Novel protein design of Protein Phosphatase 2A (PP2A) mimic to combat Alzheimer’s disease

Summary While therapeutic options for Alzheimer’s disease (AD) are abundant, the causes of AD have yet to be elucidated, hindering the development of AD treatments. One hypothesis that has received considerable attention is the tau hypothesis which states that hyperphosphorylation of tau proteins promotes microtubule degradation, suspected to initiate the AD disease cascade. Protein Phosphatase 2A (PP2A) is a trimeric protein (structural A, catalytic C, and regulatory B subunits) that is abundant in tissues and regulates kinase activity, as well as the dephosphorylation of tau proteins. Unfortunately, there is no therapeutic mimic resistant to SET, an inhibitor protein supposedly involved in the initiation of the AD disease cascade. Here, we propose to (1) synthesize a robust PP2A mimic with resistance towards inhibition by SET and (2) develop a possible therapeutic treatment to reverse the affects of hyperphosphorylation. Timeline I propose a 1.5-year timeline for this project, during which we will achieve the following: Phase One (Months 1 – 6): Isolate the catalytic subunit (C subunit) of PP2A using microwell arrays coupled with semipermeable, polycarbonate membranes functionalized with fluorescently-tagged CD56 antibodies. Phase Two (Months 7 – 12): Modify the C subunit of PP2A to create a “lysine cage” around the catalytic site. Phase Three (Months 13 – 18): Test the catalytic efficiency and resistance to SET. Innovation While the functional aspects of PP2A are known, the techniques to isolate and modify its catalytic site hinder the development of robust mimics, further preventing the development of effective therapeutics. To address this challenge, we propose a platform that integrates single-cell microwell technology with a site-specific reaction paradigm to construct our PP2A mimic, resistant to inhibition by SET. This platform has the potential to be a powerful tool for isolating, developing, and elucidating the reactivity of novel proteins. Also, the successful development of a PP2A mimic will be a first-generation mimic and potential therapeutic, which could be added to the existing arsenal used to treating AD. Significance Since it’s discovery in 1902, AD has plagued countless generations, motivating the crusade to develop robust therapeutics and possibly a cure. While these efforts have produced over 400 pharmaceutical treatments, much of the underlying pathology remains elusive to scientists and physicians, complicating therapeutic development. Efforts to elucidate the epidemiology of AD have unmasked a multi-tier, hypothesis-driven paradigm, revealing that the causes of AD are still relatively unknown. One of these competing hypotheses, the tau hypothesis, has received considerable attention; it states that tau proteins, which stabilize microtubules, initiate the disease cascade after becoming hyperphosphorylated. These hyperphosphorylated tau proteins aggregate, forming neurofibrillary tangles in nerve bodies, destroying the cytoskeleton structure of cells and collapsing the neuron’s transport system. This contrasts to a healthy system, where PP2A is crucial in maintaining the structure of microtubules by dephosphorylating tau proteins, as well as regulating cellular signaling and tumor suppression. It’s believed that in AD susceptible tissue, the inhibitory SET protein is upregulated, inhibiting the reactivity of PP2A, ultimately leading to decrease phosphatase activity and making tau proteins susceptible to hyperphosphorylation. Thus, preserving the functionality of PP2A is pivotal to preventing the AD disease cascade.

