1. Undergraduate Research Day
Identifying Critical Sites for PrPC-PrPSc Interaction Through Dominant-Negative Inhibition
Arla M.A. Mistica
Senior Honors Project
Undergraduate Research Day
April 21, 2012
Good afternoon everyone. My name is Arla Mistica and today I will be presenting “Identifying the critical sites for PrPSC-PrPC interaction through dominant-negative inhibition” as my senior honors project this semester.

2. Transmissible Spongiform Encephalopathy
Prion Diseases
Creutzfeldt-Jakob disease (Humans)
Bovine Spongiform Encephalopathy (BSE; Cattle)
Scrapie (Sheep)
Chronic Wasting Disease (Cervids)
Source: Acquired, Familial & Sporadic
Universally fatal and there is no treatment
Prions are now know to be the pathogen responsible for causing fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep and goats, as well as chronic wasting disease in cervids. In addition to horizontal transmission, other prion diseases can also be acquired through genetic vertical transmission and sporadic somatic mutation (or unknown horizontal transfer). As I said before, prion diseases are fatal but effective treatments are still yet to be developed. These are pictures of animals with TSE, scrapie is characterized by intense itching associated with shedding, behavioral changes are seen in cattle such as abnormal posture, incoordination, difficulty in rising etc., this is a picture of a deer with signs of wasting due to CWD, and this is a picture of human brain with neuronal loss from kuru disease caused by prions.

3. What is a prion? Prion: Abnormally-folded Prion Protein (PrP)
PrPC: normal cellular isoform
PrPSc: Infectious isoform
Enigma: Despite its lack of genetic material
High Infectivity
Various Strains
Daggett’s Model (Spiral model)
Jacquemot et al., J Virol 2005
So what is a prion? A prion is an abnormally folded prion protein that does not contain any genetic material. PrPC is the normal cellular PrP isoform found in the body, while PrPSC is the misfolded, infectious isoform of PrP that is responsible for prion diseases. Some difference in characteristics between the two includes: more alpha helixes in normal cellular isoform and more beta strands in infectious PrP isoform, which tends to be stable in oligomers. One way to diagnose between the two isoforms is that PrPSC is Proteinase K resistant. As seen in this figure. Mice infected with normal brain homogenate showed PrP that’s sensitive to PK while mice infected with scrapie-infected brain homogenate have PK-resistant PrP.
What makes prions an enigma is that while it is highly infective and includes natural occurring strains, much like what we would expect in a virus, it does not have any genetic material, which essentially contradicts the central dogma of biology.

4. Replication of Prion (PrPSc)
Endogenous PrPC
Spontaneous generation of PrPSc
Conversion of mutant PrPC to PrPSc
Inoculation of PrPSc
Interaction between PrPC and PrPSc
Conversion of PrPC to PrPSc
What we know about PrPSC replication is that it requires PrPSC to interact with endogenous PrPC and convert PrPC to infectious PrPSC, which can be followed by accumulation of PrPSC. The conversion of PrPC to PrPSC is thought to occur by template assisted process in which PrPSC acts as a template onto which PrPC is refolded into the infectious conformation; however the conversion from PrPC to PrPSC is still not completely understood.
Accumulation of PrPSc
The conversion of PrPC to PrPSc is still not completely understood.

5. PrPSc-PrPC or PrPSc-PrPSc Interaction
Structures of PrPSc is stable in Oligomers
 PrPSc-PrPSc interaction
Strain-specific structures are inherited with High fidelity
 PrPC-PrPSc interaction
Investigation into PrP-PrP interactions are important
We are interested in looking at PrP interaction with other PrP. The stability and maintenance of PrPSC can be understood by studying PrPSC-PrPSC interactions as PrPSC is stable as oligomers. The high fidelity inheritance of strain-specific structure of PrPSC can be further understood by studying PrPC-PrPSC interactions. Investigation of PrP interactions is definitely important. But for today we will be looking at mainly PrPC-PrPSC interaction.

