TRIM21, also known as Ro52, is a member of a large family of tripartite motif proteins with a similar domain organisation that number around hundreds in the human genome. An intriguing new gene (RING) domain with E3 ubiquitin ligase activity can be found at the N-terminus of most TRIM proteins, including TRIM21. Following this is an autoregulatory B-box and a coiled-coil domain that are responsible for TRIM21 homodimerization. These domains combine to form the tripartite motif, also known as the RING-Box-coiled coil (RBCC) domain [11] (Fig. 2).

Fig. 2: TRIM21 domains.

Several techniques for the direct degradation of endogenous proteins, such as PROTAC, TRIM-AWAY, and others, have been developed by researchers using the ubiquitin-proteasome pathway. PROTAC-targeted protein degradation is being developed as a novel therapeutic technique for disorders caused by abnormal protein expression. PROTAC molecules are bifunctional small molecules that bind a target protein and an E3-ubiquitin ligase at the same time, triggering ubiquitination and proteasome destruction of the target protein. PROTAC molecules, like small molecules, have good tissue dispersion and can target intracellular proteins [12] (Fig. 3).

Fig. 3: PROTAC system composition.

Our approach

Designing a novel autoubiquitination system based on TRIM-21 and PROTACs in order to degrade intercellular misfolded tau protein which is involved in the early stage of Alzheimer’s disease. System components (Fig. 4): TRIM-21 part which is composed of truncated TRIM-21, flexible linker of (G4S)3, and DocS domain. PROTAC part which is composed of CoH2 domain, flexible linker of (G4S)3, and tau binding peptides (TBP).

Fig. 4: Snitch system composition.

The revolutionary technique involves inserting new (truncated TRIM21/ Docs) and CoH2/ TBP complex into cells, since (DocS) has a high affinity for (CoH2), it will allow the binding of (TRIM21/DocS) and CoH2/TBP, afterwards the TBP will bind to the misfolded tau, allowing TRIM21 to act on this misfolded tau and recruit the ubiquitin-26S proteasome system, leading to their destruction. As a result, we rely on the cellular protein breakdown mechanism to eliminate unaltered native proteins.

The high-temperature requirement serine protease (HTRA) family is a well-conserved collection of proteolytic enzymes (HTRA1, 2, 3, and 4) that play important roles in protein quality control as well as the regulation of numerous signalling cascades via substrate degradation [13,14].

In human beings, the domain architecture of HtrA1, 3, and 4 are the same: an N-terminal IGFBP-like module and a Kazal-like module, a protease domain with a trypsin-like fold, and a C-terminal PDZ domain. In our literature search we found that HTRA1 type is the most effective one that acts on the two protein aggregates related to Alzheimer’s disease which are tau and Aβ [16,17].

Fig. 5: Graphical illustration showing different HtrA1 domains.

HTRA1 degrades Tau

HTRA1 is a serine protease that was recently linked to tau processing. This is an ATP-independent extracellular and intracellular protease that is found everywhere. Although expression is minimal, it may be detected in numerous tissues, including the nervous system [17]. Nonetheless, this enzyme was first linked to AD because it may be involved in amyloid processing [18]. Tubulin was later discovered as a substrate for HTRA1, indicating that HTRA1 may be involved in modulating microtubule function [19,20].

A more recent study found that HTRA1 may cleave recombinant tau in vitro into numerous pieces of various sizes, as well as breakdown insoluble and fibrillarized tau [21]. This capacity to breakdown aggregates is especially fascinating given that HTRA1 has putative chaperone function due to its C-terminal PDZ domains and prefers misfolded substrates [22], Some other studies attempted to boost HTRA1 activity by attaching the PDZ domain to other protease domains such as calpain and short peptides (sequence of H1A) [23] which redeemed successfull at the end.

HTRA1 degrades Aβ

The presence of both secreted and intracellular HtrA1 indicates that HtrA1 has the ability to cleave both the extracellular and intracellular regions of C99. The mapping of the cleavage sites using mass spectrometry (MS) supports this theory. MS identified a comparable sequence of cleavage sites in Aβ-40, Aβ-42, and Amyloid precursor protein intracellular domain (AICD), and in vitro proteolysis tests verified breakdown of the pathogenic Aβ-42 peptide. HtrA1 activity might thus limit the production f Aβ deposits in human brains by competing for substrate with β-secretase and eliminating Aβ aggregations [24].

Our switchable protease approach

Because the brain is such a delicate environment, we must restrict protease activity and target it just to our misfolded proteins, tau and Aβ-42. We created a switchable device, an affinity clamp system, to regulate the protease activity of HTRA1. we created a library of tested binding peptides for both tau and Aβ misfolded proteins, and then filtered the library based on QA and Docking results until we had two Tau binding peptides (WWW, TD28rev) and one Aβ-42 binding peptide (Aβ seed 37-42) The design of the system is HtrA1 bounded with the inhibitor in its catalytic site linked by a flexible linker (L1) and peptides specific to tau and Aβ protein aggregates and a peptide called H1A bounded with the HtrA1 at its PDZ domain also from its other side linked to another linker (L3) and another peptide aslo specific to tau and Aβ, The two peptides linked together by a flexible linker (L2). The activation of the HtrA1 protease is maintained by Protease-Based Ligand Sensors when the tau or Aβ aggregates act between the two peptides since the inhibitor leaves the catalytic (proteolytic) site of the HtrA1. So, HtrA1 will degrade all the protein aggregates. When HTRA1 degrades all protein, the inhibitor will rebind with HTRA1 protease. The peptides that are used to bind with the two protein aggregates are WWW, TD28REV, and Aβ seed.

Fig. 6: Structural composition of Plug sink system.