Biology subjects

Ritz, D.

Publications and source records attributed to Ritz, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Chemical Proteomics of Residual Acute Myeloid Leukemia Cells Reveals Therapeutic Vulnerabilities

Venetoclax combined with azacitidine has improved treatment outcomes in acute myeloid leukemia (AML), yet relapse and treatment persistence remain major clinical challenges. To investigate proteomic mechanisms associated with venetoclax-azacitidine response and adaptation, we applied a multi-layer combinatorial proteome integral solubility/stability alteration analysis (CoPISA) strategy in SKM-1 AML cells. Cells were treated with venetoclax, azacitidine, their combination, or vehicle control and profiled across four orthogonal layers: short-term lysate CoPISA, short-term intact-cell CoPISA, long-term intact-cell CoPISA after 5 days of treatment, and long-term expression proteomics of surviving cells. The venetoclax-azacitidine combination induced treatment-specific protein solubility and abundance changes that were not fully reproduced by either single agent. Long-term surviving cells displayed extensive proteomic remodeling, consistent with the emergence of an adaptive drug-tolerant state, although contributions from pre-existing resilient cell populations cannot be excluded. Integration of short- and long-term solubility changes with abundance remodeling revealed distinct adaptive regimes, including retained biochemical targets, dosage-compensated targets, sensitive-state-specific targets, and remodeled adaptive targets. This framework prioritized candidate resistance-associated proteins, including NRP2, RPL18, PLP2, RPS28, and NOTCH1. NRP2 emerged as a top candidate across all proteomics layers, consistently showing increased abundance. Overall, this study shows that combining CoPISA with expression proteomics can resolve temporally distinct proteomic states of venetoclax-azacitidine response and identify candidate adaptive vulnerabilities in AML.

cancer biology↗

bioRxiv2026

Deep nanoparticle protein corona plasma proteomics resolves a stage-specific peripheral signature of Alzheimer's disease

Structured AbstractO_ST_ABSINTRODUCTIONC_ST_ABSAlzheimers disease (AD) progresses over decades, yet plasma biomarkers that resolve disease stage rather than simply detect disease remain scarce. This distinction is clinically consequential because effective AD intervention depends on identifying patients before disease biology has progressed beyond a therapeutically responsive stage. METHODSWe used small-molecule-modulated protein corona proteomics to profile plasma from 90 individuals in the Australian Imaging, Biomarker and Lifestyle cohort, stratified by Centiloid (CL) A{beta}-amyloid burden (30 amyloid- negative, CL < 15; 30 moderate-to-high, CL 26 to 100; 30 very high, CL > 100). We quantified 3,176 proteins and applied differential abundance and actual causality analyses to identify stage-specific and candidate causal proteins. RESULTSDifferential protein abundance was exclusively captured during the moderate-to-high AD transition, revealing a discrete proteomic "switch." The switch was marked by accumulation of the autophagy receptor CALCOCO1, together with coordinated depletion of the S100A8/S100A9 calprotectin complex and core erythroid-cytoskeletal network structural markers (e.g., SPTA1, SPTB, ANK1). Adhesion G protein-coupled receptor G6 (ADGRG6) showed a significant moderate positive monotonic association with absolute CL burden, providing a proportional molecular anchor for cumulative disease burden. Actual causality analysis identified COL6A2, FOXRED2, P3H1, PRR4, and GOLGA5 as candidate upstream drivers linking matrix remodeling, Golgi trafficking, and collagen processing to AD progression. DISCUSSIONThese findings suggest a candidate blood-accessible framework for staging AD by active disease biology, which, if replicated in independent cohorts, may have implications for therapeutic selection and mechanism-guided clinical trials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/740710v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ead718org.highwire.dtl.DTLVardef@cfa523org.highwire.dtl.DTLVardef@62a367org.highwire.dtl.DTLVardef@1d5c9a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Antizyme regulates polyamine uptake via ATP13A3

