Engineering

Unsuccessful Ideas

I. Aptamers

Design

Our original idea was to enhance the recruitment of proteasomes to these aggregations by tagging them with an external polyubiquitin tail. This could be done by conjugating the tau targeting domain to a ubiquitin tail. The whole complex shall be tagged with our ubiquitin tail upon binding between tau and the domain. The rest of the degradation cascade will proceed to recruit 26S proteasomes and eliminate these harmful aggregations.

Our main goal was to construct a fusion protein composed of a Tau binding domain specific for NFTs inside Alzheimer’s brain connected to the polyubiquitin tail by a linker. Thus our design stage was divided into two main parts.

●Targeting: Based on our search for a suitable binding domain, DNA aptamers seemed to be a perfect fit as of their very high specificity and selectivity towards their target, in addition to their non-immunogenic characteristics. DNA aptamers are small oligonucleotide sequences with different, much better features than antibodies. They are currently used in biosensors to target protein biomarkers at very small concentrations in different fluids.

●Tagging: To determine the right amount of ubiquitin to be used

Fig. 1: Graphical illustration showing The Aptamers system

Failure of cycle

After the search to complete the system design, there was an issue with both parts of the stage that directed us to stop the cycle and look for alternatives

ATP Problem: All eukaryotic cells contain ubiquitin, a highly conserved peptide that is attached to the proteins that the proteasome needs to target [1]. There are three steps in this process. The ubiquitin-activating enzyme (E1) first activates a ubiquitin monomer in an ATP-dependent process (E1). A ubiquitin-conjugating enzyme (E2) receives ubiquitin after that (E2). In the last step, a ubiquitin ligase (E3) transfers the ubiquitin to the target protein (E3). The target protein and the complex E2-ubiquitin are both bound by the E3 ligase, which also aids in the creation of a covalent connection between the ubiquitin monomer produced by the E2 enzyme and the target protein [2]. Since our process include an aptamer conjugated with ubiquitin, which needs ATP, ATP is proved to stabilise tau aggregations as according to Tau can bind ATP, and this binding causes tau to self-assemble into filaments that mimic the Paired Helical Filaments (PHF) that have been isolated from Alzheimer's Disease brains. These filaments appear to establish lateral contact, bundling, and twisting. Because the nonhydrolyzable analog of ATP produces the same assembly, ATP-induced self-assembly is not energy reliant [3]

Aptamers Problem: Our main concern was the ability of protease to degrade the complex in the presence of the DNA aptamer, as they are only degraded by nucleases, so this reason in addition to the ATP problem led to a search for alternatives. [4,5,6]

II. Macrophage:

Design

The idea was to trigger the microglia (macrophage in the CNS) to engulf the plaques and degrade them (phagocytosis).

Failure of cycle The activated microglia can cause synapse loss and damage healthy neurons. In addition, receptor expression in the wet lab wasn’t an easy option for us. [7]

III. Chaperon-mediated autophagy (CMA):

Design

It is a selective autophagy that can degrade toxic proteins, these proteins are recognized by chaperon HSC70 at KFERQ-like motif then their complex binds to lysosome-associated membrane protein type 2A (lamp2a) leading to their entrance to the lysosome and degradation

Failure of cycle In Alzheimer’s disease, the lysosomes become dysfunctional. So, it cannot be used as in the late AD stage.
In our project, our aim was to degrade Tau and amyloid-beta with a method that produces fewer side effects on the brain. After the mentioned need for ATP for activation of the HSP70 function and its proven ability to stabilize Tau and amyloid-beta aggregates, we excluded this method and stop on literature search phase.[8]

IV. Cathepsin D

Design

Cathepsin D is a lysosomal aspartic protease that is important in the control of neuronal homeostasis, cell migration, and interneuron communication. It can degrade misfolded/damaged proteins, including Aβ and Tau, and has no inhibitor to stop its activity.

