RUBITAV first aims to answer the following question: what are the differences in the ubiquitinome landscape associated with fibrotic (women-specific) and calcific (men-specific) AV remodeling?

For this part, we will characterize the ubiquitinome landscape of fibrotic and calcified sections of the AV and uncover the sex differences using a mass spectrometry (MS)-based proteomics approach. We are firmly convinced that this approach will identify new sex-specific therapeutic targets to hinder AVS progression, while giving insights into the molecular underpinnings of the fibrotic and calcific transformation of the valve. AVs will be collected from patients undergoing AVR (n>40). For the exploratory analysis of AVS ubiquitinome, 10 patients (5 men, 5 women) will be matched to minimize the effect of confounders (age, comorbidities, and cardiovascular risk factors) and maximize the translational value of the analysis. AVs will be dissected into non-diseased (ND), fibrotic, and calcific sections, and the proteins will be extracted from these sections. ND sections will be used as in-patient controls. Ubiquitinated proteins will be enriched from 30 matched fractions (3 sections/patient) and analyzed by MS. We will focus on polyubiquitinated species, because polyubiquitination is the canonical signal for proteasomal degradation, while mono-ubiquitination serves other non-proteolytic regulatory functions,31 and PROTACs specifically hijack the ubiquitin–proteasome axis. The effect of sex on the difference in polyubiquitinated proteins between fibrotic or calcific tissue and ND controls will be gauged by analysis of covariance (ANCOVA) while adjusting for the unmatched variables. Bioinformatics will prioritize the putative therapeutic targets (ubiquitinated proteins, E3UL or deubiquitinases, DUB).

The specific aims of Part A are to:

• 1.

  Recruit at least 40 AVS patients undergoing elective surgical valve replacement. Collect demographics, echocardiographic, and CCT-derived calcium score data. Obtain the AV from surgery [Task 1];

• 2.

  Match at least 10 patients for age, comorbidities, and disease severity [Task 1];

• 3.

  Dissect the valves into three sections (non-diseased, fibrotic, and calcific sections) [Task 1];

• 4.

  Extract the proteins, enrich polyubiquitinated proteins with TUBEs (tandem ubiquitin binding entities) [Task 2];

• 5.

  Digest and quantify the ubiquitinated proteins by MS and identify the dysregulated proteins (fold-change in expression and the estimated effect by sex) in fibrotic and calcific sections [Task 2];

• 6.

  Prioritize 3 therapeutic targets for fibrosis and 3 for calcification by integrated bioinformatics analysis, encompassing functional enrichment analysis, protein-protein interaction analysis, prediction of E3UL and DUB, and prediction of their druggability by PROTACs [Task 3];

• 7.

  Validate the differentially expressed ubiquitinated proteins, regarded as top-tier targets, with an independent approach (pull-down assay of the target proteins and subsequent Western blotting (WB) for ubiquitin and vice-versa) [Task 4].

We hypothesize that fibrosis and calcification, the two main disease hallmarks, can be mitigated by modulating dysregulated ubiquitinated proteins (uncovered in Part A) using PROTACs, drugs designed to redirect the UPS towards the degradation of specific proteins.

In this part, we will assess the effect of targeting specific polyubiquitinated proteins or regulatory enzymes on fibrosis and calcification. We plan to use PROTACs to modulate the levels of the dysregulated proteins. PROTACs are available for >440 targets32 but have not yet been trialed in the cardiovascular setting. The primary outcome is defined as the reduction (%) in fibrosis or calcification in cells treated with the drug relative to cells treated in parallel with the respective vehicle for the same time. The effect of PROTACs will be tested in vitro, as in vivo models such as the murine and leporine do not precisely replicate human AVS and require the use of cholesterol-rich diets or gene knockout strategies, which introduce confounding factors (e.g., by stimulating coronary atherosclerosis).33 Although closer to humans, swine models, which develop AVS with age, require a lengthy protocol or, alternatively, the implementation of a high-cholesterol diet for faster phenotype development.33 Therefore, we have grounds to believe that utilizing human AV-derived VICs is an optimal strategy to bypass inter-species confounding factors and facilitate the translation to clinics. Specifically, we believe that subjecting male and female VICs to pro-fibrotic and pro-osteogenic stimuli, and modulating the targets derived from Part A, will provide the necessary proof-of-concept for identifying novel sex-specific therapeutic targets for AVS, which can subsequently be tested in vivo.

The specific aims of Part B are to:

• 1.

  Assess the effect of targeting specific proteins with PROTACs on fibrosis in male and female VICs grown under pro-fibrotic conditions (levels of the target protein, extracellular matrix accumulation, and fibrosis markers) [Task 5].

• 2.

  Assess the effect of targeting specific proteins with PROTACs on calcification in male and female VICs grown under osteogenic conditions (levels of the target protein, hydroxyapatite mineral accumulation, and calcification markers) [Task 5].

