Biology subjects

Toczyski, D. P.

Publications and source records attributed to Toczyski, D. P..

4 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

The PRC2.1 Subcomplex Opposes G1 Progression through Regulation of CCND1 and CCND2

Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenetic approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued proliferation inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in antagonizing G1 progression in a diversity of cell linages, including CML, breast cancer and immortalized cell lines.

molecular biology↗

bioRxiv2020

The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae

The S. cerevisiae ISR1 gene encodes a putative kinase with no ascribed function. Here, we show that Isr1 acts as a negative regulator of the highly-conserved hexosamine biosynthesis pathway (HBP), which converts glucose into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the carbohydrate precursor to protein glycosylation, GPI-anchor formation, and chitin biosynthesis. Overexpression of ISR1 is lethal and, at lower levels, causes sensitivity to tunicamycin and resistance to calcofluor white, implying impaired protein glycosylation and reduced chitin deposition. Gfa1 is the first enzyme in the HBP and is conserved from bacteria and yeast to humans. The lethality caused by ISR1 overexpression is rescued by co-overexpression of GFA1 or exogenous glucosamine, which bypasses GFA1s essential function. Gfa1 is phosphorylated in an ISR1-dependent fashion and mutation of ISR1-dependent sites ameliorates the lethality associated with ISR1 overexpression. Isr1 contains a phosphodegron that is phosphorylated by Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex, largely confining Isr1 protein levels to the time of bud emergence. Mutation of this phosphodegron stabilizes Isr1 and recapitulates the overexpression phenotypes. As Pho85 is a cell cycle and nutrient responsive kinase, this tight regulation of Isr1 may serve to dynamically regulate flux through the HBP and modulate how the cells energy resources are converted into structural carbohydrates in response to changing cellular needs. Author SummaryProtein phosphorylation is an essential regulatory mechanism that controls most cellular processes, integrating a variety of environmental signals to drive cellular growth. Yeast encode over 100 kinases, yet many remain poorly characterized. The S. cerevisiae gene ISR1 encodes a putative kinase with no ascribed function. Here, we show that ISR1 decreases the synthesis of a critical structural carbohydrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), by mediating inhibition of one of the enzymes responsible for its synthesis, Gfa1. UDP-GlcNAc is the precursor to protein glycosylation, GPI anchor formation, and chitin synthesis, the first two of which are essential and conserved in humans. Throughout the cell cycle, and in response to changing environmental conditions, the cell must balance its use of glucose for energy production and generation of these structural carbohydrates. Here we show that Isr1 is regulated by both cell cycle and nutrient changes, and is rapidly degraded in a phosphorylation dependent manner. Isr1-mediated inhibition of UDP-GlcNAc synthesis may serve as a mechanism of dynamically regulating how the cell utilizes glucose in response to its environment.

genetics↗