6. Dominant-Negative Inhibition
When a conversion-incompetent PrP (incPrP) co-exists with a conversion-competent PrP, the former inhibit the conversion of the latter.
Efficiencies of the inhibition varies between incPrPs
Due to variation in Binding affinity of incPrPs to PrPSc?
PrPC
There is a phenomenon called dominant-negative inhibition. This phenomenon occurs when a conversion-incompentent PrP co-exists with a conversion competent PrP and the conversion-incompetent PrP inhibits the conversion of the conversion-competetent PrP into the infectious isoform. [Show animation: normally, PrPC and PrPSC interaction causes the conversion of PrPC to PrPSC but with the presence of conversion-incompetent PrP, the interaction between PrPSC and PrPC is inhibited]. The efficiency of inhibition varies based on incPrP. This variation might be dependent upon the binding affinity of inPrP to PrPSC, much like how an enzyme inhibitor inhibits an enzyme by mimicking the substrate. We expect that if inPrP binds efficiently to PrPSC, then we will see increased dominant inhibition and less PrPSC as opposed to an inPrP that does not bind to PrPSC efficiently.
PrPSC
PrPSC
PrPSC
PrPC
incPrP

7. Hypothesis
If efficiency of dominant-negative inhibition depends on binding affinity of inPrP to PrPSc, the region necessary for PrPC-PrPSc interaction can be identified by making a series of Internal-deletion mutants and evaluating their inhibitory effects.
(3F4)MoPrP
MoPrP -YRYPNQVYYRPVDQYSNQNNFVHD-
Δ159 -YRYPN VYYRPVDQYSNQNNFVHD-
Δ YRYPN PVDQYSNQNNFVHD-
Δ YRYPN YSNQNNFVHD-
Δ YRYPN NNFVHD-
Δ YRYPN HD-
We hypothesize that if the efficiency of dominant-negative inhibition depends upon the binding affinity of inPrP to PrPSC, then we can identify the region necessary for PrPC-PrPSC interaction by making a series of internal deletion mutants.
This is a diagram of 3F4mouse PrP plasmid. We created internal deletion PrPs by deleting proteins using site-directed mutagenesis. We deleted from 1 amino acid, position 159 to 17 amino acids beginning from 159 to 175 spanning between the first and second alpha helixes and including the beta strand indicated in blue.

8. Methods
Create Internal-deletion PrPs (ΔPrPs) by site-directed mutagenesis
Amplify the plasmids encoding ΔPrPs in competent cells and purify the plasmids
Transfect the plasmids to scrapie-infected cultured cells (Co-express conversion-competent PrP with ΔPrP).
Harvest, SDS-PAGE & immunoblot to evaluate dominant-negative inhibition
After we created the internal deletion PrPs, we amplified the plasmids encoding the deletions in competent cells and purified the plasmids. These plasmids were transfected into scrapie-infected cultured cells, which allows for co-expression of conversion-competent PrPs with internal deletion PrP we just created in the same cell infected with PrPSC. We then harvested the PrPs expressed in the cell, digested the proteins with Proteinase K, used SDS-PAGE to separate the proteins, and visualized the proteins using 3F4 antibody by immunoblot.
Transfection to cultured cells
Expression of mutant PrPs
Results

9. concentration (µg/ml)
Results
∆PrPs showed insolubility to detergent & mildly protease-resistant unlike wild-type PrP
4% Sarcosyl
Non-infected
Sup Pellet
4% Sarcosyl
Sc-Infected Non-infected
Sup Pellet Sup Pellet ∆
∆159 ∆
∆159
4H11 (Endogenous Wild-type PrP)
3F4 (Transfected ∆PrPs)
∆159 ∆
Chymotrypsin
concentration (µg/ml)
We also looked at detergent solubility of internal deletion PrPs. The upper right figure shows the presence of PrPs that is detected by 3F4 antibody after being treated with 4% sarcosyl. The figure on the upper left shows PrPs detected by 4H11 antibody. What we see here is that PrPSs from deletion mutants are completely insoluble after 4% sarcosyl treatment compared to a non infected WT, suggesting that deletion mutant PrPs are the infectious isoform different from WT PrPs which is completely soluble. The third figure at the bottom indicates partial protease resistance in which we digested the deletion mutant PrPs with increasing concentrations of protease and this tells us that essentially PrPs from 159 deletion and deletion has no difference.
In addition to PK resistance, we also looked at detergent solubility of internal deletion PrPs (as detergent insolubility is a character of infectious isoform PrPSC). Again, using 3F4 antibody to visualize by immunoblot, we found that internal deletion PrPs are insoluble to detergents. Taken together, PK-resistance and detergent insolubility suggests that internal deletion PrPs are likely to aggregate like the infectious isoform PrPSC.
These findings suggests that ∆PrPs might be different from wild-type PrP but there was no difference among ∆PrPs.