We considered encapsulating Cathepsin D in a nanoparticle capable of passing the blood-brain barrier and delivering CSTD to Tau and/or beta-Amyloid aggregates, where it would be released. After its release, CSTD can degrade both beta-amyloid and Tau, leading to plaques removal and restoration of healthy neurons extracellular matrix

Failure of cycle The idea stopped in the designing step due to the drawbacks of CSTD as it works at pH below 5, can cleave structural and functional proteins and peptides, its overexpression is associated with metastatic breast cancer and needs further processing by other enzymes at acidic pH after expression since it is expressed as proenzyme. [9,10,11]

V. Bacterial two-hybrid system

Design

Using a bacterial two-hybrid system, we wanted to evaluate the binding affinity between the Coh2 and DocS domains. The bacterial two-hybrid system comprised of our target protein is fused to a DNA-binding domain that localises upstream of a reporter cassette. The binding protein is joined with a component of RNA polymerase. Interaction between the binding protein and the target protein recruits RNA polymerase to the reporter cassette's promoter region and triggers the reporter gene's production. Using antibiotic resistance as a reporter gene, the output of the bacterial two-hybrid system should result in the survival of E. coli cells containing a good binding protein and the death of all cells containing a poor binding protein.[12]

Build The dry lab team started to assemble the desired proteins fused with bacterial two-hybrid system components.

Failure of this iteration When the wet lab team attempted to construct the experimental design of this system, they discovered numerous issues that would contribute to the system's failure. The co-transformation of two plasmids would make bacterial stress, and the optimal spacing between the two expressed domains must be a specified number of nucleotides; otherwise, the expression would be ruined, and incorrect findings would be obtained.

The four cycles of Trim System

Literature

The accumulation of tau inside and outside the cells is one of the main reasons for AD pathogenesis. Therefore, reduction of its accumulations is essential. The ubiquitin–26s-proteasome system (UPS) is the major intracellular protein catabolism-and-anabolism control system in eukaryotic cells [13]. Hence, we chose it to degrade the accumulations intracellularly. It is reported that tau can be degraded by UPS [14]. However, the Ubiquitination cascade activity in AD is reduced which decreases the ubiquitination of these misfolded proteins and consequently increases their accumulation intra- and extracellularly.

I. Design

To properly ubiquitinated tau using UPS, we used the approach, developed by iGEM 2020 team: NUDT_CHINA [15], termed GFP PrePro that uses truncated Trim21 (tTrim21), an E3 ligase, where its PRYSPRY domain and igG-Fc are replaced with DocS and Coh2 to trigger predator and protac system. In our system we replaced the GFP nanobody with tau binding peptide (TBD) to achieve tau specificity.

The following figure highlights the overall system design, where tTrim21-DocS binds to Coh2-TBD to which tau fused, after that tTrim21 ubiquitinates tau using E2 conjugating enzyme. After ubiquitination, tau is degraded using 26S proteasome.

Fig.2: Graphical illustration showing mechanism of action of Snitch system to degrade hyperphosphorylated tau protein. (1) formation of the aggregations of hyperphosphorylated tau protein intracellularly, (2) CoH2-Tau binding peptide (TBP) binds to the aggregated protein, (3) CoH2-TBP-Tau complex binds to Truncated TRIM21 part through the binding of CoH2 and DocS pair proteins forming a new complex, (4) This complex recruits ubiquitin-proteasome system by tagging the bounded tau with ubiquitin molecules, (5) Then the 26S proteasome degrades the tagged protein producing non-toxic protein fragments.

II. Build For the dry lab section, we used the same sequences of tTrim21, Docs, the linker, and Coh2 with some modifications to fit our project, we get the sequences of 19 TBPs and built their structural model [16-22] to choose from them according to literature and modelling results.

III. Test We decided to start the wet lab experiments with two peptides: WWW and TD28rev [18,19]. Regarding Docs basic part, it was mentioned that the expression of DocS alone results in some issues of low stability and yield. However, when it is expressed with another part these issues are reduced [23]. Consequently, we decided to express DocS basic part with GST tag one time , which was reported before to enhance its yield [24], and another time with 6xHis. We built their structure models and the quality assessment results showed that GST-Coh2 and GST-DocS are more stable than when tagged to 6xHis.

The final step was to assemble the parts; 6xHis-tTrim21-linker-Docs and GST-Coh2-TBP then build their assembled protein structure models for further dry lab tests.

In the dry lab:

We tested the binding of ‘GST-Coh2 vs 6xHis-Docs’ and ‘GST-Docs vs 6xHis-Coh2’, the highest binding affinity were -14.653 and -14.026 kcal/mol respectively. The affinity of ‘6xHIS-tTrim21-(G4S)3-DocS’ to ‘GST-Coh2-TD28rev/WWW’ was -18.729 and -15.235 respectively. then docking their complex with tau, afterward molecular dynamics simulations of the parts and complexes to determine their stability with time.

