Biology subjects

Fitzgerald, M. E.

Publications and source records attributed to Fitzgerald, M. E..

3 recordsLinked to original sources

bioRxiv2026

Site-specific combinatorial ubiquitination drives the targeted degradation of plasma membrane proteins

Cullin-RING E3 ligases (CRLs) ubiquitinate their substrates by recruiting them to complexes containing ubiquitin-conjugating enzymes (UCEs). Ubiquitination proceeds through the transfer of ubiquitin from UCEs to substrate lysines, but the specificity of lysine ubiquitination has not been widely investigated in cells. In this study, we use global ubiquitination profiling to identify sites in the kinase domains of receptor tyrosine kinases (RTKs), EGFR and Her2, that undergo CRL-mediated ubiquitination in cells treated with heterobifunctional degrader molecules. We find that VHL- and Cereblon-dependent degraders trigger the ubiquitination of a common subset of accessible lysines. Using mutagenesis, we show that the number of available ubiquitination sites specifies the extent of RTK degradation. Furthermore, introduction of non-native sites mostly fails to rescue clearance of lysine-deficient RTK mutants. These results highlight the specificity with which UCEs ubiquitinate their substrates and suggest that the ubiquitination of multiple sites governs the efficacy of degraders targeting plasma membrane proteins.

cell biology↗

bioRxiv2024

Dual targeting of KDM1A and antioxidants is an effective anticancer strategy

AbstractLysine Specific Demethylase 1 (KDM1A / LSD1) regulates mitochondrial respiration and stabilizes HIF-1A (hypoxia-inducible factor 1A). HIF-1A modulates reactive oxygen species (ROS) levels by increasing cellular glucose uptake, glycolysis, and endogenous antioxidants. The role of KDM1A in cellular ROS response has not previously been described. We determined the role of KDM1A in regulating the ROS response and the utility of KDM1A inhibitors in combination with ROS-inducing cancer therapies. Our results show that KDM1A inhibition sensitized cells to oxidative stress and increased total cellular ROS, which was mitigated by treatment with the antioxidant N-acetyl cysteine. KDM1A inhibition decreased basal mitochondrial respiration and impaired induction of HIF-1A after ROS exposure. Overexpression of HIF-1A salvaged cells from KDM1A inhibition enhanced sensitivity to ROS. Thus we found that increased sensitivity of ROS after KDM1A inhibition was mediated by HIF-1A and depletion of endogenous glutathione. We also show that KDM1A-specific inhibitor bizine synergized with antioxidant-depleting therapies, buthionine sulfoximine, and auranofin in rhabdomyosarcoma cell lines (Rh28 and Rh30). In this study, we describe a novel role for KDM1A in regulating HIF- 1A functions under oxidative stress and found that dual targeting of KDM1A and antioxidant systems may serve as an effective combination anticancer strategy.

cancer biology↗

bioRxiv2017

A minimal RNA ligand for potent RIG-I activation in living mice

We have developed highly potent synthetic activators of the vertebrate immune system that specifically target the RIG-I receptor. When introduced into mice, a family of short, triphosphorylated Stem Loop RNAs (SLRs) induces a potent interferon response and the activation of specific genes essential for antiviral defense. Using RNAseq, we provide the first in-vivo genome-wide view of the expression networks that are initiated upon RIG-I activation. We observe that SLRs specifically induce type I interferons, subsets of interferon-stimulated genes (ISGs), and cellular remodeling factors. By contrast, poly(I:C), which binds and activates multiple RNA sensors, induces type III interferons and several unique ISGs. The short length (10-14 base pairs) and robust function of SLRs in mice demonstrate that RIG-I forms active signaling complexes without oligomerizing on RNA. These findings demonstrate that SLRs are potent therapeutic and investigative tools for targeted modulation of the innate immune system.

immunology↗