Biology subjects

Johnson, J. L.

Publications and source records attributed to Johnson, J. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Dynamic visualization of physiological CaMKII activity using sensitive FRET biosensors

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key mediator of complex physiological processes throughout the body, from the brain to the reproductive system, where CaMKII translates spatiotemporally dynamic calcium elevations into specific biological functions. Directly visualizing CaMKII activity dynamics in living cells using genetically encoded fluorescent biosensors can thus provide crucial insights into the molecular regulation of health and disease. Yet the ability to sensitively and specifically monitor endogenous CaMKII activity in physiologically relevant contexts is limited by the lack of sensors that can achieve robust, quantitative visualization of CaMKII responses. Here, we leveraged a recent serine/threonine kinome-wide substrate atlas to rationally engineer a powerful suite of Forster resonance energy transfer (FRET)-based CaMKII kinase activity reporters with high specificity, sensitivity, and signal-to-noise ratio. Using these biosensors, we were able to sensitively and robustly visualize endogenous CaMKII activity dynamics in both cultured cell lines and primary cells, including cardiomyocytes, oocytes, and neurons. We further utilized 2pFLIM imaging of organotypic hippocampal slices to quantitatively track LTP-induced CaMKII activity within single dendritic spines, highlighting a major advance in the study of physiological CaMKII signaling.

bioengineering↗

bioRxiv2026

An expedient, biology-laboratory-compatible method for preparing functional perfluoropolyether fluorosurfactants for droplet microfluidics

Biocompatible fluorosurfactants are essential for many droplet microfluidic workflows but are often obtained from commercial sources because published syntheses of perfluoropolyether (PFPE)-based surfactants typically require acid chloride intermediates and chemistry-oriented purification methods. These requirements can limit access for biology and clinical laboratories seeking low-cost or customizable surfactant systems. Here we describe a practical method for preparing functional PFPE-based fluorosurfactant materials by direct carbodiimide coupling of functionalized PFPE carboxylic acids(Krytox 157 FSH) to amine-containing head groups under laboratory-accessible conditions. Using this approach, we prepared a PFPE-polyethylene-glycol (PFPE-PEG) material from Jeffamine ED900 and a PFPE-Tris material from Tris base. Because these products were not fully structurally characterized, we present them as functional reaction products and evaluate them by use in biomicrofluidic workflows rather than by definitive compositional assignment. PFPE-Tris was useful for generating relatively uniform small droplets, whereas the PFPE-PEG preparation supported a broader range of biological applications. These materials were used in genomic library screening for {beta}-glucosidase activity, thermocycling-associated droplet workflows, and protein crystallization experiments. In addition, the PFPE-PEG preparation improved emulsion behavior in many protein crystallization screens that were unstable with a commercial droplet oil used in our laboratory. This method reduces the practical barrier to in-house fluorosurfactant preparation and allows biology-focused laboratories to explore head-group chemistry, oil composition, and operating conditions without complete reliance on commercial reagents. The results support this workflow as a useful entry point for biomicrofluidics laboratories, while also highlighting the need for careful interpretation of thermocycled droplet assays and for future analytical characterization of the resulting materials. Significance statementDroplet microfluidics relies on fluorosurfactants that are often costly and difficult to synthesize outside of chemistry-focused settings. We describe a simple, biology-laboratory-compatible approach for generating functional perfluoropolyether-based fluorosurfactant materials using direct carbodiimide coupling and straightforward cleanup. The resulting materials supported multiple biomicrofluidic workflows in our laboratory, including enzymatic screening and protein crystallization, and provide a practical route for groups seeking lower-cost and more customizable surfactant systems.

synthetic biology↗

bioRxiv2025

Targeting tumor antioxidant pathways with novel GSTP1/M2 inhibitors for cancer treatment