Fig.3: Graphical illustration showing A.GST-Coh2 vs 6xHis-Docs B.GST-Docs vs 6xHis-Coh2.>

In the wet lab:

We built our experiments to test the efficacy of each part of the system separately and then collectively to do the main function which is ubiquitination. The following will illustrate the techniques used accordingly

· UV Spectrophotometer Its purpose is to determine how much light a chemical substance absorbs [25]. It was used to estimate concentration of samples by detecting the amount of light absorbed, samples such as (bacterial culture Optical density, DNA concentration & Purity and protein concentration.

· Agarose Gel electrophoresis Its purpose is to separate DNA and RNA based on their molecular size [26]. In our project it was used to check size of plasmids & DNA fragments and to confirm if experiments such as (Restriction – Ligation – Plasmid mini prep) happened or not.

· Bradford Assay It is an accurate method for determining protein concentration under acidic conditions by using Coomassie brilliant blue dye [27]. In our project, this technique was used before and after affinity chromatography to confirm the efficiency of protein purification.

· SDS-PAGE It is a technique for separating proteins based on their molecular mass.[28] It can be used to determine if the protein was induced or not and to check its size and confirm results from assays like in vitro ubiquitination, aggregates formation and results of pulldown assay.

· Native PAGE It is a technique used to separate proteins according to their sizes and charges.[29] So, it will help in confirming protein-protein interactions.

. Pull down assay it is an invitro technique used to detect physical interactions between two or more proteins.[30] It will be used in detection the interaction between many proteins like

His-DOCS - COH2-GST
His-COH2 - DOCS-GST
His-Tau-WWW-L-COH2-GST
His-Tau-TD28REV-L-COH2-GST
His-Trim-L-DOCS-WWW-L-COH2-GST
His-Trim-L-DOCS-TD28REV-L-COH2-GST
His-Tau-His-T-L-D-(one TBP)-COH2-GST

· In vitro Ubiquitination It is an ATP dependent covalent attachment of ubiquitin to a target substrate [31]. This technique was used in our project by Using ubiquitin due to its ability to bind to trim21 system. which will help in orienting proteasomes to degrade target protein (Tau or ß amyloid).

Expression of the PROTAC system

Problem: The expression of the protac system dockerin and cohesin has a very low yield; however, after some research, we discovered that fusing the Cohesin with a folding protein may enhance the expression rate.

I. Design: To achieve a greater expression rate, we will fuse Cohesin with GST protein and use it for purification and the pulldown experiment.

II. Build: We modelled the Cohesin-GST protein complex and filtered the output models to get the best model according to the quality assessment structures.

III. Test: Drylab: We sorted the models and chose the top one with a QA of 5 out of 6 according to our QA code, then docked these models against Dockerin to ensure that the binding affinity did not change. The his-Coh had affinity of -13.153 kcal/mol and GST-Coh of -14.026 kcal/mol. Finally, we built a mathematical model to predict each part's expression rate and compare it to wetlab data.

Fig.4: The figure is showing the docking of Dockerin against A.GST-Coh2 B.His-Coh2.

Fig.5: The figure is showing the predicted expression rate of A.GST-Coh2 B.His-Coh2.

Wetlab: We transformed (GST-COH) and (His-COH) Into DH5Alpha bacterial cells to amplify it. For (GST-COH) the colonies appear in the plate showing transformation done well with efficiency of 〖80×10〗^4 while (His-COH) had lower value 〖17.2×10〗^4

Fig.6: The figure is showing the transformation of A. GST-Coh, B.His-Coh into DH5Alpha

Fig.7: The figure is showing comparison between induced and non-induced protein absorption of A. GST-Coh part, B.His-Coh part

Fig.8: The figure is showing the interaction of GST-Coh and His-Doc through The pulldown assay

The four cycles of HTRA1 system

Literature

The buildup of Amyloid-beta (A) plaques and neurofibrillary tangles (NFTs) in the entorhinal cortex and hippocampus is thought to induce Alzheimer's disease, resulting in neuronal impairment and, eventually, neuronal death. [32] The insulin-like growth factor binding domain, a kazal domain, a trypsin-like peptidase domain, and a PDZ domain of the serine protease HTRA1 have recently been linked to tau and Aβ processing. This is an ATP-independent extracellular and intracellular protease that is found everywhere.[33]

As a result, we chose it to destroy Aβ and tau accumulations both intra and extracellularly. However, in order to optimise HTRA1 action, we determined to attach it to a switchable system composed of inhibitor and binding peptides for both Aβ and Tau coupled as a clump with linker.

