Bioengineered microbes as a platform for discovering chemical rescuers of disease-related misfolding of integral membrane proteins

Project Information

ProMisMe

Grant agreement ID: 101130862

DOI

10.3030/101130862

Project closed

EC signature date 19 October 2023

Start date 1 January 2024

End date 30 June 2026

Funded under

• Widening participation and spreading excellence

Total cost

No data

EU contribution

€ 211 658,40

Coordinated by

EREVNITIKO KENTRO VIOIATRIKON EPISTIMON ALEXANDROS FLEMINGK
Greece

Periodic Reporting for period 1 - ProMisMe (Bioengineered microbes as a platform for discovering chemical rescuers of disease-related misfolding of integral membrane proteins)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

Protein misfolding diseases (PMDs) constitute a major and growing global health challenge, affecting hundreds of millions of people worldwide and imposing a substantial socio-economic burden on healthcare systems. These disorders, including Alzheimer’s disease, cystic fibrosis and type 2 diabetes, share a common molecular origin: the misfolding of specific proteins that prevents them from acquiring their functional three-dimensional structure. Despite their clinical diversity, this shared mechanism provides a unique opportunity for therapeutic intervention. However, effective treatments remain limited, particularly for diseases caused by the misfolding of integral membrane proteins (MPs), which represent a critically underexplored yet highly significant class of targets.
Membrane proteins represent more than 30% of the human proteome and are involved in essential cellular processes, while over half of currently approved drugs target MPs. Nevertheless, PMDs associated with MP misfolding remain largely incurable. This is primarily due to intrinsic technical challenges in studying these proteins and the lack of efficient discovery platforms for identifying pharmacological chaperones—small molecules or peptides that can restore proper folding, trafficking and function. As a result, there is a clear unmet need for innovative technologies that enable the systematic identification of such compounds.
To overcome these limitations, ProMisMe aims to develop a transformative and broadly applicable platform for the discovery of chemical rescuers of MP misfolding. The central objective is to establish an integrated, high-throughput microbial system capable of generating and screening ultra-diverse libraries of drug-like cyclic peptides for their ability to rescue MP misfolding. This will be achieved through three main directions: (i) biosynthesis of highly diverse cyclic peptide libraries in both bacterial and yeast systems; (ii) development of innovative ultrahigh-throughput screening strategies for identifying bioactive molecules that restore MP folding and stability; and (iii) functional validation of selected candidates using biochemical and cell-based assays.
As a proof-of-concept, the project mainly targets two clinically relevant MPs, Clarin-1 (CLRN1) and peripheral myelin protein-22 (PMP22), whose misfolding is associated with Usher syndrome III and Charcot–Marie–Tooth disease, respectively. Both conditions represent severe, currently incurable disorders with significant impact on quality of life. Successful identification of pharmacological chaperones for these targets will not only generate promising therapeutic leads but will also demonstrate the general applicability of the platform across a wide range of MP-related diseases.
The expected impact of ProMisMe is significant at multiple levels. Scientifically, it will expand the toolbox available for studying and targeting membrane protein misfolding, a long-standing bottleneck in biomedical research. Technologically, it will deliver a scalable and versatile discovery platform with potential applications across academia and industry. From a societal and healthcare perspective, it will contribute to accelerating the development of novel therapies for rare and common diseases, thus supporting the EU’s objectives in improving patient outcomes and fostering innovation-driven growth in the life sciences sector.
In summary, ProMisMe addresses a critical unmet need by bridging the gap between fundamental understanding of protein misfolding and the development of novel therapeutic strategies, offering a clear and credible pathway to scientific, technological and societal impact.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The researcher successfully generated yeast vector libraries for the biosynthesis of ultra-diverse combinatorial cyclic oligopeptides (tetra- to octapeptides) with the general formula cyclo-NuX1X2X3–X7, where Nu is a nucleophilic residue (Cys, Ser, or Thr) and X represents any natural amino acid. Peptide-encoding sequences from the pre-constructed pEcoSICLOPPS-NuX1X2X3–X7 library were subcloned or PCR-amplified into the yeast centromeric vector pAG415 under the constitutive GAP promoter, enabling intracellular expression in Saccharomyces cerevisiae. Following transformation, library integrity and expression were assessed by SDS–PAGE and Western blot analysis, confirming production of the full-length precursor and its processed form, indicative of intein-mediated splicing and cyclic peptide formation.
