Biology subjects

Wassarman, D. R.

Publications and source records attributed to Wassarman, D. R..

7 recordsLinked to original sources

bioRxiv2026

A Dimeric Rocaglate Promotes Multivalent eIF4A-RNA Assembly

Ligand dimerization represents a powerful strategy to enhance avidity, potency, and selectivity. Leveraging the natural-product molecular glue Rocaglamide (RocA), we identified BisRoc, a dimeric rocaglate ligand that potently and durably suppresses translation and exhibits greater specificity across a cancer cell line panel than the monomeric RocA. CRISPRi screening revealed that BisRoc activity is influenced by cellular context, including IFITM-mediated uptake, ABC-type efflux transporters, and the translation initiation factor eIF4A2. Mechanistic studies showed that the paralogs eIF4A1 and eIF4A2 are differentially sensitive to BisRoc-induced dimerization. Owing to the presence of multiple binding sites on RNAs, BisRoc-bridged eIF4A-RNA motifs assemble into higher-order complexes that promote stress-granule formation more efficiently than monomeric RocA. Given the widespread multivalency of RNA-RBP interactions, this ligand dimerization strategy may be extended to modulate the higher-order assembly of other RNA-binding proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/710667v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f0ce69org.highwire.dtl.DTLVardef@7f7fadorg.highwire.dtl.DTLVardef@976e81org.highwire.dtl.DTLVardef@1d7eaf2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Structural basis of QueC-family protein function in qatABCD anti-phage defense

QueC proteins are nucleoside biosynthesis enzymes required for production of the 7-deazaguanine derivative queuosine. Recently, QueC-family proteins were also shown to catalyze a deazaguanylation protein-nucleobase conjugation reaction in type IV CBASS bacterial anti-phage defense. Here we determine the structural basis of QueC-family protein function in a distinct bacterial immunity system named qatABCD. We demonstrate that the QueC-family protein QatC forms a specific complex with the immunity protein QatB and that this complex is minimally required for qatABCD defense. Crystal structures of the QatBC complex enable direct comparison of qatABCD and type IV CBASS defense and support a shared role for QueC-family proteins in targeting protein substrates for N-terminal modification. We show that the QatB unstructured N-terminus and N-terminal glycine motif are essential for qatABCD defense in vivo, suggesting a modification occurs analogous to CBASS deazaguanylation. These findings highlight broad roles of QueC proteins beyond nucleoside biosynthesis and suggest that adaptation of QueC-like proteins for specialized biochemical functions is a common strategy in bacterial anti-phage immunity.

biochemistry↗

bioRxiv2025

Activating and inhibiting nucleotide signals coordinate bacterial anti-phage defense

The cellular nucleotide pool is a major focal point of the host immune response to viral infection. Immune effector proteins that disrupt the nucleotide pool allow animal and bacterial cells to broadly restrict diverse viruses, but reduced nucleotide availability induces cellular toxicity and can limit host fitness(Ahmad et al., 1998; Goldstone et al., 2011; Hsueh et al., 2022; Itsko & Schaaper, 2014; Tal et al., 2022). Here we discover a bacterial anti-phage defense system named Clover that overcomes this tradeoff by encoding a deoxynucleoside triphosphohydrolase enzyme (CloA) that dynamically responds to both an activating phage cue and an inhibitory nucleotide immune signal produced by a partnering regulatory enzyme (CloB). Analysis of Clover phage restriction in cells and reconstitution of enzymatic function in vitro demonstrate that CloA is a dGTPase that responds to viral enzymes that increase cellular levels of dTTP. To restrain CloA activation in the absence of infection, we show that CloB synthesizes a dTTP-related inhibitory nucleotide signal p3diT (5'-triphosphothymidyl-3'5'-thymidine) that binds to CloA and suppresses activation. Cryo-EM structures of CloA in activated and suppressed states reveal how dTTP and p3diT control distinct allosteric sites and regulate effector function. Our results define how nucleotide signals coordinate both activation and inhibition of antiviral immunity and explain how cells balance defense and immune-mediated toxicity.

microbiology↗

bioRxiv2025

Deazaguanylation is a nucleobase-protein conjugation required for type IV CBASS immunity

7-deazapurines are nucleobase analogs essential for nucleic acid modifications in nearly all cellular life. Here, we discover a role for 7-deazapurines in protein modification within type IV CBASS anti-phage defense and define functions for CBASS ancillary proteins Cap9 and Cap10 in nucleobase-protein conjugation. A structure of Cap10 reveals a tRNA transglycosylase-family enzyme remodeled to bind the modified N-terminus of a partner cGAS/DncV-like nucleotidyltransferase linked to a 7-amido-7-deazaguanine (NDG) nucleobase. The structure of Cap9 explains how this QueC-like enzyme co-opts a 7-deazapurine biosynthetic reaction mechanism for NDG conjugation. We demonstrate that Cap9, Cap10, and NDG conjugation are essential for host defense against phage infection. Our results define a previously unknown 7-deazapurine protein modification and explain how nucleobase biosynthetic machinery has been repurposed for antiviral immunity.

microbiology↗

bioRxiv2022

Cellpanelr: identify predictive biomarkers from cell line panel response data

SummaryCellpanelr is an open-source R package and web application for analyzing user-generated cell panel screens using DepMap data sets. Cellpanelr can be used to identify mutation and expression biomarkers of cell line response, increasing the value and accessibility of cell panel experiments such as relative sensitivities to cancer drugs. Availability and implementationHosted web application is available from shinyapps.io (https://dwassarman.shinyapps.io/cellpanelr). Source code and installation instructions are available from GitHub (https://github.com/dwassarman/cellpanelr). Contactdwassar@gmail.com

bioinformatics↗

bioRxiv2022

Tissue-Restricted Inhibition of mTOR Using Chemical Genetics

mTOR is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product Rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a Rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this Rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in S. cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a Rapamycin-induced developmental delay in Drosophila. Significance StatementmTOR plays a number of critical organismal roles, including in cell growth, development, immunity and aging, but dissecting the tissue-specific influences of mTOR has proven challenging. This work describes a simple system for identifying the specific tissues and cells responsible for the diverse functions of mTOR, and we show that our system can be used in organisms ranging from yeast to humans.

biochemistry↗

bioRxiv2020

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of -synuclein, a dynamic protein linked to Parkinsons disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of -synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to -synuclein biology and pathobiology. We found that the conformation of -synuclein previously shown to drive yeast toxicity--an extended, membrane-bound helix--is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress -synuclein toxicity. Moreover, we find that sequence determinants of -synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that -synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

biochemistry↗