Intracellular polyamine levels are tightly regulated and frequently elevated in cancer. While the regulation of polyamine synthesis is well characterized, the regulation of polyamine uptake is poorly understood. Here we identify ATP13A3 as a plasma membrane polyamine transporter. An increase in intracellular polyamine levels, due to polyamine supplementation or induction of polyamine synthesis, causes rapid internalization of the transporter and inhibition of polyamine uptake. Mechanistically, increased polyamine concentrations lead to expression of antizyme (AZ) which binds ATP13A3 and triggers its internalization. Mutations in the AZ binding site of ATP13A3 prevent AZ binding and lead to polyamine toxicity through uncontrolled polyamine influx. These findings establish ATP13A3 as a key polyamine transporter and provide a framework for targeting polyamine metabolism in cancer.

cell biology↗

bioRxiv2026

TORC1 spatial segregation and hysteresis are regulated by the small GTPase Arf1

TORC1 is a central regulator of growth, integrating inputs from kinases and signaling proteins. How TORC1 activity is regulated, is still not completely understood. The small GTPase Arf1 has been implicated in TORC1 regulation, but the underlying mechanism remains elusive. Here, we show that Arf1 controls TORC1 in an allele-specific manner under nutrient and heat stress in yeast. Unexpectedly, both the hyperactive mutant arf1-11 and the loss-of-function mutant arf1-18 reduce TORC1 activity, while other loss-of-function alleles did not. We reveal two distinct functions of Arf1: first, it ensures Golgi-to-vacuole/lysosome transport for the functional maintenance of these organelles; second, it regulates TORC1 activity at the ER-Golgi-vacuole interface. Hyperactive Arf1-11 drives sequestration of Kog1 and Tor1 into cytoplasmic foci, to which Arf1-11 can be equally recruited. Importantly, Arf1 also promotes TORC1 re-activation during stress recovery - so-called hysteresis. Our data reveal an unexpected dual function for Arf1 as a negative and a positive regulator of TORC1 activity in a context-dependent manner. TeaserThe small GPTase Arf1 acts as a dual switch for the key cellular growth regulator TORC1 by increasing or decreasing TORC1 activity depending on nutritional status and stress.

cell biology↗

bioRxiv2026

Molecular architecture of the ciliary base in mammalian multiciliated cells

Multiciliated epithelial cells (MCCs) generate tens to hundreds of motile cilia to drive fluid flow in diverse physiological contexts. While the axonemal structure of motile cilia has been described extensively in recent years, the molecular architecture of the transition zone, basal body, and surrounding ciliary environment of MCCs remain more elusive. Here, we use cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to obtain in situ 3D views of the ciliary base within intact MCCs from mammalian trachea, complemented by in situ cross-linking mass spectrometry (XL/MS) and ultrastructure expansion microscopy (U-ExM) for molecular identification. Our data reveal spatially-defined modifications of microtubule architecture from the proximal centriole to the early axoneme, including transition zone-specific features such as an A-B linker bridging microtubule doublets and a helical assembly of microtubule inner proteins (MIPs). We show that the ciliary necklace, a feature observed in many motile cilia, is spatially aligned with the transition zone and quantify its regular organization within the membrane. Our in situ data capture rarely observed events, including intraflagellar transport (IFT) trains connecting to ciliary vesicles tethered to undocked centrioles. The surrounding ciliary environment contains intermediate filaments that encircle the basal bodies and bundled actin filaments that elaborate microvilli structures between the cilia. Integration of XL/MS and U-ExM identified novel microtubule associated proteins (MAPs), MIPs, and membrane-associated proteins localized to these distinct subdomains. This work provides a molecular and structural map of the mammalian MCC ciliary base, revealing architectural principles that underlie its assembly, organization, and function.