In parallel, several clinically relevant membrane proteins (MPs), including PMP22, CLRN1, MC4R, GnRHR and V2R, were evaluated as potential reporters for the development of a yeast-based screening system. FLAG-tagged wild-type and mutant variants were expressed in Saccharomyces cerevisiae, with expression confirmed by SDS–PAGE and Western blot analysis. Although most MP variants were successfully produced, functional assessment using spheroplast-based immunofluorescence and flow cytometry analyses revealed limited differences in plasma membrane localisation between wild-type and mutant proteins, restricting their immediate applicability for high-throughput screening. In addition, fluorescence-based detection using anti-FLAG antibody labeling did not provide sufficient signal resolution for all tested MPs, while inhibition of proteasomal degradation with MG132 did not significantly improve fluorescence levels, indicating that proteasome-mediated degradation was not the primary limiting factor under the tested conditions.
To overcome these limitations, alternative strategies were explored to improve membrane targeting, detection sensitivity and assay robustness. A split fluorescent reporter system was developed, in which the target MP was fused to the mNeonGreen2(11) fragment, while the complementary mNeonGreen2(1–10) fragment was anchored to the plasma membrane. Validation of this system using the adenosine A2A receptor as a reporter protein demonstrated efficient membrane localisation and successful signal reconstitution. Furthermore, fusion of MPs to a deubiquitination (dUB) signal significantly improved plasma membrane localisation, highlighting a promising strategy for enhancing the performance and robustness of the yeast-based screening platform.
Finally, a new bacterial screening system available in the host laboratory was applied to identify cyclic peptides capable of rescuing the misfolding and ER retention phenotype of the disease-associated CLRN1(N48K) mutant. Following iterative fluorescence-activated cell sorting (FACS), progressive enrichment of fluorescent populations was observed over consecutive screening rounds, indicating potential successful selection of candidate rescuing molecules. Subsequent next-generation sequencing (NGS) analysis identified multiple peptide candidates for further functional evaluation regarding their ability to restore correct folding, membrane localisation, and trafficking of CLRN1(N48K).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The results obtained during ProMisMe contribute to advancing the state of the art in the field of membrane protein misfolding through the development and optimisation of complementary microbial approaches for the discovery of pharmacological rescuers of misfolded MPs.
The project generated ultra-diverse intracellular libraries of drug-like cyclic peptides in yeast suitable for downstream functional screening and explored innovative fluorescence-based screening strategies to monitor membrane protein localisation and trafficking. In addition, the identification of peptide candidates capable of rescuing the misfolding phenotype of the disease-associated CLRN1(N48K) mutant demonstrates the potential of these approaches for the discovery of novel pharmacological chaperones. The optimization strategies investigated during the project, including split fluorescent reporters and dUB-mediated enhancement of membrane localisation, also provide valuable methodological advances for improving the expression and detection of challenging membrane protein targets in microbial systems. Importantly, the developed cyclic peptide libraries constitute a broadly applicable resource that can be adapted for the screening of additional classes of therapeutic targets beyond membrane proteins, thereby expanding the potential utility of the platform across multiple areas of biomedical and biotechnological research.
These results establish important methodological and experimental foundations for the future implementation of high-throughput microbial screening systems targeting membrane protein misfolding. Further work will focus on functional validation of the identified peptide candidates, optimization of screening sensitivity and robustness, and establishment of proof-of-concept screening systems for additional disease-associated membrane proteins. Overall, these results identify key experimental components and optimisation strategies required for the future implementation of microbial screening platforms for discovering chemical rescuers of disease-related misfolding of integral membrane proteins.