Biology subjects

Mills, C. E.

Publications and source records attributed to Mills, C. E..

15 recordsLinked to original sources

bioRxiv2026

Screening metatranscriptomes for ultrastable RNA secondary structures reveals hidden bacteriophages and novel capsid nanomaterials

Metatranscriptomics has transformed our view of RNA bacteriophage diversity, revealing vast numbers of single-stranded RNA (ssRNA) phages whose protein capsids can be engineered for biotechnology applications. However, many ssRNA phages remain hidden from current detection methods, which require protein-level similarity to known phages. Here we show that RNA structure provides an additional signal for the detection of ssRNA phages in metatranscriptomes, including hidden phages missed by prior protein-based methods. By computationally folding each contig and screening for exceptionally stable RNA secondary structures, we find evidence of thousands of previously unrecognized phages encoding novel coat proteins. We express a library of 12,000 such coat proteins in E. coli and find that most assemble into nuclease-resistant capsids. We determine the 3D structure of one such capsid by cryo-electron microscopy and demonstrate that it can be disassembled and reassembled in vitro to package heterologous RNA--a key step toward repurposing these particles as RNA delivery vehicles. We compile the newly discovered ssRNA phages with previously known ones into a database that contains sequence and structural information for over 460,000 unique RNA molecules and over 100,000 distinct coat proteins, providing a comprehensive resource for microbiology and nanomaterials research.

biochemistry↗

bioRxiv2026

Polypharmacology of an Optimal Kinase Library

Despite decades of research, current understanding of the spectrum of targets bound by kinase inhibitors remains incomplete. This complicates mechanism of action studies, drug repurposing, and development of new therapies. Here, we describe kinome-wide profiling of an optimal kinase library (OKL) comprising 192 small molecules selected based on stage of clinical development, chemical diversity, and target coverage. Our results show that polypharmacology is widespread and independent of regulatory approval. The generally understood ("assigned") targets of approved molecules are not necessarily the most potently inhibited and off-targets include multiple understudied kinases. Moreover, median selectivity has not increased over time We illustrate how an OKL in combination with detailed kinome profiling can be used to identify potential toxicity targets, repurpose anti-inflammatory drugs for neurodegenerative and infectious diseases, and perform chemical genetic studies. Our studies also highlight how much remains to be discovered about the chemistry and biology of one of the largest classes of human therapeutics.

pharmacology and toxicology↗

bioRxiv2025

Determinants of Protein-Level Parameters Governing MS2 VLP Reassembly

MS2 virus-like particles (VLPs) are widely used as protein nanocages for cargo encapsulation, yet in vitro disassembly-reassembly protocols remain poorly standardized, and reassembly yields are reported inconsistently. As a result, the same experiments reported in literature produce widely divergent yields, limiting reproducibility and cross-study comparability. Here, we introduce a cargo-specific, quantitative framework for standardized MS2 VLP reassembly yield determination. We evaluate commonly used disassembly and post-disassembly processing methods and identify practical trade-offs between protein recovery, accessibility, and reproducibility. Reassembly yield is quantified using size exclusion chromatography calibrated against purified VLP standards, enabling robust, cargo-specific yield measurement. Using this framework, we apply a full factorial design of experiments to quantify the individual and combined effects of coat protein concentration, ionic strength, buffer pH, and molecular crowding on reassembly yield. The resulting statistical model explains more than 99% of the explainable variance and its linear fit to the experimental data indicates that optimal reassembly conditions extend beyond those tested to date. Protein concentration and ionic strength dominate reassembly yield, whereas pH and osmolyte concentration contribute more modestly within the tested ranges. Finally, we propose practical guidelines for standardized MS2 VLP disassembly, reassembly, and yield reporting, defining a transferable operating envelope for MS2 VLP reconstruction. While demonstrated here using a single nucleic acid cargo (tr-DNA), the framework is readily extensible to alternative cargos and coat protein variants.

biophysics↗

bioRxiv2025

Integrative single-cell and spatial mapping of oxidative stress response uncovers GCLC+ mesenchymal tumor cell state linked with favorable outcomes in triple negative breast cancer