cell biology↗

bioRxiv2026

CoPISA: Combinatorial Proteome Integral Solubility/Stability Alteration analysis

Combination therapies are widely used in acute myeloid leukemia (AML), but systematic datasets capturing proteome-wide responses to multi-drug perturbations remain limited. Here we present CoPISA (Combinatorial Proteome Integral Solubility/Stability Alteration), a quantitative proteomics assay designed to profile protein solubility and stability responses to single and combined drug treatments. The dataset includes two AML drug pairs (LY3009120-sapanisertib and ruxolitinib-ulixertinib) applied to four AML cell lines (MOLM-13, MOLM-16, SKM-1, and NOMO-1) under control, single-agent, and combination conditions in both lysate and intact-cell formats. Thermal solubility profiling coupled with TMT-based multiplexed LC-MS/MS generated 16 TMT16-plex experiments comprising 192 LC-MS/MS raw files, providing deep proteome coverage across treatments and biological contexts. The resource includes raw and processed proteomics data, detailed experimental metadata in Sample and Data Relationship Format (SDRF), and reproducible analysis scripts for reporter normalization, protein-level aggregation, statistical modeling, and classification of combinatorial response patterns. The experimental design enables identification of proteins responding uniquely to combination treatments as well as overlapping single-agent effects. Technical validation demonstrates reproducible quantification across multiplex experiments and assay formats. All data are publicly available through the PRIDE repository (PXD066812) together with analysis code, enabling independent reanalysis and method development. This dataset provides a benchmark resource for studying proteome responses to drug combinations, comparing lysate and intact-cell perturbation profiles, developing computational approaches for combinatorial target inference, and supporting training in computational proteomics.

bioinformatics↗

bioRxiv2026

A systemic circadian nicotinic acid riboside (NaR) signal engages the unfolded protein response and adipogenesis via the prefoldin complex

Daily light-dark cycles impose predictable environmental fluctuations that require coordinated temporal regulation of cellular physiology. This coordination is mediated by the circadian clock, which operates as a network of tissue oscillators; however, the molecular signals that convey circadian information between organs remain incompletely defined. Here, we identify nicotinic acid riboside (NaR) as a circulating metabolite whose rhythmicity depends on the liver clock. In differentiating 3T3-L1 adipocytes, NaR engages unfolded protein response (UPR) gene programs and modulates adipogenic competence. Proteome-wide stability profiling implicates the prefoldin complex as a molecular target of NaR signaling, linking NaR exposure to altered proteostasis. Functionally, NaR-induced UPR signaling converges on the adipogenic transcription factor CEBPA, which is a central regulator of adipogenesis. Importantly, sustained NaR exposure suppresses adipocyte lipid deposition, whereas temporally restricted NaR stimulation enhances adipogenesis, indicating that NaR acts in a time-dependent manner. Together, these findings identify NaR as a liver clock-controlled circulating metabolite that couples systemic circadian metabolism to adipocyte proteostasis and differentiation, revealing a mechanism by which temporal metabolic signals shape tissue-specific physiological outcomes. HighlightsO_LIThe circadian clock is required for rhythmic regulation of circulating nicotinic acid riboside (NaR). C_LIO_LIThe liver clock is sufficient to generate NaR rhythmicity. C_LIO_LINaR engages the prefoldin complex to regulate unfolded protein response signaling and Cebpa expression. C_LIO_LITime-dependent NaR exposure differentially regulates CEBPA levels and adipocyte lipid deposition. C_LI

systems biology↗

bioRxiv2026

Within-host population structure, migration, and parallel adaptive evolution of Pseudomonas aeruginosa in cystic fibrosis lung disease

Pseudomonas aeruginosa infections in adults with cystic fibrosis (CF) are comprised of heterogeneous populations, most often tracing ancestry back to a single recent common ancestor. What is not clear is the physical spatial structure within the lung infection population, its stability over time and whether this physical structure leads to different evolutionary trajectories in different adaptive environments. To compare the P. aeruginosa populations across a single lung, we performed whole genome sequence analyses of 450 isolates recovered from lavage samples of the three different lobes of the right lung from a person with mild-to-moderate CF lung disease at three time points over the course of [~]1.5 years. We found that isolates fell into five distinct phylogenetic lineages with evidence for repeated translocation of isolates from different lineages across lobes and loss-of-function mutations in lasR and mucA were present in all 450 isolates. The well-resolved phylogenetic analyses revealed a structured population in which we find the coexistence of a slowly evolving lineage and more rapidly evolving lineages. There is also support for numerous migration events. Further, strong evidence for parallel adaptive mutations in multiple genes revealed distinct evolutionary paths affecting mucoid phenotypes and genetic variation in antibiotic resistance-associated pathways across coexisting populations within a single individual over time. These results provide an example of within-host evolution leading to microheterogeneity that may be useful to consider in future study of infection metapopulations dynamics over the course of chronic infection. IMPORTANCEIndividuals with cystic fibrosis (CF) commonly have chronic lung infections that contain clonally derived Pseudomonas aeruginosa populations with genotypic and phenotypic diversity. This study describes a substantial dataset containing 450 isolates from different lobes of the right lung across three timepoints from an individual with mild-to-moderate CF lung disease. Some regional enrichment for specific lineages with parallel mutations among individual lobes of the lung was observed, but longitudinal analysis also demonstrated that compartmentalization is not strictly maintained and that isolates migrate between lobes of the lung over time. Perspectives on within lung evolution will be important for understanding the pathogen populations in chronic respiratory infections in CF and other diseases.

