What is TRIM21?
Tripartite motif containing-21 (TRIM21) is an antibody receptor and cytosolic ubiquitin ligase that acts as
the body's last line of defense against incoming viruses. It accomplishes this by functioning as a sensor,
intercepting antibody-coated viruses that have escaped extracellular neutralization and entered the cell [1].
As described in (Fig.1) , the TRIM21 protein is composed of a RING domain with the activity of E3 ligase attached
to an autoregulatory B-box and a domain responsible for TRIM21 homodimerization called the coiled-coil domain at the N-terminus.
The PRYSPRY domain found at the C-terminus is responsible for the TRIM21 interaction with other proteins,
especially the Fc domains of the antibody [2].
Fig.1: A graphical illustration showing the domains of TRIM21.
Types of TRIM21 degradation systems
The degradation system, depending on TRIM21, can be divided into three generations:
1st generation (TRIM-Away system)
This system consists of TRIM21 protein bound to antibodies.
The system can be specific to the target by expressing antibodies specific to the targeted protein/pathogen (Fig.2) [2].
2nd generation (Predator system)
This system is updated by NUDT 2019 team. It depends on using the
IgG Fc domain instead of using the whole antibody. This part is bounded to a targeting module by a linker (Fig.2) This team improved the system by replacing the PRYSPRY domain with different types of pair proteins [3].
3rd generation (Snitch system)
This system is considered a modified version of the predator system.
It consists of two parts: truncated TRIM21 bounded to a protein called DocS.
The truncation process was done by removing the PRYSPRY domain.
The other part (PROTAC part) is composed of CoH2 protein bounded to tau binding peptides (TBP) by a
flexible linker (GGGGS)3 (Fig.2).
Fig.2: TRIM21 degradation systems.
The system is used to detect tau protein aggregates by binding Truncated TRIM21-DocS, CoH2-TBP, aggregated Tau complex, which activates the normal ubiquitin-proteasome degradation cascade and initiates the degradation of bounded tau by the 26S proteasome. (fig. 3). pathway (Fig.3).
Fig.3: 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.
Genetic circuits
The Snitch system parts were constructed using the T7 promoter induced by IPTG, lac operator,
different ribosomal binding sites (RBSs) according to the expressed protein, and T7 terminator.
It was cloned into our cloning and expression plasmids pUC-IDT and pGS-21a, respectively.
A. Expression of Truncated Trim21-DocS
Fig.4: Schematic representation of Truncated Trim21-DocS expression system.
B. Expression of CoH2-TBP
We choose two TBP (TD28rev and WWW) to be expressed in our system bounded to CoH2 protein by a flexible linker (GGGGS)3.
Fig.5: Schematic representation of (a) CoH2-TD28rev and (b) CoH2-WWW expression systems.
C. Expression of Tau (0N4R)
Fig.6: Schematic representation of Tau protein (0N4R) expression system.
D. Expression of E2 enzymes (UBE2N, UBE2V2, and UBE2W)
Fig.7: Schematic representation of (a) UBE2W, (b) UBE2N, and (c) UBE2V2 expression systems
E. Expression of ubiquitin C
Fig.8: Schematic representation of Ubiquitin C expression system
What is HTRA1?
HTRA1, a serine protease that has recently been linked to tau processing, is an ATP-independent extracellular protease found inside the body. Despite its low expression, it has been detected in several tissues, including the nervous system. The insulin-like growth factor binding domain, a kazal domain, a trypsin-like peptidase domain, and a PDZ domain compose the HTRA1. To achieve optimal expression, we exclusively expressed the trypsin-like peptidase domain and the PDZ domains.
A switchable protein circuit.
Because the brain is such a critical environment, we must limit HTRA1 protease activity and direct it to just operate on our misfolded proteins, tau, and Aβ42. To modulate the protease activity of HTRA1, we developed a switchable device, the so-called affinity clamp system.
