Biology subjects

Crawford, M. C.

Publications and source records attributed to Crawford, M. C..

4 recordsLinked to original sources

bioRxiv2024

Transfer RNA acetylation regulates in vivo mammalian stress signaling

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2 phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

developmental biology↗

bioRxiv2024

Paralogue-selective degradation of the lysine acetyltransferase EP300

The transcriptional coactivators EP300 and CREBBP are critical regulators of gene expression that share high sequence identity but exhibit non-redundant functions in basal and pathological contexts. Here, we report the development of a bifunctional small molecule, MC-1, capable of selectively degrading EP300 over CREBBP. Using a potent aminopyridine-based inhibitor of the EP300/CREBBP catalytic domain in combination with a VHL ligand, we demonstrate that MC-1 preferentially degrades EP300 in a proteasome-dependent manner. Mechanistic studies reveal that selective degradation cannot be predicted solely by target engagement or ternary complex formation, suggesting additional factors govern paralogue-specific degradation. MC-1 inhibits cell proliferation in a subset of cancer cell lines and provides a new tool to investigate the non-catalytic functions of EP300 and CREBBP. Our findings expand the repertoire of EP300/CREBBP-targeting chemical probes and offer insights into the determinants of selective degradation of highly homologous proteins.

biochemistry↗

bioRxiv2023

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

biochemistry↗

bioRxiv2020

The tubovesicular network in Plasmodium vivax liver-stage hypnozoites and schizonts associates with host aquaporin 3

The apicomplexan Plasmodium parasites replicate in the liver before causing malaria. P. vivax can also persist in the liver as dormant hypnozoites and cause relapses upon activation. The host water and solute channel aquaporin-3 (AQP3) has been shown to localize to the parasitophorous vacuole membrane (PVM) of P. vivax hypnozoites and liver schizonts, along with other Plasmodium species and stages. In this study, we use high-resolution microscopy to characterize temporal changes of the tubovesicular network (TVN), a PVM-derived network within the host cytosol, during P. vivax liver-stage infection. We demonstrate an unexpected role for the TVN in hypnozoites and reveal AQP3 associates with TVN-derived vesicles and extended membrane features. We further show AQP3 recruitment to Toxoplasma gondii. Our results highlight dynamic host-parasite interactions that occur in both dormant and replicating liver-stage P. vivax forms and implicate AQP3 function during this time. Together, these findings enhance our understanding of AQP3 in apicomplexan infection.

microbiology↗