microbiology↗

bioRxiv2026

Recalibrating Nanoparticle Protein Corona Analysis for Accurate Biological Identity and Soluble Plasma Proteome Profiling

The nanoparticle (NP) protein corona is considered the biological identity that determines NP fate, safety, targeting, and therapeutic effectiveness in biofluids. Nonetheless, standard corona isolation workflows assume the recovered protein signature originates primarily from plasma proteins adsorbed directly onto the NP surface, while largely overlooking co-isolation of endogenous nanoscale biological structures such as extracellular vesicles (EVs). This oversight can distort the apparent "biological identity" of the NP. Here, we show that EVs are a major hidden contributor to the perceived protein corona composition in human plasma. Using highly monodispersed polystyrene NPs (50-1000 nm) and superparamagnetic beads, we compared corona formation in standard human plasma and plasma depleted of an EV- enriched sedimentable fraction by ultracentrifugation at 100,000 x g for 2 h, with the recovered vesicles subsequently characterized by MACSPlex immunoaffinity analysis. Mass spectrometry revealed that EV depletion reduced the number of proteins identified on polystyrene NPs by 60-75% and on magnetic beads by 45-50%, demonstrating a substantial fraction of the conventionally assigned corona proteome arises from EV- associated carryover. EV depletion also restructured the apparent abundance hierarchy, increasing the relative prominence of soluble plasma proteins such as albumin and shifting dominant signals away from intracellular cytoskeletal component proteins that are characteristic of EV carryover towards genuine soluble plasma proteins and complement factors. These results highlight that standard corona workflows can inadvertently co-isolate a vast array of EV-associated material and thereby yield inaccurate assignments of protein origin. Distinguishing proteins adsorbed from the soluble phase from EV-surface and intravesicular material is essential for accurate interpretation of NP-biofluid interactions, biomarker discovery, and therapeutic targeting because molecular compartment determines both analytical meaning and drug accessibility. Significance StatementThe nanoparticle (NP) "protein corona" defines how engineered nanomaterials interact with living systems, influencing therapeutic safety, efficacy, and diagnostic utility. Conventionally, corona isolation workflows assume that recovered proteins were adsorbed directly from the fluid phase. This study reveals a major, previously overlooked source of analytical distortion: standard separation techniques routinely co-isolate endogenous extracellular vesicles (EVs), drastically distorting the perceived biological identity of NPs. Depletion of an EV-enriched sedimentable fraction by ultracentrifugation reduced identified corona proteins by up to 75% and restructures the apparent proteomic hierarchy. Distinguishing true soluble adsorbates from vesicular carryover is essential for accurately predicting NP behavior in vivo and prevents false positives in nano- diagnostics, establishing a critical new standard for high-fidelity biomarker discovery and nanomedicine.

