The development of an inclusive world, where no one deserves to be left behind, calls for research on rare illnesses often overlooked by medicine and research, life-changing diseases that deserve thorough study. In fact, an inclusive world is a place where no one is left alone.

This is the reason why our Uni-Padua-IT iGEM team 2025 chose to work on a rare illness. After reviewing many rare diseases, we chose to focus our project on an innovative strategy against Progeria, inspired by our fellow student at the University of Padua, Sammy Basso. Not only was he affected by this disease, but he also became a dedicated researcher on Progeria, fully aware that he would not personally benefit from the results of his work. We recognized iGEM as a way to raise awareness on rare illnesses and more specifically on Progeria.

Hutchinson–Gilford Progeria Syndrome (HGPS) is a rare genetic disorder most often caused by a de novo point mutation in the LMNA gene. It is estimated to affect 1 in 20 million newborns, with about 400 affected children known worldwide.

aberrant splicing of LMNA gene in Progeria
Figure 1. How is progerin synthesized? Progerin synthesis occurs because of a single base mutation in the LMNA gene exon 11. This mutation causes aberrant splicing, which is then responsible for incorrect post-translational modifications. The final protein is shorter and retains a farnesyl group. Made with Biorender.com

LMNA encodes lamin A and lamin C, key components of the nuclear lamina that support nuclear structure and function. In normal processing, prelamin A undergoes: (1) farnesylation of the CaaX cysteine; (2) –AAX cleavage; (3) carboxymethylation; (4) final endoproteolytic removal of the last 15 amino acids by ZMPSTE24 to yield mature lamin A.

The classic HGPS mutation (c.1824C>T; p.G608G) activates a cryptic splice donor site in exon 11 of the lamin A transcript, deleting 150 nucleotides (50 amino acids). This deletion removes the sequence required for the final ZMPSTE24 cleavage (4) of prelamin A, producing a truncated, permanently farnesylated protein called progerin[1].

Progerin accumulation in the inner nuclear membrane disrupts nuclear architecture, causing abnormal nuclear morphology, loss of peripheral heterochromatin, transcription/replication defects, and contributes to DNA-damage phenotypes.

Clinically, the symptoms are widespread to the whole body on different levels. Patients show alopecia, loss of subcutaneous fat, growth delay, skeletal abnormalities, and cardiovascular disease. Life expectancy without treatment is typically in the mid-teens (around 14.5 years), though rare individuals live longer [2].

Progeria symptoms
Figure 2. Progeria (HGPS) pathophysiology. Progeria symptoms are widespread to the whole body and are particularly severe on the cardiovascular tissues, but they do not include neurological complications. Created with BioRender.com

ProgERASE - Uni-Padua-IT 2025 iGEM project

At the beginning of our project, we were aware that research on this disease was very limited. For this reason, we carefully reviewed the available literature.

Our findings brought us to the design of ProgERASE: a PROTAC-like tool capable of targeting progerin and inducing its degradation through ubiquitination.

Let’s put it together: the Ring Bait System

The studies concerning progerin interactors brought us together with the implementation of the Ring Bait System, a recently developed technique for inducing proximity-dependent protein degradation. This method relies on recruiting a protein of interest to the proteasome through a custom-designed interaction with a bait protein fused to a degradation domain.

Ring Bait System mechanism
Figure 3. ProgERASE in action. Schematic overview of the system promoting progerin polyubiquitination and degradation. Progerin-specific interactors (magenta) bind progerin (turquoise), enabling RING domain (light green) dimerization and subsequent ubiquitin transfer (Ub, pink), which triggers proteasome-mediated degradation. Created with BioRender.com.

In order to be functional, ProgERASE must be able to bind progerin. For this reason, we focused on the development, study, and validation of progerin interactors. Building ProgERASE required more than a single line of research: the challenge we chose to address led us to pursue three parallel approaches: computational, yeast-based, and mammalian cell culture-based.

The main problems we faced are the following:

  • The absence of a determined 3D structure of progerin, which required us to rely on computational modeling to generate structural predictions;
  • The need to experimentally test the interaction between progerin and its designed interactors, which led us to implement assays such as Yeast Two Hybrid and NanoBiT complementation;
  • The lack of prior studies on the progerin phenotype in yeast, which made it necessary to establish our own S. cerevisiae model before performing interaction assays;
  • The uncertainty of Y2H results translating to human cells, highlighting the need for further validation of predicted interactions in mammalian systems;
  • The requirement for biologically relevant validation, which led us to test our approach in human cell lines to obtain concrete results

On top of that, we were also led to reflect on what our work meant on a human level: we are scientists working toward a more inclusive world, but how can we make science more inclusive? For this reason, alongside our targeted degradation system, we developed E.A.S.Y.: a tool designed to make scientific papers more accessible to everyone.

