Biology subjects

Brandman, O.

Publications and source records attributed to Brandman, O..

14 recordsLinked to original sources

bioRxiv2026

Transcriptional Response to Tumor Mutational Burden Is Consistent Across Cancer Types

Tumor mutational burden (TMB) shapes tumor transcriptional state, but studies typically describe this response as an average effect pooled across cancer types. Whether that average reflects a consistent response present within individual cancer types, or is an artifact of merging heterogeneous, tissue-specific responses, remains unresolved. Here we analyze ~9,100 tumors across 32 TCGA cancer types to test whether the transcriptional response to TMB is genuinely consistent across tissues. We construct a TMB axis score from TMB-associated genes upregulated with increasing TMB, yielding a sample-level measure of response strength, and subsequently decompose it at the component and pathway/complex levels. The pooled transcriptional response to TMB stays largely consistent within each cancer type, and no single cancer is driving the pooled signal. This consistency was also observed at the component and pathway/complex levels. These findings support TMB as a promising tissue-agnostic signature, with implications for tissue-agnostic therapeutic targeting.

cancer biology↗

bioRxiv2026

Nup42 safeguards heat-induced mRNAs from nuclear condensation tosupport chaperone synthesis

Cells exposed to acute stress selectively express stress-adaptive genes while repressing growth-related genes. Upon heat shock, most pre-existing mRNAs localize to translationally repressed biomolecular condensates. How heat-induced mRNAs evade condensation and remain translationally competent remains unclear. Here, we show that ribosomal protein-coding transcripts preferentially accumulate in condensates during heat shock, whereas heat-induced chaperone mRNAs are selectively excluded and preferentially translated. Using a whole-genome CRISPRi screening platform, Fractionation of Reporter-Seq (FRep-Seq), we identify the nucleoporin Nup42 as the strongest suppressor of heat-induced mRNA condensation. Loss of Nup42 triggers temperature- and transcription-dependent nuclear condensation of chaperone mRNAs, which are exported but remain translationally incompetent, leading to impaired chaperone production and thermosensitivity. Co-transcriptional mRNP packaging is a critical determinant of condensation in the absence of Nup42. Together, our findings reveal a nuclear, translation-independent layer of mRNP solubility control that enables heat shock gene expression.

molecular biology↗

bioRxiv2024

Mechanochemical forces regulate the composition and function of CAT tails

The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acids (CAT tails) in an mRNA-free, non-canonical elongation process. CAT tail composition includes Ala, Thr, and potentially other residues. The relationship between CAT tail composition and function has remained unknown. Using biochemical approaches in yeast, we discovered that mechanochemical forces on the NC regulate CAT tailing. We propose CAT tailing initially operates in an "extrusion mode" that increases NC lysine accessibility for on-ribosome ubiquitination. Thr in CAT tails enhances NC extrusion by preventing formation of polyalanine, which can form -helices that lower extrusion efficiency and disrupt termination of CAT tailing. After NC ubiquitylation, pulling forces on the NC switch CAT tailing to an Ala-only "release mode" which facilitates nascent chain release from large ribosomal subunits and NC degradation. Failure to switch from extrusion to release mode leads to accumulation of NCs on large ribosomal subunits and proteotoxic aggregation of Thr-rich CAT tails. One sentence summaryMechanochemical forces regulate the composition of CAT tails to extrude or release stalled nascent chains and recycle ribosomes.

biochemistry↗

bioRxiv2022

Diffusive lensing as a mechanism of intracellular transport and compartmentalization

While inhomogeneous diffusivity has been identified as a ubiquitous feature of the cellular interior, its implications for particle mobility and concentration at different length scales remain largely unexplored. In this work, we use agent-based simulations of diffusion to investigate how heterogeneous diffusivity affects movement and concentration of diffusing particles. We propose that a nonequilibrium mode of membraneless compartmentalization arising from the convergence of diffusive trajectories into low-diffusive sinks, which we call "diffusive lensing," is relevant for living systems. Our work highlights the phenomenon of diffusive lensing as a potentially key driver of mesoscale dynamics in the cytoplasm, with possible far-reaching implications for biochemical processes.

biophysics↗

bioRxiv2021

ReporterSeq reveals genome-wide determinants of proteasome expression

The ubiquitin-proteasome system (UPS) is critical for cellular and organismal health. To uncover mechanisms regulating the UPS in normal and stress conditions, we systematically probed the genome of the eukaryotic model system Saccharomyces cerevisiae for modulators of the UPS master regulator Rpn4 under basal and stress conditions using the reverse genetic method ReporterSeq. The top UPS regulators were the thioredoxin reductase Trr1 and proteins of the large ribosomal subunit, both of which had no previously known role in UPS regulation. Unlike all known mechanisms for Rpn4 regulation which regulate Rpn4 levels, we found that Trr1 modulates the molecular activity of Rpn4 and does so in response to oxidative stress. Our work illuminates the genetic landscape through which cells regulate the UPS, and provides insight into how cells combat proteotoxicity.