bioengineering↗

bioRxiv2026

Towards the development of an insulin degradation test

People with diabetes rely on exogenous insulin to reduce blood glucose levels, compensating for insulin resistance or impaired pancreatic {beta}-cell function. Despite being essential for diabetes management, insulin formulations exhibit inconsistent performance due to their relatively fragile stability. This instability carries significant cost implications: some individuals spend over $1,000 USD per month on insulin, and these high prices influence one in six Americans with diabetes to ration their insulin supplies. Environmental stressors can induce conformational changes that cause insulin to misfold and aggregate into fibrils, which are inactive structures that contribute to long-term diabetic complications. Although insulins instability is well-documented, no test currently exists outside of laboratory settings to determine whether an insulin formulation has degraded. Here, we compare biochemical techniques for assessing bioactivity and structural integrity in three commercial insulin analogs exposed to physiologically relevant stress conditions, showing that fibril formation precedes measurable loss of bioactivity in insulin and that fibrillation depends on both the stressor type and the insulin formulation tested. We then demonstrate proof-of-concept testing for antibody-based degradation detection using commercial monoclonal antibody candidates. Together, these findings underscore the critical need for accessible insulin quality testing and demonstrate the feasibility of antibody-based detection of insulin fibrillation. ImportanceInsulin remains one of the most essential yet fragile biopharmaceuticals used in modern medicine. Globally, 150 million people with diabetes depend on exogenous insulin to regulate blood glucose levels, but the proteins inherent instability can cause degradation during storage or transport. These degradation events can reduce insulins potency and safety, yet patients and healthcare providers currently have no practical means to assess insulin quality before injection. This knowledge gap contributes to inconsistent therapeutic outcomes and increases the risk of complications associated with degraded insulin. Our work directly addresses this unmet clinical and public health need by identifying the molecular changes that occur when insulin analogs are exposed to everyday environmental stressors and by completing proof-of-concept testing for a device to detect insulin fibrillation. We demonstrate that structural transitions to fibrils precede measurable loss of bioactivity and that fibrillation behavior depends on both the insulin analog and the stressor type. By combining biochemical characterization with antibody-based detection, this study establishes a foundation for a low-cost, accessible method to verify insulin integrity outside the laboratory. Such a tool could prevent the use of degraded insulin, improve treatment consistency, and empower patients to ensure the quality of their medication. More broadly, this approach exemplifies how protein stability monitoring can be integrated into biotherapeutic quality assurance, improving safety, efficacy, and trust in life-sustaining biologic medicines.

biochemistry↗

bioRxiv2026

Ca2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

Lysosomes are essential in maintaining cellular health. Endocytic lysosome reformation (ELR) regenerates functional lysosomes following degradation of endocytic cargo, yet the mechanisms driving this process remain largely unknown. Here, we define the molecular machinery underlying ELR. We find that unlike autophagic lysosome reformation (ALR), ELR proceeds independently of mTOR and dynamin 2, but requires the mitochondrial fission GTPase DRP1. DRP1 mediates scission of endolysosomal tubules at contact sites with the endoplasmic reticulum (ER) and mitochondria. Disruption of DRP1 function or ER-endolysosome contact results in elongated tubules, indicating defective lysosome reformation. Moreover, mitochondrial activity is essential for tubule initiation, and Ca2+ transfer from endolysosomes to mitochondria is crucial for ELR onset. Our findings reveal a dual role for mitochondria in ELR: first in ELR initiation and second in DRP1-dependent tubule fission at ER-mitochondria-endolysosome tripartite contact sites, uncovering the previously unappreciated role of mitochondria in endolysosome remodeling and fission.

cell biology↗

bioRxiv2025

Exercise Engages Coordinated Neuron-Glia Signaling to Shape Spinal Cord Plasticity

Physical activity induces systemic benefits for brain and muscle function, but how the healthy spinal cord adapts to exercise remains largely unknown. Here, we combine bulk proteomics, single-nucleus RNA sequencing, and cellular communication inference to map exercise- induced molecular adaptations in the mouse lumbar spinal cord. Endurance training elicited robust baseline remodeling, dominated by glial transcriptional changes. Acute exhaustive exercise triggered biphasic responses: widespread metabolic and synaptic gene upregulation at 6 h followed by balanced suppression at 24 h, with trained animals exhibiting enhanced amplitude and faster resolution. Cell-cell communication analysis revealed that exercise reshaped signaling networks in both magnitude and composition. While glia emerged as primary transcriptional responders, cholinergic neurons--despite minimal transcriptional changes--were central signaling hubs, engaging various pathways with astrocytes, oligodendrocyte precursor cells, and oligodendrocytes in a training-dependent and temporally restricted manner. Glial-derived communication further diversified these responses, with astrocytes, oligodendrocytes, and microglia shifting toward pathways supporting synaptic remodeling, axon guidance, and growth factor signaling while dampening inflammatory cues. Together, these findings identify neuron-glia communication as potential driver of spinal cord adaptation to exercise, suggesting pathways through which glial plasticity may serve as a key mediator linking motor activity to spinal cord resilience. Significance StatementExercise confers broad benefits for neural and motor system health, yet how the healthy spinal cord adapts to physical activity is poorly understood. Using multi-omic profiling, we show that endurance training and acute exercise elicit coordinated transcriptional and signaling responses in spinal cord glia, with cholinergic neuron-glia communication emerging as a key upstream driver. These findings identify glial plasticity and neuron-glia crosstalk as a potential mechanism of spinal cord adaptation, providing a foundation for leveraging exercise-based strategies in neurological health and disease.

physiology↗

bioRxiv2025

Core Clock Protein Subcellular Dynamics Coordinate Local and Global Circadian Control in Syncytia.