References:
  • [1] Luo, Y. B., Mastaglia, F. L., & Wilton, S. D. (2014). Normal and aberrant splicing of LMNA. Journal of medical genetics, 51(4), 215–223. https://doi.org/10.1136/jmedgenet-2013-102119
  • [2] Merideth, M. A., Gordon, L. B., Clauss, S., Sachdev, V., Smith, A. C., Perry, M. B., Brewer, C. C., Zalewski, C., Kim, H. J., Solomon, B., Brooks, B. P., Gerber, L. H., Turner, M. L., Domingo, D. L., Hart, T. C., Graf, J., Reynolds, J. C., Gropman, A., Yanovski, J. A., Gerhard-Herman, M., … Introne, W. J. (2008). Phenotype and course of Hutchinson-Gilford progeria syndrome. The New England journal of medicine, 358(6), 592–604. https://doi.org/10.1056/NEJMoa0706898

In our project, the ultimate goal is to selectively degrade progerin, the mutant and toxic form of lamin A responsible for Hutchinson–Gilford Progeria Syndrome, while preserving the normal lamin A protein.

To achieve this high level of specificity, we used bioinformatics tools to design bait proteins capable of binding exclusively to progerin. By leveraging structure-based modeling, interface prediction, and sequence comparison, we generated and evaluated candidate interactors that specifically recognize the unique features of progerin’s altered C-terminal region.

Our computational approach integrates the AI-based models AlphaFold3, RFdiffusion, and ProteinMPNN, in combination with the molecular docking platforms HADDOCK and ClusPro and affinity estimation with PRODIGY. The pipeline begins with structural modeling and refinement of the target, proceeds through binder backbone and sequence design, and concludes with docking simulations and estimation of binding affinity.

bioinformatics pipeline
Figure 4. Schematic representation of the developed computational pipeline. Made with Biorender.com

How to test protein-protein interactions with an easy tool?
This is one of the questions we asked ourselves when we started working on ProgERASE. And the answer was the simplest existing eukaryote: Saccharomyces cerevisiae.

S. cerevisiae offers an amazing platform for the study of protein interactions called Yeast Two Hybrid (Y2H), a commonly known test that makes use of two plasmids co-transformed into the same cells (in our case pGAD and pGBK). If interaction occurs, a specific molecular pathway is activated, allowing us to observe the interaction.

yeast two hybrid mechanism
Figure 5. Yeast two hybrid (Y2H) molecular mechanisms. The two plasmids, pGADT7 and pGBKT7, are co-transformed into the same S. cerevisiae Y190 cell. If the proteins interact, the histidine synthesis pathway is activated, allowing yeast to grow in histidine-deficient medium. Made with Biorender.com

We implemented the Y2H test by testing it firstly on a known progerin interactor, BUBR1. This protein provides a critical contribution to the Progeria phenotype which arises from progerin’s interaction with BUBR1, a core component of the spindle assembly checkpoint (SAC):

  • The C-terminus of progerin binds strongly to the N-terminal region of BUBR1 (BUBR1-N);
  • This tethering mislocalizes BUBR1 to the nuclear membrane in interphase cells;
  • Consequently, BUBR1 cannot localize to the kinetochore or interact with other SAC proteins

The result is impaired checkpoint activity, accelerated cellular senescence, and severe mitotic defects, including chromosome mis-segregation and aneuploidy.

After implementing the Y2H test on BUBR1, we further validated our findings by testing the peptides predicted by our bioinformatics analysis

We also implemented an S. cerevisiae model of progeria; to do so, we made use of pYES2 plasmid to express progerin in a galactose-dependent manner. Our results show that progerin limit yeast cell growth and provides the basis for further studies regarding phenotype rescue, for example through the usage of a chemical library.

Our degradation system evolves from the RING-Bait system [1] , an advanced version of earlier models like TRIM-Away [2] and PROTACs. These systems use the RING domains of E3 ligases to mediate protein degradation via the Ubiquitin-Proteasome System (UPS), which degrades 90% of cellular proteins. PROTACs (Proteolysis-Targeting Chimeras) are engineered molecules that hijack the UPS to selectively degrade disease-causing proteins. Inspired by this concept, we focused on a PROTAC-like system based on the RING domain of TRIM proteins, particularly TRIM21 [3].

