Biology subjects

Garaffo, N.

Publications and source records attributed to Garaffo, N..

3 recordsLinked to original sources

bioRxiv2025

Cul5Wsb2 uses BCL2 proteins as co-receptors to target Bim for degradation.

Anti-apoptotic BCL2 family proteins (e.g. BCL-XL) protect cells by binding and inhibiting pro-apoptotic proteins (e.g. BIM). Although CUL5WSB2 was linked to apoptosis regulation, its substrate and mechanism were unknown. We find that BCL2 proteins recruit BIM to CUL5WSB2 for degradation. WSB2 recognizes BCL-XL through a motif conserved between BCL-XL, BCL-W and BCL2, but not MCL1. Disruption of this interaction through mutation of either BCL-XL or WSB2 blocks the binding of WSB2 to the BCL-XL/BIM dimer. WSB2 also associates with the MCL1/BIM dimer through a separate WSB2 interface, suggesting that WSB2 has evolved independent two means to target BIM. While WSB2 is not essential in most cells, it is essential in cells derived from tumors of the nervous system, and knockdown of WSB2 in these lines causes death and apoptosis. This work uncovers a novel mechanism of apoptosis regulation, with implications for developing therapies against neuroblastomas and other cancers reliant on this pathway for survival.

biochemistry↗

bioRxiv2025

The regulation of Protein Phosphatase 4 by FBXO42 is required for cancer cell survival.

FBXO42 is a poorly characterized F-box protein that is essential in 15% of cancer cell lines from diverse tissue types. FBXO42 has been implicated in the regulation of mitosis and p53 signaling. High-throughput approaches indicate that FBXO42 function correlates with that of CCDC6, and that the two proteins interact physically, but the relationship between these proteins is not understood. Through a genome-wide CRISPR knockout screen, we found that mutation of FBXO42 is synthetically lethal with mutations in the {gamma}- tubulin ring complex proteins MZT1 and MZT2B, suggesting that cells with centrosome and/or mitotic spindle assembly dysfunction are more sensitive to FBXO42 loss. Furthermore, we found that FBXO42 and CCDC6 contribute to p53 activation in response to centrosome depletion. Using mass spectrometry-based proteomics, we found that FBXO42 binds, is required for the ubiquitination of, and negatively regulates the expression of PPP4C (protein phosphatase 4 catalytic subunit). FBXO42s interaction with PPP4C was independent of CCDC6. Similarly, we found that CCDC6 physically interacts with PPP4C independently of FBXO42 and does not affect PPP4C ubiquitination. Knockdown of PPP4C reduced FBXO42-CCDC6 interactions, suggesting that FBXO42 and CCDC6 may bind to and regulate PPP4C through separate mechanisms. Using gene knockdown rescue experiments, we confirmed that aberrant expression of PPP4C is a major driver of cell death in an FBXO42-essential Neuroblastoma cell line. These findings shed light on the function of two poorly understood proteins in regulating PP4 activity, p53 signaling, mitosis and cancer cell survival. A better understanding of FBXO42 and CCDC6 could inform the development of targeted cancer therapeutics.

cell biology↗

bioRxiv2024

p53 protein abundance is a therapeutic window across TP53 mutant cancers and is targetable with proximity inducing small molecules

TP53 mutant cancers are associated with approximately half of cancer deaths. The most common mechanism of p53 inactivation involves missense mutations. Such mutations in TP53 result in a robust upregulation of the p53 protein. Here, we demonstrate an induced proximity approach to selectively kill TP53 mutant cells. This approach uses the increased abundance of p53 protein in TP53 mutant cancer cells to concentrate toxic molecules in these cells. We demonstrate the first generalizable strategy using a small molecule to selectively kill TP53 mutant cells. This molecule binds the Y220C mutant of p53 and concentrates a PLK1 inhibitor in cells harboring TP53 Y220C mutations. Together, these data demonstrate that the abundance of p53 protein provides a therapeutic window for TP53 missense mutant cancers that can be translated into a cell death signal using proximity-inducing small molecules.

biochemistry↗