HTRA1 system is based on switchable design principle. Connecting a protease domain (P1) to an autoinhibitory (AI) domain that binds and blocks the active site of the protease yields a simple signal transducer. A conformational change of the linker dislodges the attached inhibitor from the active site, thereby activating the protease. In its simplest form, such a rearrangement is caused by the force that binding peptide clamp exerts upon attachment to its target, which separates the AI domain from the transducer protease, so relieving autoinhibition and activating the transducer protease. After the targeted protein is completely degraded, the binding clamp is free so that the linker tension will be released, and the inhibitors will return back to their previous place, once again making HTRA inactive.
It was crucial to collectively find binding peptides, linkers, and inhibitors that allow the system switchability during the presence or absence of the target proteins. For that, we created a library of each to decide on the appropriate collection of the linkers, inhibitors, and binding peptides to connect the switch to the HTRA1.
Our libraries
First, we created a library of tested binding peptides for both tau
and amyloid beta misfolded proteins and then filtered the library based
on QA and Docking findings until we had two Tau binding peptides (WWW, TD28rev)
and one Aβ42 binding peptide (seed37-42)
Second, we looked for an HTRA1 binding peptide since it is the connection between
the switch system and HTRA1; thus, we filtered based on binding energy and QA and
found one peptide called (H1A), There are two reasons behind using H1A peptide,
first it boosts HTRA1 proteolytic activity, second it links the switchable domain
and HTRA1, so we hypothesised that the binding energy between H1A and HTRA1 must
have the highest binding affinity of all domains which interact with HTRA1.
Third, because we are only expressing proteolytic and PDZ domains, we looked for
proteolytic domain inhibitors and screened them based on affinity and inhibitory
impact. We chose two inhibitors from our inhibitor library based on two rounds
of exclusion criteria: first, our QA ranking code, which rates the quality of
the predicted 3D structure. During the second phase, we screened the inhibitors
based on their binding affinity scores and chose SPNK8 and WAP4, the inhibitors
with the highest and lowest scores, respectively, in order to compare the activity
of each inhibitor with HTRA1.
Finally, we examined numerous linkers of varying lengths and sequences to assure system switching,
which is mostly dependent on linker tension. We have made different size variants of two linkers
((GSGS)n (GGGSG)n`), whereas their length range changed according to the switch assembly.
Nevertheless, we added all of the tested inhibitors, the binding peptide of the target protein,
and the HTRA1 PDZ domain as a part collection to ease the further usage of our novel system by the future iGEM teams.
Fig.1: Graphical illustration showing mechanism of action of Plug sink system. (1) formation of protein aggregates (Tau and amyloid-beta (Aβ), (2) HtrA1 become inactivated while the targeted proteins are not found, (3) the targeted protein bound to HtrA1 swith clamp, (4) the HtrA1 inhibitor detached from the catalytic domain of HtrA1 making it active, (5) the activated HtrA1 degrades the targeted protein and after degrading all the proteins it becomes inactivated again.
Genetic circuit
To express the system, we divided it into two circuits, one for the HTRA1 and one
To express the system, we divided it into two circuits, one for the HTRA1 and one for the switch, with a T7 promoter, a lac operator, and a T7 terminator to regulate the expression through induction, but with distinct RBSs to control the expression of each portion.
For cloning, we utilized pJEt plasmid, and for expression, we used pGS-21a plasmid.
A. Expression of HtrA1 monomer
Fig.2: Schematic representation of HtrA1 monomer expression system.
B. Expression of HtrA1 switches
We expressed 4 different switches (HtrA1 switch 10, 12, 15, and 18) with different HtrA1 inhibitors, linkers and Tau and Aβ binding peptides for targeting them.
Fig.3: Schematic representation of HtrA1 switch expression system.
C. Expression of amyloid-beta (Aβ)
Fig.4: Schematic representation of amyloid-beta (Aβ) expression system.
D. Expression of Tau (0N4R)
Fig.5: Schematic representation of Tau protein (0N4R) expression system.