Targeted-Protein Silencing Tools: Overview and Future Perspectives

Prozzillo, Yuri; Fattorini, Gaia; Santopietro, Maria Virginia; Suglia, Luigi; Ruggiero, Alessandra; Ferreri, Diego; Messina, Giovanni

doi:10.20944/preprints202011.0061.v1

Cited by 7 publications

References 48 publications

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Rapid, efficient auxin-inducible protein degradation inCandidapathogens

Milholland,

Gregor,

Hoda

et al. 2023

Preprint

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A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation AID2 systems work efficiently and rapidly in the human pathogenic yeasts Candida albicans and Candida glabrata. We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid (5-Ad-IAA). Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens.

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Rapid, efficient auxin-inducible protein degradation inCandidapathogens

Milholland,

Gregor,

Hoda

et al. 2023

Preprint

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A Roadmap of CAR-T-Cell Therapy in Glioblastoma: Challenges and Future Perspectives

Montoya,

Gallus,

Phyu

et al. 2024

Cells

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Glioblastoma (GBM) is the most common primary malignant brain tumor, with a median overall survival of less than 2 years and a nearly 100% mortality rate under standard therapy that consists of surgery followed by combined radiochemotherapy. Therefore, new therapeutic strategies are urgently needed. The success of chimeric antigen receptor (CAR) T cells in hematological cancers has prompted preclinical and clinical investigations into CAR-T-cell treatment for GBM. However, recent trials have not demonstrated any major success. Here, we delineate existing challenges impeding the effectiveness of CAR-T-cell therapy for GBM, encompassing the cold (immunosuppressive) microenvironment, tumor heterogeneity, T-cell exhaustion, local and systemic immunosuppression, and the immune privilege inherent to the central nervous system (CNS) parenchyma. Additionally, we deliberate on the progress made in developing next-generation CAR-T cells and novel innovative approaches, such as low-intensity pulsed focused ultrasound, aimed at surmounting current roadblocks in GBM CAR-T-cell therapy.

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A Uniform Benchmark for Testing SsrA-Derived Degrons in the Escherichia coli ClpXP Degradation Pathway

Klimecka

Izert

et al. 2021

Molecules

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The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder the development of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.

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Targeted-Protein Silencing Tools: Overview and Future Perspectives

Cited by 7 publications

References 48 publications

Rapid, efficient auxin-inducible protein degradation inCandidapathogens

Rapid, efficient auxin-inducible protein degradation inCandidapathogens

A Roadmap of CAR-T-Cell Therapy in Glioblastoma: Challenges and Future Perspectives

A Uniform Benchmark for Testing SsrA-Derived Degrons in the Escherichia coli ClpXP Degradation Pathway

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