Experimental Design Phase One (Months 1 – 6): During these initial months, we will isolate and purify the C subunit of PP2A. To accomplish this, we will use a polydimethylsiloxane (PDMS) microwell platform to capture PP2A (Figure 1). Using a healthy, neuronal cell line (HCN-2, CRL-10742) we will physically separate cells into each well, achieving single cell resolution. After separation, the microwell chip will be sealed with our functionalized membrane and cells will be lysed. The membrane will allow lysis buffer to diffuse into the wells, however, confine single cell debris. Following cell lysis, the arrays will be rinsed with a high salt crowding solution (2M NaCl, 1X PBS, and 50% PEG) that will crowd cellular debris, primarily onto the sides of the wells, as well as on the CD56 antibodies functionalized to the membrane. Since CD56 has a high affinity for PP2A, we suspect it will preferentially bind our protein of interest – this will be confirmed with a fluorescent study. After confirmation of the CD56/PP2A binding, the functionalized membrane, containing the CD56/PP2A complex, will be removed from the chip and dissolved in dichloromethane (DCM) at 40C. Following the dissolution of the membrane, our complex will be purified, followed by another fluorescent study to confirm we still have our complex. During phase one of this project, the lysis buffer and DCM solution pose the greatest potential complications. For instance, if the lysis buffer is too harsh, it could potentially denature our protein of interest, preventing the formation of the CD56/PP2A binding complex. Thus, we will have to fine-tune our lysis buffer to ensure PP2A fidelity. Also, the use of DCM to dissolve our polycarbonate membrane could potentially undergo side reactions, possibly dissociating the protein complex. Since DCM is fairly stable at temperatures below 40C, we will use a temperature gradient to test the reactivity of DCM, as well as conduct a fluorescent study to ensure only the membrane is being dissolved and our complex is not dissociating. Phase Two (Months 7-12): After isolating the CD56/PP2A complex, we will modify the catalytic site of PP2A (Figure 2). To do this, we will biotinylate the C subunit, dissociate the CD56/PP2A complex, and perform a streptavidin pull down on nanoparticles while simultaneously dissociating the PP2A into it’s three subunit – this will allow us to isolate only the C subunit of PP2A. Following streptavidin pull down, we will perform as series of site-specific reactions converting targeted amino acids, in the alpha helices surrounding the catalytic site, into lysines – these lysines will create a “cage,” protecting the catalytic site from SET’s acidic C terminus, it’s primary reaction site (Figure 3). This will provide our PP2A mimic (PP2Am) with resistance to SET, thus maintaining the fidelity of its reactivity. To confirm successful insertion of lysines, we could perform X-ray Crystallography and compare our PP2Am diffraction pattern to that of a normal PP2A diffraction pattern. The diffraction patterns would obviously be different, and quantifying these differences would confirm whether or not the lysines were successfully

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Figure 1: Microwell device for single cell capture and interrogation.

Figure 2: Catalytic subunit of PP2A (Shi, Cell Press, 139, 2009)

inserted. Although a plethora of variables could contribute to differences in diffraction patterns, the high abundance of lysines would alter the electron density of the catalytic site through electronic effects, potentially making the differences obvious. If this fails, we could also do a fluorescent study with fluorescein isothiocyanate (FITC) to quantify the presence of lysine before and after amino acid conversion. A possible complication that could arise is unsuccessful biotinylation of the C subunit - without this, the C subunit cannot be successfully isolated and modified. A resolution to this complication would be to use modified proteases that would digest specific amino acid strands maintaining the ionic interactions between CD56 and PP2A, as well as the interactions between the A, B, and C subunits of PP2A, allowing successful isolation of the C subunit. Phase 3 (Months 13-18): In this final phase, we will validate the activity of PP2Am, as well as its resistance to SET. Validation of this enzyme mimic will be carried out with a modified pyrosequencing method. While pyrosequencing is often used to sequence DNA, it can be reconfigured to detect the activity of PP2Am. In this activity assay, which will be performed in microwells, hyperphosphorylated tau proteins are covalently bound to the base of each well with ATP sulfurylase, adenosine 5’ phosphosulfate (A5P), luciferin, and luciferase present in solution. If the PP2Am is active, when added to the mixture, it will dephosphorylate the tau proteins, generating pyrophosphates (PPi). ATP sulfurase will convert these PPis to ATP in the presence of A5P. Following ATP production, the ATP will act as a substrate for the luciferase-mediated conversion of luciferin to oxyluciferin which will generate light of varying intensity that is dependent on the quantity of ATP (more ATP = brighter light) – the light generated will be captured and analyzed using a camera. With this assay, we will assess the efficacy of our enzyme – if the mimic works, light will be generated. We will also use this assay to compare the reactivity efficiency between our mimic and normal PP2A. Finally, we will assess our mimic’s resistance to SET. We hypothesize that, when this assay is ran with our mimic and SET, there should be no change in light intensity, compared to our control (mimic w/o SET). However, running the same assay with normal PP2A should show a decrease in light intensity compared to our control (mimic w/ SET) because the tau proteins are not being dephosphorylated. Using transmission electron microscopy (TEM), we will confirm our PP2Am’s secondary structure conformation. A possible complication could be side reactions while using the pyrosequencing chemistry. These reactions could inhibit the formation of ATP, preventing detection of PP2A and our mimic’s activity. To resolve this, we propose a microfluidic platform, where reagents can be flowed into the reaction chamber sequentially, as opposed to concerted in the microwell layout – this would minimize the opportunity for side reactions, however, introduce more complexity into our experimental design. Upon successfully synthesis of our mimic, we could move into drug trials and begin the long process of getting this protein in the clinic. Ideally, our robust mimic would one day be the first line of defense against AD, ultimately preventing the initial disease cascade.

Figure 3: Overview of the chemical modification of the catalytic subunit. Not shown here are the biotin/streptavidin complex, as well as nanoparticle scaffold.

Blue:  amino  acids  converted  into  lysine  to  form  the  cage.