genomics↗

bioRxiv2021

Oxaliplatin kills cells via liquid-liquid demixing of nucleoli

Platinum (Pt) compounds such as oxaliplatin are amongst the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here, we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically-relevant concentrations by interfering with the interaction networks that organize nucleoli. This biophysical defect leads to cell cycle arrest, impaired rRNA processing and shutdown of PolI-mediated transcription, ultimately resulting in cell death. We propose that the mechanism of action of oxaliplatin provides a blueprint for the therapeutic targeting of the increasing number of cellular processes being linked to biomolecular condensates.

cell biology↗

bioRxiv2020

Genome-wide, time-sensitive interrogation of the heat shock response under diverse stressors via ReporterSeq

Interrogating cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single cell isolation. Here, we used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. We measured genome-wide HSR regulation in budding yeast across thirteen stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.

genetics↗

bioRxiv2019

Protein products of non-stop mRNA disrupt nucleolar homeostasis

Mutations that cause ribosome stalling or impair the cells protective response to stalling have been demonstrated to cause neurodegeneration, yet the mechanisms underlying these pathologies remain poorly understood. Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the Ribosome-associated Quality Control (RQC) E3 ligase LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tail of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress. Changes in nucleolar morphology are consistent with a simple physical model in which LTN1 impairment enhances the inter-molecular interactions of nucleolar components. Our work elucidates how failure to degrade the protein products of stalled translation may affect distal cellular processes and will inform studies on the pathology of neurodegenerative disease.

cell biology↗

bioRxiv2019

The ubiquitin-proteasome system productively adapts to proteasomal and protein folding stressors via independent mechanisms

Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.

cell biology↗

bioRxiv2019

Viscoadaptation controls intracellular reaction rates in response to heat and energy availability

Summary/AbstractCells must precisely orchestrate thousands of reactions in both time and space. Yet reaction kinetics are highly dependent on uncontrollable environmental conditions such as temperature. Here, we report a novel mechanism by which budding yeast influence reaction rates through adjustment of intracellular viscosity. This \"viscoadaptation\" is achieved by production of two carbohydrates, trehalose and glycogen, which combine to create a more viscous cellular environment in which biomolecules retain solubility. We demonstrate that viscoadaptation functions as both an acute response to temperature increase as well as a homeostatic mechanism, allowing cells grown at temperatures spanning from 22{degrees}C to 40{degrees}C to maintain equivalent rates of intracellular diffusion and diffusion-controlled chemical reactions. Multiple conditions that lower ATP trigger viscoadaptation, suggesting that viscoadaptation may be a general cellular response to low energy. Viscoadaptation reveals viscosity to be a tunable property of cells through which they can regulate diffusion-controlled processes dynamically in response to a changing environment.

cell biology↗

bioRxiv2019

Rqc2 levels alter CAT tail composition, function, and toxicity in S. cerevisiae

The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.

cell biology↗

bioRxiv2019

Chaperone-Mediated Reflux of Secretory Proteins to the Cytosol During Endoplasmic Reticulum Stress

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such \"ER stress\", we employed an ER-targeted, redox-responsive, green fluorescent protein--eroGFP--that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress agents cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to \"reflux\" back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in S. cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and co-chaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER-protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.\n\nSIGNIFICANCEApproximately one third of eukaryotic proteins are synthesized on ribosomes attached to the endoplasmic reticulum (ER) membrane. Many of these polypeptides co- or post-translationally translocate into the ER, wherein they fold and mature. An ER quality-control system proofreads these proteins by facilitating their folding and modification, while eliminating misfolded proteins through ER-associated degradation (ERAD). Yet, the fate of many secretory proteins during ER stress is not completely understood. Here, we uncovered an ER-stress induced \"protein reflux\" system that delivers intact, folded ER luminal proteins back to the cytosol without degrading them. We found that ER protein reflux works in parallel to ERAD and requires distinct ER-resident and cytosolic chaperones and co-chaperones.

cell biology↗

bioRxiv2019

MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here we describe a phenomenon termed MISTERMINATE (mitochondrial stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs including complex-I 30kD subunit (C-I30) mRNA, occurring on mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C-terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally-extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinsons disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

cell biology↗

bioRxiv2018

CAT tails drive on- and off-ribosome degradation of stalled polypeptides

Stalled translation produces incomplete, ribosome-associated polypeptides that Ribosome-associated Quality Control (RQC) targets for degradation via the ubiquitin ligase Ltn1. During this process, the Rqc2 protein and large ribosomal subunit elongate stalled polypeptides with carboxy-terminal alanine and threonine residues (CAT tails). Failure to degrade CAT-tailed proteins disrupts global protein homeostasis, as CAT-tailed proteins aggregate and sequester chaperones. Why cells employ such a potentially toxic process during RQC is unclear. Here, we developed quantitative techniques to assess how CAT tails affect stalled polypeptide degradation in Saccharomyces cerevisiae. We found that CAT tails improve Ltn1s efficiency in targeting structured polypeptides, which are otherwise poor Ltn1 substrates. If Ltn1 fails, CAT tails undergo a backup route of ubiquitylation off the ribosome, mediated by the ubiquitin ligase Hul5. Thus, CAT tails functionalize the carboxy-termini of stalled polypeptides to drive their degradation on and off the ribosome.

cell biology↗