Inhibiting oxidative stress response (OSR) proteins has been suggested as a therapeutic strategy in triple negative breast cancer (TNBC). However, the cell type specificity and spatial distribution of OSR genes and proteins, such as GCLC and NQO1, is unknown. Using single cell and spatial transcriptomics datasets we found that OSR genes were highly expressed in TNBC tumor cells, which localized in spatial clusters. Multiplex immunofluorescence imaging of 345 TNBC samples from 186 patients demonstrated that OSR proteins GCLC and NQO1 exhibit distinct expression profiles across tumor, immune, and stromal cell populations and are elevated in inflamed histological regions. Tumor cell OSR protein expression was associated with the composition of the adjacent cellular neighborhood. Furthermore, we identified GCLC and vimentin positive (GCLC+VIM+) mesenchymal-like tumor cells, residing near immune cells and exhibiting increased proliferation and decreased anastasis signatures, suggesting sensitivity to chemotherapy. Across a panel of thirteen TNBC cell lines, GCLC expression was positively correlated with sensitivity to cisplatin. Cox regression analysis revealed that patients with higher proportions of GCLC+VIM+ tumor cells had a longer overall survival. Collectively, our results demonstrate that individual OSR proteins are expressed in distinct microenvironments and tumor cell states, potentially contributing to patient outcomes.

cancer biology↗

bioRxiv2025

Self-assembling protein materials with genetically programmable morphology and size

Materials are challenging to synthetically program down to the atom level. Nature, however, excels at creating hierarchical materials from nanoscale building blocks, a feat that remains a major challenge in synthetic systems. A deeper understanding of the molecular rules governing self-assembly would unlock the potential for designing genetically programmable materials with atomic precision. Hexameric bacterial microcompartment (BMC-H) proteins offer a powerful model system for exploring this question. These sequence-defined proteins naturally assemble into complex architectures and can be expressed biologically, making them ideal candidates for studying how minor sequence variations influence supramolecular structure. In this work, we leverage cell-free protein synthesis (CFPS) alongside immunostaining and super-resolution microscopy to investigate the self-assembly behavior of two BMC-H proteins, PduA and PduJ. We find that both proteins form micro-to millimeter scale structures when expressed in vitro. Further, we demonstrate how single point mutation changes lead PduA and PduJ to form significantly different supramolecular structures when produced using CFPS. These studies support the future exploration of self-assembling proteins as programmable scaffolds in broad materials applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/666636v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3376faorg.highwire.dtl.DTLVardef@c81730org.highwire.dtl.DTLVardef@6a6a5borg.highwire.dtl.DTLVardef@6cb4ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitutes the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs.

synthetic biology↗

bioRxiv2022

Paired evaluation defines performance landscapes for machine learning models

The true accuracy of a machine learning model is a population-level statistic that cannot be observed directly. In practice, predictor performance is estimated against one or more test datasets, and the accuracy of this estimate strongly depends on how well the test sets represent all possible unseen datasets. Here we present paired evaluation, a simple approach for increasing the robustness of performance evaluation by systematic pairing of test samples, and use it to evaluate predictors of drug response in breast cancer cell lines and of disease severity in patients with Alzheimers Disease. Our results demonstrate that the choice of test data can cause estimates of performance to vary by as much as 30%, and that paired evaluation makes it possible to identify outliers, improve the accuracy of performance estimates in the presence of known confounders, and assign statistical significance when comparing machine learning models.

bioinformatics↗

bioRxiv2021

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

bioengineering↗

bioRxiv2021

Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. ImportanceMCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here we provide evidence of a potential mechanism inS. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

synthetic biology↗

bioRxiv2021

Multiplexed and reproducible high content screening of live and fixed cells using the Dye Drop method

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g. CellTiter-Glo(R)). Here we describe a public domain "Dye Drop" method that uses sequential density displacement and microscopy to perform multi-step assays on living cells. We use Dye Drop cell viability and DNA replication assays followed by immunofluorescence imaging to collect single-cell dose-response data for 67 investigational and clinical-grade small molecules in 58 breast cancer cell lines. By separating the cytostatic and cytotoxic effects of drugs computationally, we uncover unexpected relationships between the two. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.

