Budding yeast Saccharomyces cerevisiae is an ideal model organism to study membrane trafficking pathways. The ESCRT (endosomal sorting complexes required for transport) pathway was first identified ...in this organism. Upon recognition of endocytosed ubiquitinated membrane proteins at endosomes, ESCRTs assemble at these organelles to catalyze the biogenesis of multivesicular bodies (MVBs). Formation of MVBs leads to the trafficking of these membrane proteins to vacuoles for degradation. Here, we describe genetic and biochemical approaches to study ESCRT function. We outline in vivo endocytosis assays using two model cargoes in Saccharomyces cerevisiae and also describe an in vitro approach to analyze ESCRT-III polymerization on lipid monolayers.
An in vitro effect of (+)MK-801 (dizocilpine), an inhibitor of the glutamate/NMDA and nicotinic acetylcholine receptors, on the Aβ1–42 and Aβ1–40 peptides is described and compared to that of ...memantine. Memantine has been approved by the U.S. Food and Drug Administration for the treatment of mild-moderate Alzheimer’s disease. Both compounds accelerated the formation of a β-sheet structure by Aβ1–42, (+)MK-801 more rapidly than memantine, as observed in a thioflavin T fluorescence assay. The acceleration was followed by a decrease in the fluorescence signal that was not observed when the ligand was absent. Nuclear magnetic resonance spectra of the soluble peptides in the presence and absence of (+)MK-801 demonstrated that the monomeric form did not bind (+)MK-801 and that in the presence of (+)MK-801 the concentration of the monomeric form progressively decreased. Small angle X-ray scattering confirmed that the presence of (+)MK-801 resulted in a more rapid and characteristic transition to an insoluble form. These results suggest that (+)MK-801 and memantine accelerate the transition of Aβ1–42 and Aβ1–40 to ThT-negative insoluble forms.
HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report ...here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.
Abstract Although precision medicine strategies for the treatment of human cancers are clinically successful, they have also revealed that only a small fraction of cancers carry actionable ...alterations in oncogenic drivers. To expand the druggable genome while maintaining an emphasis on genetically targeted therapies, recent efforts have focused on exploiting synthetic lethal relationships. Paralogous protein pairs are particularly compelling targets in this context due to the clear mechanistic basis for the synthetic lethality. As such, functional genomics screens as well as work from Ogiwara et al. have shown compelling rationale for targeting the histone acetylase transferase p300 in the context of CBP-deficient cancer types. We discovered potent heterobifunctional degraders with demonstrated selectivity for p300 over CBP. Our compound exhibits degradation of p300 within 2 hours (DC50 < 10nM, Dmax > 90%) with minimal impact on CBP through 48 h (DC50 >1uM, Dmax = 30%). Utilizing CRISPR-generated isogenic cell lines depleted for either CBP or p300, we observed significant growth inhibition in CBP knockout (KO) cells (gIC50 = 17nM) with minimal impact on growth of p300 KO (gIC50 = >10uM) or parental wild-type cells (gIC50 > 10uM). This translated to downstream pharmacology, where we observed a rapid and potent inhibition of global H3K27 acetylation in the CBP deficient context but a significantly attenuated response in p300 deleted or parental cells. We subsequently employed computational methods to identify and characterize CBP alterations, defining a putative loss-of-function (LoF) phenotype and confirming this sensitivity pharmacologically in cancer cell types harboring these endogenous mutations. Results mirrored what we observed with CBP depletion in these endogenous CBP LoF cell lines, with potent growth inhibition seen in multiple cell lines including LK-2 (gIC50 = 8nM), H1703 (gIC50 = 9nM), and TE-8 cells (gIC50 = 23nM). To confirm the in vitro results in an in vivo setting, we evaluated the response in mice harboring H1703 xenografts. Once daily oral administration of our compound led to almost complete degradation of p300 within H1703 xenograft tumors, resulting in a pronounced inhibition of tumor growth. Lastly, we profiled p300 degradation in human bone marrow derived myeloid progenitor colony-forming assays where we observed a significant reduction in toxicity (IC50 = 3.9uM) compared to a dual CBP/p300 inhibitor (IC50 = 121nM) or degrader (IC50 = 16nM), supporting the hypothesis that a p300-specific mechanism will offer an improved therapeutic index while maintaining anti-tumor efficacy in a biomarker population Taken together, our results suggest that a p300-selective degrader has the potential to serve as an effective therapeutic modality in CBP-mutated cancers. Citation Format: Mike R. Russell, Cassandra L. Lowenstein, Xuqing Zhang, Jeremy Roach, Jianing Song, Rakesh Nagilla, Nathan Kendsersky, Shreyas Joshi, Peter Orth, Matt Tudor, Qiaolin Deng, Clemente Aguilar-Bonavides, Elham Behshad, Sudeep Banjade, Zhihua Sui, Corey Strickland, Larry Jolivette, Helai P. Mohammad. Discovery and characterization of a p300-selective degrader with potent anti-tumor activity in CBP mutant cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6043.
Abstract
Signaling through the T cell receptor (TCR) mediates T cell activation, differentiation, and proliferation. Previous microscopy studies have shown the spatial reorganization of signaling ...molecules into microclusters on the plasma membrane after TCR activation. However, how microclusters form and what roles these structures play in signal transduction remain unclear. Importantly, direct comparison of the biochemical activities of signaling molecules in the clustered versus unclustered state has been lacking. Here, we address these questions by reconstituting a TCR signaling pathway in vitro with 12 purified proteins, beginning with TCR phosphorylation and culminating in actin polymerization. We show that upon initiation of phosphorylation, multivalent protein-protein interactions drive the phase separation of signaling molecules into microclusters that display liquid-like properties. These clusters enrich kinases but exclude phosphatases, thus sustaining protein phosphorylation. We also directly demonstrate that the reorganization of actin regulatory proteins into these clusters substantially enhances actin filament assembly at the membrane. These results establish that microclusters create unique physical and biochemical environments that promote reactions in TCR signaling. The principles revealed here likely apply to other systems that exploit phase separation to regulate biochemical reactions on membranes, in the cytoplasm, or the nucleus.
Citation Format: Xiaolei Su, Jon Ditlev, Enfu Hui, Sudeep Banjade, Julia Okrut, Jack Taunton, Mike Rosen, Ron Vale. Phase separation of signaling molecules promotes T cell receptor signal transduction. abstract. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A087.
Cells are organized on length scales ranging from Angstroms to microns. However, the mechanisms by which Angstrom-scale molecular properties are translated to micron-scale macroscopic properties are ...not well understood. Here we show that interactions between diverse, synthetic multivalent macromolecules (including multi-domain proteins and RNA) produce sharp, liquid-liquid demixing phase separations, generating micron-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to valency of the interacting species. In the case of the actin regulatory protein, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) interacting with its established biological partners Nck and phosphorylated nephrin
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, the phase transition corresponds to a sharp increase in activity toward the actin nucleation factor, Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions are likely used to spatially organize and biochemically regulate information throughout biology.
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