The TRIM Protein Family

TRIM (Tripartite Motif) proteins share a conserved N-terminal tripartite structure:

  • RING domain: E3 domain that interacts with E2 enzymes, catalyzing polyubiquitination (mainly K48- and K63-linked).
  • B-Boxes: modulate RING activity, sometimes with independent E3 ligase roles.
  • Coiled-coil domain: mediates dimerization and structural stability.
The variable C-terminal domains classify TRIMs into 11 subfamilies, influencing localization and interaction partners. TRIM proteins regulate transcription, cell cycle, DNA repair, and protein quality control (PQC).

Spotlight on TRIM21

Structure of the active TRIM21 protein
Figure 6. Structure of the active TRIM21 protein. The image shows the structure of two dimerized TRIM21 molecules bound to an antibody. It can be observed that the PRY/SPRY domains are responsible for the interaction with the Fc region, while the Coiled-Coil regions are essential for dimerization – although these are still predicted regions. Finally, the image highlights how the B-Box region inactivates the RING domain unless phosphorylated.Jones et al. TRIM21/Ro52 – Roles in Innate Immunity and Autoimmune Disease. Front Immunol. 2021

TRIM21 is unique because it functions as both:

  • An E3 ubiquitin ligase;
  • A cytosolic antibody receptor, via its PRY/SPRY domain binding Fc antibodies;
This dual activity allows TRIM21 to target antibody-bound proteins for proteasomal degradation, a powerful mechanism in antiviral defense.

The RING-Bait System for Progeria: Concept and Mechanism

The RING-Bait system fuses the TRIM21 RING domain with a protein-specific “Bait.” Once the Bait binds its target, clustering of RING domains occurs, triggering ubiquitination and proteasomal degradation. This system was first developed for degrading Tau aggregates in neurodegenerative diseases.

Our four-step strategy

  1. Confirming RING’s nuclear activity

    We tested the ability of RING to dimerize and induce degradation by co-transfecting RING-SpyCatcher and SpyTag-mEmEGFP-SpyTag constructs into MRC-5 cell lines, exploiting the interaction between SpyCatcher and SpyTag, two known self-assembling peptides. In this setup, the binding of RING-SpyCatcher to both SpyTags brings two RING domains into proximity, triggering proteasomal degradation of mEGFP. Transfection experiments in 24-well plates, followed by FACS analysis, will allow us to assess mEGFP degradation. Meanwhile, cell viability has to be monitored with AlamarBlue assays to ensure that fluorescence decrease is due to degradation rather than toxicity.

  2. Testing progerin degradation

    To evaluate progerin degradation, we focused on key pathological features of HGPS, such as increased ROS, reduced viability, and nuclear abnormalities. After confirming progerin expression through Western blot, we compared proliferation and viability between progerin-expressing cells and controls. Co-transfections with RING-SpyCatcher were then performed to determine whether degradation of progerin could reverse disease-associated phenotypes.

  3. Validating peptide–progerin interactions

    To test synthetic peptide binding to progerin, we used the NanoBiT® complementation assay. By fusing progerin and the candidate peptides to NanoLuc fragments, protein–peptide interactions could be monitored in living fibroblasts through luminescence. This strategy provided direct experimental evidence supporting our in silico predictions.

  4. Assessing degradation in HGPS patient-derived cells

    Finally, the most promising interactors were expressed in primary fibroblasts derived from a Progeria patient. This step provided a biologically relevant validation, allowing us to assess the potential of our RING-based system in a disease-specific cellular context.

yeast two hybrid mechanism
Figure 7. Final system validation. Schematic representation of the RING-based degradation assay for progerin. The RING–interactor complex binds to progerin aggregates, triggering RING activation and ubiquitin-dependent recruitment to the proteasome. Progerin degradation is evaluated through Western blot quantification and by assessing cell vitality and reactive oxygen species (ROS) production over time. Created with Biorender.com

  1. Nuclear Localization:
    • Progerin accumulates in the nucleus;
    • While TRIM21 can shuttle into the nucleus, the PRY/SPRY domain (important for nuclear translocation) is absent from RING-Bait constructs
  2. Specificity vs. Off-target Effects:
    • Progerin is nearly identical to lamin A;
    • Risk: RING-Bait may also degrade wild-type lamin A;
    • However, HGPS mouse models lacking both progerin and lamin A showed a better phenotype than those expressing both, suggesting that lamin A degradation might be tolerable if progerin is effectively removed
  3. Clustering Requirements:
    • Tau forms large homogeneous aggregates that enable efficient clustering;
    • Progerin aggregates incorporate lamin A and other proteins, possibly limiting RING activation
  4. Delivery and Immune Response:
    • Adeno-Associated Viruses (AAV vectors) can deliver RING-Bait systemically and even cross the blood-brain barrier. However, AAV can trigger immune responses in adults;
    • In pediatric HGPS patients, reduced immune priming might make therapy more feasible