Glutathione transferase (GSTP1 and GSTM2) are tractable targets for anticancer drug development. In this work, a series of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) based analogues were designed, synthesized, and evaluated both theoretically and experimentally as GSTP1 and GSTM2 inhibitors. Among the synthesized compounds, 3h showed selective inhibition toward GSTP1 while 5b showed selective inhibition of GSTM2. Compounds 5b and 5c exhibited stronger potency while compound 3h showed slightly lower potency against the tested cancer cells than its parent molecule NBDHEX. Comprehensive biological studies were conducted on the effect of 3h, 5b and 5c towards breast cancer MDA-MB-231 and pancreatic MiaPaCa-2 cell lines revealed that 3h, 5b and 5c could activate JNK pathway and induce cell apoptosis. Furthermore in vivo experiments using NSG mice demonstrated that 5b significantly reduced tumor growth when administered in combination with gemcitabine, effectively overcoming gemcitabine resistance in the MiaPaCa-2 cell model through targeted inhibition of GSTM2. These findings suggests that, 5b could become a promising candidate for further development as a potential antitumor agent in cancer therapy.

cancer biology↗

bioRxiv2025

Nitroxoline-O-protected derivatives inhibit MetAP2 and activate ATF4 through mTORC1 to inhibit cancer cell growth

Reprogrammed cancer cell proliferation requires high levels of protein synthesis and concomitant folding and processing. N-terminal methionine amino peptidases (MetAP) are a class of enzymes that cleave the initiator methionine amino acids to allow for peptide maturation and co-translational processing. Specifically, based on its role in protein synthesis, MetAP2 has been found to be upregulated in cancer cells and has been explored as a potential anticancer target. Cellular perturbations that impinge on protein synthesis activate cellular stress pathways, including the integrated stress response and mTORC1. Nitroxoline, a MetAP2 inhibitor has been explored as an anticancer agent but is hampered by poor pharmacokinetic properties. Here, we synthesize a few O-substituted silyl and nonsilyl nitroxoline analogs to diversify the nitroxoline template to reduce metabolic vulnerability. In vitro MetAP2 and cancer cell proliferation inhibition assays demonstrate that synthesized analogs retain potency when compared to the parent nitroxoline. Mechanistically, we show that the lead candidate compound 3 and nitroxoline activate ATF4 mediated stress responses through non-canonical mTORC1. These results further implicate MetAP2 protein processing in mTORC1 nutrient sensing pathways and provide novel synthetic analogs of nitroxoline for potential cancer treatment.

cancer biology↗

bioRxiv2025

A Structure-Guided Kinase-Transcription Factor Interactome Atlas Reveals Docking Landscapes of the Kinome

AbstractProtein kinases orchestrate cellular processes through phosphorylation, yet the structural basis for their specific binding partner interactions remains largely unmapped. Here, we present a structure-guided atlas of the human and Drosophila kinome, built by applying a new interface-aware scoring framework (iLIS) to AlphaFold-Multimer predictions. The resulting atlas recapitulates hallmark sequence preferences, confirms previously reported and functionally related protein-protein interactions, and uncovers unrecognized docking interactions. Notably, our analysis predicts a potentially widespread docking motif on homeodomain transcription factors that mediates interactions with basophilic kinases. Furthermore, we map putative allosteric interaction hotspots across the kinome and provide proof-of-concept evidence that targeting these surfaces can inhibit kinase activity. Finally, we demonstrate the physiological utility of the atlas by identifying a novel regulatory mechanism between Sgg/GSK3 and Hnf4 that controls lipid metabolism in vivo. This resource provides a blueprint for dissecting signaling networks and for the rational design of docking-site-specific kinase modulators.

bioinformatics↗

bioRxiv2025

Collaboration between two conserved sequence motifs drives ATPase stimulation of Hsp90 by Aha1

