Targeted Protein Degradation Tools: Overview and Future Perspectives

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

doi:10.3390/biology9120421

Cited by 40 publications

(37 citation statements)

References 52 publications

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“…The reduction seen using MAPF is comparable to other methods for selective depletion of proteins that are currently in use. [77][78][79] These EMILIN1 and AGRN depleted hydrogels are additional tools that will assist in isolating the role of biochemical cues from physical cues. One possible conclusion that could be drawn from these results is that neither AGRN nor EMILIN1 are directly associated with the physical properties of the ovary.…”

Section: Discussionmentioning

confidence: 99%

The matrisome contributes to the increased rigidity of the bovine ovarian cortex and provides a source of new bioengineering tools to investigate ovarian biology

Henning

Laronda

2021

Preprint

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The gonadotoxic effects of some cancers significantly increase the risk of developing infertility and cessation of ovary hormones (premature ovarian insufficiency, POI). Fertility preservation in the form of ovarian tissue cryopreservation (OTC) is offered to pediatric and adolescent cancer patients who cannot undergo oocyte retrieval and egg cryopreservation. The cryopreserved ovarian tissue can be transplanted back and has been found to restore fertility in 20 - 40% of transplants and restore hormone function for an average of 3 to 5 years. However, some individuals have primary or metastatic disease within their ovarian tissue and would not be able to transplant it back in its native form. Therefore, there is a need for additional methods for hormone and fertility restoration that would support a safe transplant with increased successful livebirths and long-term hormone restoration. To support this goal, we sought to understand the contribution of the ovarian microenvironment to its physical and biochemical properties to inform bioprosthetic ovary scaffolds that would support isolated follicles. Using atomic force microscopy (AFM), we determined that the bovine ovarian cortex was significantly more rigid than the medulla. To determine if this difference in rigidity was maintained in isolated matrisome proteins from bovine ovarian compartments, we cast, and 3D printed hydrogels created from decellularized bovine ovarian cortex and medulla slices. The cast gels and 3D printed bioprosthetic ovary scaffolds from the cortex was still significantly more rigid than the medulla biomaterials. To expand our bioengineering toolbox that will aide in the investigation of how biochemical and physical cues may affect folliculogenesis, we sought to confirm the concentration of matrisome proteins in bovine ovarian compartments. The matrisome proteins, COL1, FN, EMILIN1 and AGRN were more abundant in the bovine ovarian cortex than the medulla. Whereas VTN was more abundant in the medulla than the cortex and COL4 was present in similar amounts within both compartments. Finally, we removed proteins of interest, EMILIN1 and AGRN, from decellularized bovine ovarian cortex materials and confirmed that this specifically depleted these proteins without affecting the rigidity of cast or 3D printed hydrogels. Taken together our results indicate the existence of a rigidity gradient in the bovine ovary, that this rigidity gradient is maintained in resulting engineered materials strongly implicating a role for matrisome proteins in contributing to the physical properties of the bovine ovary. By establishing additional engineering tools, we will continue to explore mechanisms behind matrisome-follicle interactions.

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Section: Discussionmentioning

confidence: 99%

The matrisome contributes to the increased rigidity of the bovine ovarian cortex and provides a source of new bioengineering tools to investigate ovarian biology

Henning

Laronda

2021

Preprint

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“…Given that transcriptional changes occur rapidly, traditional genetic approaches have failed to effectively define direct targets of sequence-specific transcription factors, and therefore, have inadequately defined mechanisms of transcriptional control by these proteins 9,10 . Thus, CRISPR-based addition of degron tags to endogenous transcription factor proteins has provided a technological breakthrough that is greatly aiding the study of transcription factor function 7,8,34,35 .…”

Section: Discussionmentioning

confidence: 99%

“…Genetic inactivation coupled with RNA-seq and chromatin immunoprecipitation has suggested that DNA-binding transcription factors can control the expression of thousands of genes, but faster methods of transcription factor inactivation suggest that this is an over estimation due to indirect and compensatory transcriptional changes 7,8 . Small molecule "degraders" coupled with the analysis of nascent transcripts and proteomics offer an opportunity to greatly refine these transcription factor networks and define mechanisms of transcriptional control 9,10 .…”

mentioning

confidence: 99%

PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

Zhang¹,

Wang

Liu

et al. 2021

Preprint

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Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues1. Thus, it is difficult to achieve a mechanistic understanding of transcription factor function using traditional genetic deletion or RNAi methods, because these slow approaches make it challenging to distinguish direct from indirect transcriptional effects. Here, we used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO12-6 to define how the t(2;13)(q35;q14) disrupts normal gene expression programs to trigger cancer. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses, we identified a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Moreover, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. The activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective and often only a single element within a complex super-enhancer was affected. In addition, fusion of the endogenous PAX3-FOXO1 with APEX2 identified proteins in close proximity with PAX3-FOXO1, including ARID1A and MYOD1. We found that continued expression of PAX3-FOXO1 was required to maintain chromatin accessibility and allow neighboring DNA binding proteins and chromatin remodeling complexes to associate with this small number of regulated enhancers. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network.

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“…to obtain an accurate and efficient protein degradation, such as deGradFP, Auxin-inducible Degradation (AID) and degradation TAG (dTAG) which aim to achieve proteolysis of POI exploiting the powerful of degradation signal peptide sequences (tags) to hijack POI to E3 ubiquitin ligases for ubiquitylation and consequentially proteasomal degradation by recruitment of the ubiquitinproteasome system [2]. deGradFP exploits the proteasome-based pathway to achieve direct depletion of GFP-tagged proteins, while AID needs of Auxin and transgenic OsTIR1 adapter to trigger POI depletion.…”

Section: Opinionmentioning

confidence: 99%