systems biology↗

bioRxiv2021

Integrating multi-omics data reveals function and therapeutic potential of deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. Inhibition of DUBs promises to make classically undruggable targets such as the tumor suppressor TP53 and oncogene c-Myc amenable to regulation by small molecules. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper, which are browsable online via the DUB Portal, promise to improve understanding of DUBs as a family as well as the activities of specific DUBs such as USP14, UCHL5 and USP7, which have been targeted with investigational cancer therapeutics.

cancer biology↗

bioRxiv2021

Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper

MotivationIdentifying gene clusters of interest in phylogenetically proximate and distant taxa can help to infer phenotypes of interest. Conserved gene clusters may differ by only a few genes, which can be biologically meaningful, such as the formation of pseudogenes or insertions interrupting regulation. These qualities may allow for unsupervised clustering of similar gene clusters into bins that provide a population-level understanding of the genetic variation in similar gene clusters. ResultsWe developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster in 435 genomes containing mixed taxa. In a subsequent application investigating the diversity and impact of gene complete and incomplete LT2 Pdu gene clusters in 1130 S. enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When replicated in vivo, disruption of pduN with a frameshift mutation negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering both distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements. AvailabilityGeneGrouper software and code are publicly available at https://github.com/agmcfarland/GeneGrouper.

bioinformatics↗

bioRxiv2021

Computational and experimental approaches to controlling bacterial microcompartment assembly

Bacterial microcompartments compartmentalize the enzymes that aid chemical and energy production in many bacterial species. These protein organelles are found in various bacterial phyla and are postulated to help many of these organisms survive in hostile environments such as the gut of their hosts. Metabolic engineers are interested in repurposing these organelles for non-native functions. Here, we use computational, theoretical and experimental approaches to determine mechanisms that effectively control microcompartment self-assembly. As a model system, we find via multiscale modeling and mutagenesis studies, the interactions responsible for the binding of hexamer-forming proteins propanediol utilization bacterial microcompartments from Salmonella and establish conditions that form various morphologies. We determine how the changes in the microcompartment hexamer protein preferred angles and interaction strengths can modify the assembled morphologies including the naturally occurring polyhedral microcompartment shape, as well as other extended shapes or quasi-closed shapes. We demonstrate experimentally that such altered strengths and angles are achieved via amino acid mutations. A thermodynamic model that agrees with the coarse-grained simulations provides guidelines to design microcompartments. These findings yield insight in controlled protein assembly and provide principles for assembling microcompartments for biochemical or energy applications as nanoreactors.

biophysics↗

bioRxiv2019

The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

cell biology↗

bioRxiv2017

Therapeutically advantageous secondary targets of abemaciclib identified by multi-omics profiling of CDK4/6 inhibitors

FDA approval of multiple drugs differing in chemical structures but targeting the same protein raises the question whether such drugs have sufficiently similar mechanisms of action to be considered functionally equivalent. In this paper we compare three recently approved inhibitors of the cyclin-dependent kinases CDK4/6 - palbociclib, ribociclib, and abemaciclib - that are becoming important therapies for the treatment of hormone-receptor positive breast and potentially other cancers. We find that transcriptional and proteomic changes induced by the three drugs differ significantly and that abemaciclib has unique cellular activities including induction of cell death (even in pRb-deficient cells), arrest in the G2 phase of the cell cycle, and reduced drug adaptation. These activities appear to arise from inhibition of kinases other than CDK4/6 including CDK2/Cyclin A/E and CDK1/Cyclin B.\n\nSIGNIFICANCEThe target profiles of most drugs are established relatively early in their development and are not systematically revisited at the time of approval. Scattered reports suggest that palbociclib, ribociclib, and abemaciclib differ in pharmacokinetics, dosing, and adverse effects but the three drugs are generally regarded as similar. Our finding that the drugs differ substantially in mechanism of action - abemaciclib retains activities of the earlier-generation drug alvocidib - suggests the potential for different uses in the clinic: in particular, abemaciclib may show activity in patients progressing on palbociclib or ribociclib. More generally, our approach relying on data from five distinct phenotypic and biochemical assays strongly suggests that a multi-faceted approach is necessary to get a reliable picture the target spectrum of kinase inhibitors.

cancer biology↗