AbstractHsp90 is a dimeric molecular chaperone essential for the folding, stabilization, activation, and maturation of hundreds of client proteins, which are critical for cellular function. Co-chaperones, such as Aha1, play a key role in regulating the ATP-dependent Hsp90 client activation cycle by modulating Hsp90s ATPase activity and controlling progression through the cycle. Two highly conserved motifs in Aha1--the NxNNWHW and RKxK motifs--are known to regulate specific aspects of the Hsp90 ATPase cycle. In this study, we demonstrate that the K60 residue within the RKxK motif facilitates the structural organization of the NxNNWHW motif prior to ATP hydrolysis. Mutation of the K60 residue partially impairs the in vivo functionality of yeast Aha1. Additionally, we reveal that each individual residue within the NxNNWHW motif modulates the ATPase rate and apparent affinity for ATP of Hsp90. These findings provide new insights into how conserved regions of Aha-type co-chaperones influence Hsp90 kinetics and its regulation of client protein folding.

biochemistry↗

bioRxiv2025

Structural Identification of Major Molecular Determinants for Phosphotyrosine Recognition in Tyrosine Kinases Reveals Tumour Promoting and Suppressive Functions

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

biochemistry↗

bioRxiv2025

SURFS and AlphaFold Reveal Ribosome Footprint Shift Caused by EF-Tu D81 Mutation

Protein synthesis relies on accurate mRNA decoding by tRNA, a process guided by EF-Tu. We investigated how mutations at a conserved residue, aspartate 81, affect EF-Tu function using GTPase assays, AlphaFold modeling, and quantum-sensing-based super-resolution force spectroscopy (SURFS). All D81 variants retained GTPase activity but impaired tRNA release, revealed by sub-nucleotide ribosome footprinting. AlphaFold3 modeling suggests that D81 mutations disrupt magnesium coordination and interaction with the sarcin-ricin loop in the GTP-bound state. AlphaFold2-based sequence-structure analysis indicates that D81 anchors coevolutionary constraints, and its mutation enables cryptic structural variation. These results show how a single conserved residue links catalytic coordination, allosteric communication, and evolutionary constraint, offering mechanistic insight into translation fidelity and demonstrating the utility of an unconventional force spectroscopy in probing ribosome dynamics.

biophysics↗

bioRxiv2025

Global Landscape of Human Kinase Motifs in Viral Proteomes

Viruses are classically viewed as targets of host sensing, yet whether they also sense and respond to host cues remains largely unexplored. We propose that host-driven post-translational modification of viral proteins allows viruses to dynamically sense host cellular states. We annotated human kinase motifs in 1,505 viral proteomes and discovered an enrichment for stress, inflammation, and cell-cycle kinases. Mapping kinase motifs onto 21,606 viral protein structures and integrating with phosphoproteomics of infected cells revealed surface-accessible residues were preferentially phosphorylated, showed greater kinase specificity, and were under positive selection for stress and immune kinase motifs. Temporal phosphoproteomics of alphavirus-infected cells confirmed stress kinase activation and viral protein phosphorylation, and MAP kinase inhibition reduced alphavirus replication and phosphorylation of ERK and JNK motifs on viral proteins. Our findings suggest that viruses evolved as biosensors of the host signaling state, unveiling new antiviral opportunities aimed at disrupting virus decision-making.

microbiology↗

bioRxiv2025

Atlas of the Bacterial Serine-Threonine Kinases expands the functional diversity of the kinome

Bacterial serine-threonine protein kinases (STKs) regulate diverse cellular processes associated with cell growth, virulence, and pathogenicity. They are evolutionarily related to the druggable eukaryotic STKs. However, an incomplete knowledge of how bacterial STKs differ from their eukaryotic counterparts and how they have diverged to regulate diverse bacterial signaling functions presents a bottleneck in targeting them for drug discovery efforts. Here, we classified over 300,000 bacterial STK sequences from the NCBI RefSeq non-redundant and UniProt protein databases into 35 canonical and seven non-canonical (pseudokinase) families based on the patterns of evolutionary constraints in the conserved catalytic domain and flanking regulatory domains. Through statistical comparisons, we identified distinguishing features of bacterial STKs, including a distinctive arginine residue in a regulatory helix (C-Helix) that dynamically couples ATP and substrate binding lobes of the kinase domain. Biochemical and peptide-library screens demonstrated that constrained residues contribute to substrate specificity and kinase activation in the Mycobacterium tuberculosis kinase PknB. Collectively, these findings open new avenues for investigating bacterial STK functions in cellular signaling and for the development of selective bacterial STK inhibitors.