Circadian rhythms are pervasive among eukaryotes, and the underlying clocks share a common regulatory architecture - a negative feedback loop. A wealth of genetic and biochemical data underpin current perceptions of circadian oscillators but aspects of their cell biology remain cryptic, especially in syncytial systems. We employed novel microfluidic systems and a blind mutant that retains circadian function to simultaneously track multiple clock components in vivo across circadian cycles, revealing remarkable subcellular and subnuclear dynamics of clock proteins and providing insights into spatiotemporal regulation in a multinucleated system. Despite heterogeneity of clock gene (frq) expression, we find robust cycles in FRQ nuclear localization among all nuclei and document free diffusion of multiple clock components among nuclei. Within nuclei, clock components form distinct, small, highly dynamic nuclear bodies that persist throughout the cycle, occasionally co-localizing for circadian regulatory functions. This rich context of in vivo spatiotemporal information illustrates how separate nuclear clocks ensure synchronous regulation of cellular activities across a macroscopic syncytium.

cell biology↗

bioRxiv2025

Proteomics of Transglutaminase 2 polyaminated proteins

Post-translational modification of proteins by polyamines (putrescine, spermine and spermidine) is poorly studied due to technical challenges in identification. Polyamination is mainly performed by transglutaminases via transamidation of a polyamine to an acceptor glutamine-residue on the target protein. We performed polyamination reactions on whole cell lysates using 2 different polyamine probes (Biotin-pentylamine and DNP-pentylamine) and identified modified peptides by mass spectrometry. 51 protein targets modified on 66 different sites were identified, with an overlap between hits tagged with the different probes. Many of the targets are involved in translation, cytoskeletal organization or have roles in cancer.

molecular biology↗

bioRxiv2024

Antibiotics that Kill Gram-negative Bacteria by Restructuring the Outer Membrane Protein BamA

The essential outer membrane protein insertase BamA has recently emerged as a valid target for killing Gram-negative bacteria. Bamabactins, competitive inhibitors targeting the lateral gate of BamA, disrupt the substrate folding process, compromise the outer membrane integrity, and lead to bacterial cell death. Despite their promise, the full pharmacological potential of bamabactins remains underexploited. We applied phylogenetic genome mining and synthetic biology to identify xenorceptides which selectively kill Enterobacteriaceae. Mode of action studies show that xenorceptide A2 integrates itself into BamA as an additional {beta}-strand between {beta}1 and {beta}16 at the lateral gate, inducing a conformation of BamA that has not been observed before. Biological evaluation of xenorceptide A2 shows promising activity in vitro and in vivo, and limited resistance which differentiates it from other bamabactin antibiotics. Our data show that the chemical diversity of bamabactins is far greater than previously recognized and thus an attractive source for antibiotic discovery.

microbiology↗

bioRxiv2024

Exploring Mechanisms of Action in Combinatorial Therapy through Stability/Solubility Alterations: Advancing AML Treatment