Modelling

I. Design There was no human HTRA1 model with PDZ and catalytic domain. And it was difficult to represent htra1, since it is trimer, and none of the modelling tools we were using could model it.

II. Build We created the monomer using template-based methods and clustered it on the ClusPro server.

III. Test We executed a code-based quality assessment test and graded all of the structures accordingly.

IV. Learn All the structures have a very high c-beta deviation and it isn’t suitable for further testing. So we need new approach for modelling the trimer

V. Redesign We decided to try a novel strategy in which we utilise a denovo modelling tool to choose the optimal monomer and then trimerize this monomer.

VI. Rebuild We modelled the monomer in AlphaFold and picked the best model based on QA, then utilised Cluspro's cluster option.

VII. Retest We checked the model's QA before refining and found it to be 4 out of 6 and aligned it with RCSB models to ensure we have a well-structured model that is closest to those models

VIII. Relearn The RMSD between our model and the RCSB models was 0.9, indicating that our model is built right and was appropriate for further study.

We needed to express the htra1 linked to the inhibitors and binding peptides in order to control the activity of HTRA1 and target the degradation, but we ran into a problem because we needed to trimerize the HTRA1 first, so we used a switchable system consisting of clamp for targeting, inhibitor, and binding peptide to attach and activate the HTRA1.

Clamp assembly

I. Design According to the Paper, we created our binding peptide in the shape of a clamp consisting of (peptide - linker - peptide) so that we could target just Tau and Aβ and manage the switching on and off upon binding.

II. Build We created a library of binding peptides and linkers and modelled each peptide in several modelling programmes in order to achieve the best modelled structures and the highest affinity clamp, as well as to select the linker that is appropriate for the switchable system.

III. Test We evaluated the binding of each binding peptide individually by docking with both Aβ and Tau and graded them based on quality assessment and binding affinity. The clamp was then constructed with varied linker lengths to examine their influence on affinity and tested by docking with both Aβ / Tau whole filaments and seed

IV. Learn According to the Quality assessment and docking results we decided to go with TD28rev and WWW as binding peptides for tau aggregations hence having a binding affinity of -175.066 and -171.237 with` whole tau aggregates respectively and the Seed for Aβ plaques which having binding affinity of -44.19 with Aβ42. And for the clamp we chose TD28revGGSGGGGWWW and seedGGSGGGGGseed.

Fig. 9: Graphical illustration showing Docking of the Clumb with A. Aβ, Tau aggregates

Switch assembly

I. Design To fully degrade the tau and Aβ without distracting the healthy environment of the brain we used HTRA1 which is a natural protease expressed in low yield in the human body and linked it with Inhibitor to control its activity and attached this inhibitor with binding peptides to link its binding with the dissociation and association of HTRA1 and inhibitor.

II. Build We made a library of inhibitors, clamps and linkers. Then we modelled all of them and filtered according to the quality of the model. Then we assembled all the possible probabilities yielding 40 switches.

III. Test

In the dry lab:
In drylab we used the QA and docking to filter these structures and choose the best candidate. We used docking of each part alone of the switch and calculated its energy of binding,afterwards calculated the RMSD of the previously separated docked structure of HTRA1 binding peptide, Inhibitors, and clamps with the top models of the switches to configure best four that succeeded to be close to the docked structures orientation tested ahead, with maintenance of their flexibility owing to the right linker choice.

In the wet lab:
we test the inhibitory activity by protease assay, the inhibitory activity will be detected by the remaining amount of tau aggregates or beta amyloid plaques, which will be visualized by SDS-PAGE and it’s expected that HTRA1 will not show any proteolytic activity

IV. Learn:
According to docking and prodigy data, switch number 10 revealed to be approximate in meeting our requirements by SPINK8 inhibitor that has lower binding affinity than HTRA1 binding peptide and (H1A) has much lower binding affinity to our clamp than Tau and amyloid-beta. As a result, Amyloid beta and tau must bind strongly enough to dissociate the inhibitor from HTRA1, allowing protease activity and degradation to occur.

Fig.10: The figure is showing the switch 10

Description

I. Aptamers

Design

Failure of cycle

II. Macrophage:

Design

III. Chaperon-mediated autophagy (CMA):

Design

IV. Cathepsin D

Design

V. Bacterial two-hybrid system

Design

Literature

I. Design

In the dry lab:

In the wet lab:

Expression of the PROTAC system

Literature

Modelling

Clamp assembly

Switch assembly