bioinformatics↗

bioRxiv2024

Uncovering the signaling networks of disseminated glioblastoma cells in vivo with INSIGHT

Dysregulation of intracellular signaling networks underpins cancer. However, a systems-level elucidation of how signaling networks within distinct cell subpopulations drive cancer progression in vivo has been unattainable due to technical limitations. We developed INSIGHT (INvestigating SIGnaling network of specific cell subpopulation in Heterogeneous Tissue), a new platform technology combining fluorescence-activated cell sorting with ultra-sensitive mass spectrometry to enable phosphoproteomic characterization of rare and discrete cell subpopulations from fixed tissues. We demonstrated the broad utility of INSIGHT by analyzing the oligodendroglial cell-specific signaling network in the mouse brain. We then applied INSIGHT to investigate the rare, disseminated tumor cell subpopulation in glioblastoma patient-derived xenograft models. INSIGHT uncovered a global rewiring of signaling networks with tumor cell dissemination, marked by a transition from proliferation-associated signaling in the primary tumor cells to signaling associated with postsynapse, neuronal migration, and ion homeostasis in disseminated tumor cells. We reveal interconnections between signaling circuitries within the networks, with numerous proteins, including GluA2, exhibiting altered phosphorylation without protein expression changes, emphasizing the role of post-translational modifications in glioblastoma dissemination. We validated key phosphorylation changes and inferred differentially active kinases with tumor spread to offer new systems-level insights into glioblastoma dissemination mechanisms in vivo. INSIGHT is generally applicable to a wide range of biological systems without genetic engineering and provides quantitative phosphorylation and protein expression data for selected cell subpopulations from heterogeneous tissues.

systems biology↗

bioRxiv2024

PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1s consensus substrate motif, protein interactors, and how interactors, including Ca2+ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca2+-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca2+ sensor of the CREC family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca2+ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1s catalytic activity, and raise the possibility that different Ca2+ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca2+ concentrations.

biochemistry↗

bioRxiv2024

Comprehensive evaluation of phosphoproteomic-based kinase activity inference

Kinases play a central role in regulating cellular processes, making their study essential for understanding cellular function and disease mechanisms. To investigate the regulatory state of a kinase, numerous methods have been, and continue to be, developed to infer kinase activities from phosphoproteomics data. These methods usually rely on a set of kinase targets collected from various kinase-substrate libraries. However, only a small percentage of measured phosphorylation sites can usually be attributed to an upstream kinase in these libraries, limiting the scope of kinase activity inference. In addition, the inferred activities from different methods can vary making it crucial to evaluate them for accurate interpretation. Here, we present a comprehensive evaluation of kinase activity inference methods using multiple kinase-substrate libraries combined with different inference algorithms. Additionally, we try to overcome the coverage limitations for measured targets in kinase substrate libraries by adding predicted kinase-substrate interactions for activity inference. For the evaluation, in addition to classical cell-based perturbation experiments, we introduce a tumor-based benchmarking approach that utilizes multi-omics data to identify highly active or inactive kinases per tumor type. We show that while most computational algorithms perform comparably regardless of their complexity, the choice of kinase-substrate library can highly impact the inferred kinase activities. Hereby, manually curated libraries, particularly PhosphoSitePlus, demonstrate superior performance in recapitulating kinase activities from phosphoproteomics data. Additionally, in the tumor-based evaluation, adding predicted targets from NetworKIN further boosts the performance, while normalizing sites to host protein levels reduces kinase activity inference performance. We then showcase how kinase activity inference can help in characterizing the response to kinase inhibitors in different cell lines. Overall, the selection of reliable kinase activity inference methods is important in identifying deregulated kinases and novel drug targets. Finally, to facilitate the evaluation of novel methods in the future, we provide both benchmarking approaches in the R package benchmarKIN. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/601117v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@44de1org.highwire.dtl.DTLVardef@9510a3org.highwire.dtl.DTLVardef@775225org.highwire.dtl.DTLVardef@1b2256f_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

