QuPID-MS, which measures the propensity of proteins to phase separate in native cell extracts proteome-wide, finds that weak interactions drive condensation of half of the proteome in a crowding- and temperature-dependent manner and presents evidence that this proteome demixing occurs in cells.
It is demonstrated here that the interaction between H-NS and StpA fine-tunes the physico-chemical properties of nucleoid-associated compartments, thereby preserving gene repression and optimizing bacterial growth under stress.
Jian Guan, Bikash R. Sahoo, Tyler S. Brant et al.· Molecules and Cells· 0 citations
Emerging high-throughput strategies to study protein condensation and aggregation at scale are reviewed, emphasizing what they truly measure, their limitations, and how the cross-talk among these complementary approaches can provide a more accurate and mechanistic mapping of sequence-to-assembly relationships.
Mariano Martín, Alice Lissmatz, Benedetta Bolognesi· Current Opinion in Structura...· 0 citations
Intracellular biomolecular condensation forms multicomponent signaling hubs that regulate development, stress responses, and environmental adaptation. While the molecular grammar encoded within scaffold proteins defines the basal associative features driving condensation, heterotypic condensates are intrinsically dynamic, multicomponent, and far-from-equilibrium systems. Consequently, how condensates organize component composition, stoichiometry, and functional specificity in space and time under physiological conditions remains poorly understood. Addressing this challenge requires integrative frameworks that combine predictive biophysical features with experimental information on protein abundance, interaction networks, subcellular localization, and evolutionary conservation. Here, we first analyzed phase separation (PS) proteins across the Tree of Life in 1,106 species, revealing a stark contrast in computationally predicted phase-separation propensity between eukaryotes and prokaryotes, with genome size as a key determinant. Through a broad analysis of amino acid homorepeat-containing proteins (HRPs) across all species, we uncovered how phase separation evolves via a balance between functional condensation and avoidance of harmful, aggregation-prone sequences. We further identified potential signaling hubs and components across kingdoms by integrating PS-positive proteins with experimentally derived abundance and interactome data from four model eukaryotic species. Using Arabidopsis as a model, we dissect the relationships among PS propensity, condensation hub prediction, HRPs, subcellular localization, and structural conservation. Consequently, we developed PhaseHub (https://phasehub.sbs.ntu.edu.sg/), a user-friendly interface for exploring scaffold-client dynamics, PS components, sequence signatures within each PS protein, and hubs. Our work provides an evolutionary framework for understanding multicomponent PS hubs by integrating molecular grammar with physiological context, thereby facilitating hypothesis generation and rational design.
ABSTRACT Environmental fluctuations remodel RNA modification landscapes, yet the routes that connect cue detection to writer–eraser–reader control remain dispersed across disciplines. Here, we consolidate upstream mechanisms capable of driving epitranscriptomic change and organize them by response speed. At the fastest proximal level, catalytic output can be modulated through shifts in substrate and cofactor availability, redox and ionic state, temperature, and direct chemical or metal interference with enzyme active sites, although transcriptome‐wide RNA readouts may appear later. Over minutes to hours, cue‐responsive signaling can reach the machinery through post‐translational modification, partner switching, subcellular trafficking, and stress‐induced condensates that may gate access to modified transcripts. Across hours to days, regulator abundance and specificity are reshaped by transcriptional programs, translational control, and protein quality‐control pathways, enabling adaptation and, in some contexts, persistence. We propose a kinetics‐to‐sensors approach for interpreting time‐resolved epitranscriptomic datasets and prioritizing perturbations that discriminate among candidate upstream inputs. We also outline conceptual gaps and experimental practices needed to establish causal cue‐to‐mark chains.
Shayan Jalali, E. P. Ahi· Advances in Biology· 0 citations
The spatial distribution of small molecules within cells shapes their biological activity, yet these distributions are generally assumed to be governed passively by reaction-driven electrochemical gradients. Here we show that aminoglycoside antibiotics actively control their own subcellular organization by undergoing phase separation with RNAs. Combining in vitro reconstitution, bacterial assays, and molecular dynamics simulations, we discovered that aminoglycosides coacervate with RNA through multivalent electrostatic interactions, displacing and releasing RNA-bound Mg2+. This condensate-dependent Mg2+ release remodels the cytosolic labile Mg2+ pool and activates magnesium signaling. This effect dampens the magnesium-starvation regulation, sustains ribosome activity, and shifts the cellular electrochemical state, promoting bacterial fitness. Because condensation occurs only above a defined concentration threshold, it generates a non-monotonic dose-response in which higher antibiotic concentrations paradoxically enhance bacterial survival. This antibiotic condensate-dependent Mg2+ signaling confers tolerance to multiple ribosome-targeting antibiotics simultaneously even in cells lacking resistance gene, while condensate dissolution restores antibiotic efficacy. Our findings establish antibiotic-driven phase separation as a previously unrecognized mechanism to encode cellular signaling and identify antibiotic condensates as a distinct functional unit underlying drug tolerance.
Yuefeng Ma, Wen Yu, E. Moon et al.· bioRxiv· 0 citations
The reliable operation of biomolecular circuits depends on the availability of shared cellular resources such as ribosomes, whose levels can vary substantially across growth conditions and cellular contexts. Although resource competition among co-expressed genes is well recognized, the relationship between resource variation and the robustness of circuit dynamics has not been characterized quantitatively. This paper integrates a resource-aware gene expression model, contraction theory–based analytical bounds, and experimental validation to study the effect of translational resource variation on constitutive gene expression and its mitigation through feedback. We show that the constitutive circuit exhibits sensitivity to resource perturbations, and that redesigning it with negative autoregulatory feedback enhances the contraction rate and reduces the steady-state deviation bound, though at lower expression levels. Experiments in E. coli using both plasmid copy number variation and a ribosome sequestration module are consistent with these predictions, confirming that the feedback circuit maintains relatively stable expression under conditions where the constitutive circuit shows large changes. These findings offer a systematic approach for analyzing and improving the robustness of biomolecular circuits operating under variable resource conditions.