Patients with AVS flagged at the Cardiology services of both Hospital Pedro Hispano – Unidade Local de Saúde de Matosinhos (ULSM) and Hospital Centre of São João – Unidade Local de Saúde de São João (ULSSJ) and undergoing elective AVR at the Department of Cardiothoracic Surgery of ULSSJ will be invited to participate. The inclusion criteria include (1) willingness to participate; (2) >18 years; (3) echocardiographic evidence of severe degenerative AVS. The exclusion criteria comprise (1) inability to give consent; (2) severe AV insufficiency; (3) bicuspid AVs; (4) rheumatic AVS; (5) active infection or steroid therapy in the last 3 months; (6) severe kidney disease (glomerular filtration rate <30 mL/min/1.73 m2); (7) severe liver disease; (8) endocrine disorders affecting phosphocalcic metabolism.

Patients’ clinical data will be collected, including the most relevant cardiovascular risk factors (e.g., hypertension, diabetes mellitus, dyslipidemia) and comorbidities (e.g., chronic kidney disease, coronary artery disease). Disease severity (e.g., aortic jet velocity, transvalvular gradients, aortic valve area) will be characterized by echocardiography, following the European Echocardiography Association guidelines.34 The calcium score will be collected by CCT.

The valves excised during surgery will be collected in a preservative solution (Custodiol) and transported to the laboratory. As a byproduct of AVR, the collection of the AV does not compromise the patient's safety. Each valve will be dissected by gross macroscopy, ensuring safety margins, to obtain 3 sections: non-diseased (internal control, ND), fibrotic (F), and calcific sections (C). ND is transparent, F is stiff and yellowish, and C is the thickest and most irregular. To ensure correct assignment, a representative transversal section will be obtained from each section for histological analysis. Fibrosis will be assessed by Sirius Red staining and calcification will be assessed by Alizarin Red staining. For all fragments, the % of each stain will be determined in relation to the total tissue area. Following distribution analysis, specific % cutoffs will be set to define ND, F and C sections. All sections for proteomics will be stored at −80°C until further analysis.

We expect to use valves from at least 10 matched patients (50% men) from a total of >40 patients, resulting in 30 fractions (10 ND, 10 F, 10 C). The remaining valves will be used in Task 4 (validation). The total sample size (n=30) was estimated based on our previous study reporting sex differences in aortic valve tissue proteome.20 In this study we found that the proteins dysregulated by sex displayed large effect sizes (minimum Cohen's d: 0.84; mean Cohen's d: 1.00; maximum Cohen's d: 1.23). For a more conservative target of Cohen's d=0.7, with α=0.05 and 80% power, 24 fractions would suffice to determine the effect of sex by ANCOVA. However, to account for the differences between the proteome and ubiquitinome, we inflate this number by 25%, giving a total target of 30.

As a contingency plan for the lack of in-patient non-diseased controls, we will use non-fibrotic and non-calcific valve tissue sections obtained from patients undergoing AVR due to severe aortic regurgitation (without concomitant severe AVS).

The recruitment of AVS patients has been approved by the ethics committee of Unidade Local de Saúde São João (ref. CEC109-2020, reviewed on September 26th 2025). All patients willing to participate will be asked to sign an informed consent. This study will adhere to the 1964 Declaration of Helsinki and its subsequent amendments. The collection of clinical information and echocardiography data abides by the 58/2019 Portuguese Law, warranting General Data Protection Regulation UE/2016/679 regarding the protection of the singular person, including personal treatment and free movement of such data.

The AV is a challenging tissue for molecular analysis, not only due to its heterogeneity, but also due to extensive fibrosis and calcification. For this reason, the AV proteins will be extracted using a bead-based mechanical disruption method we have already optimized for this sample type.35 A lysis buffer enriched with deubiquitinase inhibitor (PR-619) will be used to preserve the modifications. Polyubiquitinated species will be enriched using magnetic Tandem-repeated Ubiquitin-Binding Entities (TUBEs). TUBEs stabilize ubiquitinated substrates and protect them from DUBs and proteasomal degradation during sample handling, thereby improving recovery and sensitivity. The efficiency of the enrichment assay will be controlled by semi-quantification of ubiquitin by WB. A technical negative control, run in triplicate, using ethanolamine-blocked TUBE matrix will control for specificity.

The proteins in the enriched fraction will be digested with LysC+trypsin and the peptides will be analyzed by nano-high-performance liquid chromatography–tandem MS in a data-independent acquisition mode to increase the quantification accuracy. Spectra will be analyzed by sourcing the SwissProt human database and defining Lys ubiquitination as a variable modification. A 5% false discovery rate will be applied for identifications, and the abundance of ubiquitinated proteins will be estimated by label-free quantification. Analysis of covariance (ANCOVA) will be used to assess the effect of sex on the difference in ubiquitinated protein abundances between fibrotic or calcific tissue and non-diseased control tissue. Furthermore, shotgun analysis of the whole proteome in, at least 3 random samples per group, will help filter off proteins whose variation in ubiquitination solely reflects variation at the total level. Data will be deposited in a public repository for proteome datasets: ProteomeXchange.