Acute myeloid leukemia (AML) remains a formidable clinical challenge due to genetic heterogeneity, high relapse rates, and toxicities associated with conventional chemotherapies. Rationally designed drug combinations offer improved efficacy, yet their selection is often empirical and lacks molecular mechanistic understanding. Here, we present CoPISA workflow (Proteome Integral Solubility/Stability Alteration Analysis for Combinations), a high-throughput proteomics workflow that captures protein solubility/stability alterations unique to combinatorial drug treatments, revealing mechanisms unattainable through single-drug analyses. Applying CoPISA to two rationally designed AML drug pairs, LY3009120-sapanisertib (LS) and ruxolitinib-ulixertinib (RU), we mapped primary (lysate) and secondary (living cell) protein target landscapes. Notably, our analysis uncovered an emergent mechanistic principle, "conjunctional targeting" (i.e., conjunctional inhibition), wherein cooperative drug actions induce treatment-specific targets not achievable individually, analogous to an AND-gate logic model. LS-specific AND-gate proteins converged on SUMOylation, chromatin condensation, and VEGF-linked adhesion, while RU-specific targets disrupted DNA-damage checkpoints, mitochondrial bioenergetics, and RNA-splicing machinery, collectively implicating synthetic-lethal vulnerabilities. Additionally, the post-translational modifications (PTMs) profiling of differential soluble proteins confirms several combination-induced modifications (e.g., acetylation, dimethylation, phosphorylation) on key AML proteins, such as NPM1. Network interrogation of AML-associated proteins showed that a high percentage of targeted proteins are unique to the combinations, including frequently mutated drivers DNMT3A, NPM1, and TP53. CoPISA exposes how drug pairs enact multi-axis pressure on AML cells through conjunctional targeting, a mechanistic layer beyond classical synergy. By pinpointing combination-exclusive protein targets and signaling pathways, CoPISA provides a blueprint for precision-guided regimen design in AML and other heterogeneous cancers. Data are available via ProteomeXchange with identifier PXD066812.

biochemistry↗

bioRxiv2024

Common regulatory mutation increases single-cell survival to antibiotic exposures in Pseudomonas aeruginosa.

Typical antibiotic susceptibility testing (AST) of microbial samples is performed in homogeneous cultures in batch environments, which does not account for the highly heterogeneous and dynamic nature of antibiotic responses. The most common mutation found in P. aeruginosa lineages evolved during chronic infections in the human lung, a loss of function of repressor MexZ, increases basal levels of multidrug efflux MexXY, but does not increase resistance by traditional minimal inhibitory concentration (MIC) assays. Here, we use single cell microfluidics to show that P. aeruginosa response to aminoglycosides is highly heterogeneous, with only a subpopulation of cells surviving exposure. In contrast, strains carrying mexZ mutations bypass the lengthy process of MexXY activation, increasing survival to sudden drug exposures and conferring a fitness advantage in fluctuating environments. Building on the data we present here, we propose a simple "Response Dynamics" assay to quantify the rate of population-level recovery to drug exposures across strains. We used this assay to profile a representative panel of 49 P. aeruginosa strains from diverse environments, showing that the presence of mexZ mutations correlates with faster population recovery from exposures to aminoglycosides, and thus confers an advantage to cells exposed to a sudden, large dose of antibiotic. We propose that the Response Dynamics assay can be used alongside MIC assays for profiling of antibiotic sensitivity to better predict clinical outcomes from in vitro sensitivity/resistance profiles. SignificanceCommon mutations affecting the regulation of antibiotic resistance in bacterial pathogens often do not increase resistance by traditional measures. However, antibiotic resistance is typically measured in stable cultures, without accounting for fluctuations in drug concentration. Here, we show that P. aeruginosa response to aminoglycosides is highly heterogeneous, and that the most common mutation found in clinical isolates improves resistance by increasing single-cell survival to drug exposures. Therefore, the success of antibiotic treatments depends on the dynamics of drug delivery and the heterogeneous activation of microbial responses. We then develop an assay to measure resistance in a dynamic context, which captures this overlooked aspect of antibiotic resistance and can be used alongside traditional measures in the profiling of clinical isolates.

microbiology↗

bioRxiv2024

Bacterial RNA promotes proteostasis through inter-tissuecommunication in C. elegans

Life expectancy has been increasing over the last decades, which is not matched by an increase in healthspan. Besides genetic composition, environmental and nutritional factors influence both health- and lifespan. Diet is thought to be a major factor for healthy ageing. Here, we show that dietary RNA species extend healthspan in C. elegans. Inherent bacterial-derived double stranded RNA reduces protein aggregation in a C. elegans muscle proteostasis model. This beneficial effect depends on low levels of systemic selective autophagy, the RNAi machinery in the germline, even when the RNA is delivered through ingestion in the intestine and the integrity of muscle cells. Our data suggest a requirement of inter-organ communication between the intestine, the germline and muscles. Our results demonstrate that bacterial-derived RNAs elicit a systemic response in C. elegans, which protects the animal from protein aggregation during ageing. We provide evidence that low stress levels are beneficial for healthspan. One-Sentence SummaryBacteria-derived dietary cues and inter-tissue communication promote proteostasis and fitness in C. elegans

cell biology↗