10. Results
Co-transfected (3F4)MoPrP with the Internal-deletion mutants and observed the levels of (3F4)PrPSc
PK-resistant core
(3F4)MoPrP+
Film ∆ ∆ ∆ ∆ ∆
Vector
∆159
; Finally, I observed dominant-negative inhibition.
; As ALL the constructs have not been made up these are still preliminary results.
; This is the very first one. As I had not done it very carefully, the results are a little strange with ~~~
; In this session, I used only Δ159 & Δ and did in triplicate.
; and got satisfactory results.
; The results were a little different between Scanning the fluorescence on Typhoon and quantification on ImageQuant and Film&ImageJ
; ImageJ gives more favorable results with the Lower & the Higher accentuated.
; But, need to do
This figure shows proteins detected by 3F4. As far as dominant inhibition goes: the left lane shows empty vector PrPs. The next two lanes: deletion of 159 and shows less amount of PrPs compared to control, which suggests dominant negative inhibition, while the rest of the internal deletion PrPs seem to be similar as the control, which suggests that dominant negative inhibition did not occur. The graph on the right shows that the amount of PrPs tend to look more similar as the control as the internal deletions become larger, ignoring del as there was a leak in the membrane.
The data suggests that the binding of deletion of 159 and to PrPSC is efficient, hence, the decreased amount of conversion from PrPC to PrPSC, which would be expected due to dominant negative inhibition. The rest of the deletions we created impaired the ability of deletion mutant PrPs to bind to PrPSC, causing the reduced PrPC-PrPSC conversion. This suggests that the rest of the deletions created after 162 are potentially critical for the binding of PrPC to PrPSC.
(Add explanation of trend to look more closely like control – graph)
*There was a leak in “Δ-164”
Vector
∆159 ∆ ∆ ∆ ∆ ∆
∆159 and ∆ showed most efficient inhibition and the inhibitory effects were reduced as the deletions were extended

11. Discussion
Reproducible differences between ∆PrPs in efficiencies of dominant-negative inhibition were observed
The differences suggests that the loop region between the second beta strand and the second alpha helix might be important for efficient inhibition
This supports the point of view that the loop region is important for propagation of prion/PrPSc
Reproducible differences between internal deletion PrPs in efficiencies of dominant-negative inhibition were observed. The differences suggests that the loop region between the second beta strand and the second alpha helix might be important for efficient inhibition…..As we have seen in previous slide, the binding affinity decreases after the region after B strand is deleted. This supports the point of view that the loop region is important for propagation of PrPSC
Potential future of this project includes:
Isolation of the critical proteins that are absolutely needed for binding (proteins following the B-sheet)
Finding efficient inPrPs that can bind to PrPSC effectively may be used therapeutically by the inhibition of PrPC to PrPSC conversion.
(3F4)MoPrP
MoPrP -YRYPNQVYYRPVDQYSNQNNFVHD-
Δ159 -YRYPN VYYRPVDQYSNQNNFVHD-
Δ YRYPN PVDQYSNQNNFVHD-
Δ YRYPN YSNQNNFVHD-
Δ YRYPN NNFVHD-
Δ YRYPN HD-

12. To be continued….
Isolation of the critical amino acids that are absolutely needed for binding (proteins following the B-sheet)
Finding efficient incPrPs that can bind to PrPSc effectively may be used therapeutically by the inhibition of PrPC to PrPSc conversion.
Potential future of this project includes:
Isolation of the critical proteins that are absolutely needed for binding (proteins following the B-sheet)
Finding efficient inPrPs that can bind to PrPSC effectively may be used therapeutically by the inhibition of PrPC to PrPSC conversion.

13. Acknowledgments Prof. Hermann Schatzl Honors Program
Dr. Yuzuru Taguchi
Rick Matlock
Dr. Sabine Gilch
Missy Stuart
Fawn Pickard
Ted John
Leah Kyle
Xiaotang Du
Shaihla Khan
Lauren Millet