Biology subjects

COPPOLA, V.

Publications and source records attributed to COPPOLA, V..

4 recordsLinked to original sources

bioRxiv2024

Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

cancer biology↗

bioRxiv2021

A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

The OsTIR1/auxin-inducible degron (AID) system allows "on demand" selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. This new tool paves the way to studying the differential roles that target proteins may have in specific phases of the cell cycle.

synthetic biology↗

bioRxiv2021

Functional differences drive the selection of NRAS mutants in melanoma

Distinct NRAS mutants are enriched in various tumor types. Here, we generated a suite of fully congenic, conditional, Nras knock-in mouse models (LSL-Nras Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R) to test the hypothesis that melanocyte transformation requires functions specific to the NRAS mutants enriched in human melanoma (Q61R and Q61K). Consistent with the rarity of NRAS codon 12 and 13 mutants in human melanoma, spontaneous melanomas were rare or absent in mice expressing NRAS G12D, G13D or G13R. Mice expressing less common codon 61 alleles (Q61H, Q61P) also developed few or no tumors. NRAS Q61R, Q61K, or Q61L expression, by contrast, induced rapid melanoma onset with high penetrance. Cohorts of heterozygous mice containing one LSL-Nras Q61R and one LSL-Nras Q61K, -L, -H, -P, or -Q allele were generated to assess potential interactions between NRAS mutants. The ability of each Nras variant to substitute for an Nras Q61R allele was consistent with its own ability to drive spontaneous melanoma formation. However, LSL-Nras Q61Q/Q61R mice rarely developed tumors. In vitro experiments in mouse embryonic fibroblasts (MEFs) highlighted activation of the MAPK pathway as a defining difference between tumorigenic and non-tumorigenic NRAS mutants. Enhanced MAPK activation was associated with the promotion of BRAF-BRAF and BRAF-CRAF dimers. These results support the development of cancer preventative strategies specific to the properties of the commonly observed RAS mutants in each tumor type.

cancer biology↗

bioRxiv2019

Tagging allows faithful tracing of expression and enhances biochemical detection of Ran Binding Protein 9 in vivo.

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a novel interaction with Nucleolin, a prominent nucleolar protein. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available tag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially reveal novel functions of this protein in vivo such as those linked to Nucleolin.

bioengineering↗