F mutant PACT proteins and PKR and the wild-type PACT sample. Three samples of cell colonies will be
used, one sample containing mutant plasmid (flag-mutant PACT protein), one sample containing wild-type plasmid (flag-
WT PACT protein), and one control sample containing an empty plasmid (no PACT). The three samples will be lysed and
suspended in PACT antibody to precipitate PACT. Ab-Sepharose beads will then be added to the three sample tubes to
bind/purify the PACT proteins from the samples of which will also extract PKR with it due to dimerization. After adding
the beads, the samples will be incubated for 24 hours. The antibody complexes will then be collected and centrifuges to
separate the protein. Finally, the beads will be washed and analyzed using SDS-PAGE and western blot analysis with anti-
PKR, anti-Flag, antibodies to detect the amount of immunoprecipitated PACT and co-immunoprecipitated PKR in each of
the three samples. This protocol has been used extensively in the Patel lab and is well established (Burnett et al. 2020;
Vaughn et al. 2015).
Project Timeline:
Task 1: Introduce two mutations into PRKRA gene that encodes PACT protein (May-August)
Task 2: Perform mammalian cell transfections and analyze PACT-PKR interaction (September-November)
Anticipated Results and Dissemination:
We expect to see a change in PACT dimerization and PACT-PKR interaction in response to cellular stress. Our
study will be presented at the Fall CIEL GLD Showcase and hopefully published as a manuscript on how these PACT
mutations affect DYT16.
Personal Statement:
Beginning January of 2022, I have had the opportunity to work alongside Dr. Rekha Patel’s research team on
discovering how the dysregulation of PACT-PKR-eIF2a pathway causes DYT16. Throughout my undergraduate studies
thus far I have built knowledge and skills while working in enrolled laboratory course environments and using equipment
and procedures such as SDS-PAGE and PCR that I will be using to perform the techniques for this project. So far, I have
been working alongside Dr. Patel’s team to get a better understanding of the research projects in the lab and how to help
maintain the lab such as autoclaving and growing cell culture. They have prepared me to start working on my own project
that will aid in a bigger picture towards the overall research goal of creating therapeutic drugs for DYT16. I am excited to
finally explore my interests of working with genes and contribute towards aiding patients of this disorder. With the help of
the Magellan Scholar Award, I will be able to live near campus during the Summer 2022 so that I can dedicate more time
to working on this project in hopes of contributing towards published research work in the Fall.
 References:
Burnett, S. B., Vaughn. L. S., Nutan, S., Ronit, K., Patel, R. C. (2020) Dystonia 16 (DYT16) mutations in PACT cause
dysregulated PKR activation and eIF2α signaling leading to a compromised stress response for PKR activation.
Neurobiology of Disease, 146, 105135.
Camargos, S. et al. (2008). DYT16, A Novel Young-onset Dystonia-Parkinsonism Disorder: identification of a
segregating mutation in the stress-response protein PRKRA. Lancet Neurol, 7(3), 207-215.
Geyer, H., & Bressman, S. (2006). The diagnosis of dystonia. Lancet Neurology.
Masnada, S. et al. (2021) PRKRA-Related Disorders: Bilateral Striatal Degeneration in Addition to DYT16 Spectrum.
Movement Disorders, 35(4), 1038-1040.
Singh, M., & Patel, R. C. (2012). Increased interaction between PACT molecules in response to stress signals is
required for PKR activation. Journal of Cellular Biochemistry, 113(8), 2754-2764.
Vaughn, L. S., Bragg, D. C., Sharma, N., Camargos, S., Cardoso, F., & Patel, R. C. (2015). Altered Activation of
Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein
PACT. Journal of Biological Chemistry, 290(37), 22543-22557.
 Magellan Scholar BUDGET FORM

Student’s Name: ___Autumn Terrelonge______

Double-click on table to enter data

Student salary Hours Rate Subtotal
Estimated number of hours student
Enter the hourly wage
will work
Research hours during semesters
when enrolled in classes 0 0 $0.00
Research hours during semesters
when NOT enrolled in classes 300 9 $2,700.00

Fringe: Student salary * student fringe rate 1 (what is fringe? See budget instructions or guidebook)
Enrolled in classes $0.00 0.55% $0.00
Not enrolled in classes $2,700.00 8.23% $222.21

Materials/Supplies Enter sub-total from below: $0.00

Travel Enter sub-total from below: $2,922.21

TOTAL: $2,922.21

Amount requested for Scholar award: $2,922.00

Budget Justification/Description
NOTE: Magellan Scholar awards are processed through “E” funds. All expenditures MUST remain compliant with E fund
procurement requirements. All budgets must be reviewed by department business managers prior to submission.

Student Salary: Indicate estimated number of student research hours per week and hourly rate separated by semesters when student is enrolled in
classes or not enrolled in classes (generally fall or spring vs summer semesters). Time during breaks (Fall, Winter or Spring break) are still hours
during semesters of enrolled classes.

• Summer (10 weeks): 30 hours/week - total 300 hours at $9.00 per hour

Materials/Supplies*: Indicate items, quantity, and estimated price. Be sure to include taxes on all purchases.
*Review guidebook for allowable/unallowable expenses.
• Are you requesting funds for participant incentives? You must attach an approval memo from business manager – see guidebook.
• ALL non-expendable items MUST be fully explained/justified, if not described in methods

• Materials and supplies provided by Dr. Patel’s research team.

Travel: Indicate location, purpose of travel, provide itemized costs (list out each cost separately: transportation, lodging, registration, etc). For
conferences, provide name of conference, dates, and explain why this conference is most appropriate. No more than $1000 is permitted for
conference travel.
No travel is anticipated for research or for presentation during summer.

*NOTE: Expenses beyond the $3000 award will be covered by student (travel).