Ultimately, we anticipate identifying differentially expressed ubiquitinated proteins between the ND, F, and C sections, as well as between men and women, and establishing sex-specific correlations with echocardiography- or CCT-derived parameters of AVS severity.

To shortlist the targets to tackle fibrosis and calcification (each down to 3), the dysregulated ubiquitinated proteins will be prioritized combining 5 main analyses: (1) Functional enrichment analysis (Gene Ontology, Reactome) to map the proteins according to disease-relevant pathways and processes (with particular attention to ubiquitination and proteasome degradation pathways), with standard overrepresentation test for hypergeometric distributions; (2) Protein–protein interaction (STRING) and network topological analyses (Cytoscape), including node degree and betweenness centrality, to assign proteins their hub/bottleneck features (prioritization based on major disease modulation activity); (3) Prediction of the E3UL/DUB responsible for shaping the fibrotic- and calcific-specific valve ubiquitinome. Databases like UbiNet and prediction tools like UbiBrowser will be used to infer E3UL-substrate relationships, with emphasis on E3ULs associated with ubiquitination-mediated proteasomal degradation pathways (e.g., SCF complex); (4) Cross-referencing with publicly available datasets reporting sensitivity to proteasome inhibition (e.g., MG132-responsive proteins), to support the identification of targets more likely to undergo proteasomal turnover; (5) Prediction of the druggability of the putative targets with a machine-learning framework such as DrugnomeAI (this tool specifically provides a scoring function for PROTACs).

This multi-level target prioritization accounts not only for disease relevance but also for the likelihood of proteasomal regulation, thereby increasing the suitability of selected targets for targeted protein degradation approaches.

After statistical and bioinformatic analyses, a final set of proteins will be selected for Tasks 4 and 5.

The final selection of the potential therapeutic targets will take into account an independent validation of the differentially expressed ubiquitinated proteins. First, target proteins will be pulled down from AV lysates by immunoprecipitation. Then, WBs targeting ubiquitin will be used to confirm the changes in the degree of ubiquitination of the target proteins in fibrotic and calcific sections from ND. To confirm this dysregulation, the inverse strategy will be applied: ubiquitinated proteins will be immunoprecipitated first with a pan-ubiquitin antibody and then the target proteins will be validated by WB. Additional WBs are also envisioned to clarify underlying pathways, such as UPS or autophagy.

Once the final targets (ubiquitinated proteins, E3UL, or DUBs) are defined in Task 4, we aim to assess the effect of modulating their expression using PROTACs in two disease hallmarks: fibrosis and calcification. To do that, VICs from both sexes will be isolated from human valves (already implemented in our laboratory, Figure 2). Briefly, valve tissue is subjected to collagenase digestion, and the VICs are cultured in DMEM, supplemented with fetal bovine serum (FBS) and antibiotics, at 37°C and 5% CO2, according to Cuevas et al.,36 with slight modifications. Then, a protocol will be implemented to induce fibrosis and calcification.

Figure 2.

Representative image of an excised aortic valve (left) and isolated valve interstitial cells (right) in culture. Cells were imaged at 40× magnification. Scale bar: 500 μm.

The major risks associated with the various tasks, along with their corresponding mitigation strategies, are presented in Table 2.

RUBITAV builds on the recent findings of our group demonstrating sexual dimorphism in AVS from a proteomics perspective.20 The AV proteome supports an alternative splicing program that contributes to the more pronounced fibrotic remodeling in females and an increased propensity for lipoprotein accumulation, immune system activation, and oxidative stress in males, which primes a more pronounced pro-calcific phenotype. With RUBITAV, we expect to build on these findings, clarifying the role of the UPS, specifically ubiquitination, in the pathological transformation of the valve. We also expect to uncover sex-specific therapeutic targets to tackle fibrosis and calcification in AVS, and to open the way for repurposing PROTACs in atherosclerotic cardiovascular disease, specifically in AVS.

Overall, this project aligns with the UN Sustainable Development Goals 2030 Agenda, specifically goal 3.4, which aims to reduce premature mortality from non-communicable diseases through prevention and treatment. This project will also contribute specifically to goal 5.1, aiming at achieving gender equality concerning the personalization of the diagnosis and treatment. In fact, women have been chronically underrepresented in many milestone trials, resulting in suboptimal treatment. By developing sex-specific therapeutic strategies to target not only valve calcification (mainly afflicting men) but also fibrosis (mainly afflicting women), we will promote equality in the treatment of AVS.