Near-Zero Photon Bioimaging

Enhancing the reliability and reproducibility of optical microscopy by reducing specimen irradiance continues to be an important biotechnology target. As irradiance levels are reduced, however, the particle nature of light is heightened, giving rise to Poisson noise, or photon sparsity that restricts only a few (0.5%) image pixels to comprise a photon. Photon-sparsity can be addressed by collecting more than 200 photons per pixel; this, however, requires extended acquisition durations and, thus, suboptimal imaging rates. Here, we introduce near-zero photon imaging, a method that operates at kHz rates and 10,000-fold lower irradiance than modern microscopy. To achieve this performance, we deployed a judiciously designed epi-fluorescence microscope enabling ultralow background and artificial intelligence that learns to reconstruct biological images from as low as 0.01 photons per pixel. We demonstrate that near-zero photon imaging captures the structure of both multicellular and subcellular targets with high fidelity, including features represented by nearly zero photons. Beyond optical microscopy, the near-zero photon imaging paradigm can be applied in remote sensing, covert applications, and biological or biomedical imaging that utilize damaging or quantum light.

biophysics↗

bioRxiv2024

Illuminating the Dark Cancer Phosphoproteome Through a Machine-Learned Co-Regulation Map of 26,280 Phosphosites

Mass spectrometry-based phosphoproteomics offers a comprehensive view of protein phosphorylation, but limited knowledge about the regulation and function of most phosphosites restricts our ability to extract meaningful biological insights from phosphoproteomics data. To address this, we combine machine learning and phosphoproteomic data from 1,195 tumor specimens spanning 11 cancer types to construct CoPheeMap, a network mapping the co-regulation of 26,280 phosphosites. Integrating network features from CoPheeMap into a machine learning model, CoPheeKSA, we achieve superior performance in predicting kinase-substrate associations. CoPheeKSA reveals 24,015 associations between 9,399 phosphosites and 104 serine/threonine kinases, including many unannotated phosphosites and under-studied kinases. We validate the accuracy of these predictions using experimentally determined kinase-substrate specificities. By applying CoPheeMap and CoPheeKSA to phosphosites with high computationally predicted functional significance and cancer-associated phosphosites, we demonstrate the effectiveness of these tools in systematically illuminating phosphosites of interest, revealing dysregulated signaling processes in human cancer, and identifying under-studied kinases as putative therapeutic targets.

cancer biology↗

bioRxiv2023

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the Glucocorticoid Receptor

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of client proteins, including the glucocorticoid receptor (GR)1-3. Previously, we revealed that GR ligand binding activity is inhibited by Hsp70 and restored by Hsp90, aided by co-chaperones4. We then presented cryo-EM structures mechanistically detailing how Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding5,6. In vivo, GR-chaperone complexes are found associated with numerous Hsp90 co-chaperones, but the most enigmatic have been the immunophilins FKBP51 and FKBP52, which further regulate the activity of GR and other steroid receptors7-9. A molecular understanding of how FKBP51 and FKBP52 integrate with the GR chaperone cycle to differentially regulate GR activation in vivo is lacking due to difficulties reconstituting these interactions. Here, we present a 3.01 [A] cryo-EM structure of the GR:Hsp90:FKBP52 complex, revealing, for the first time, that FKBP52 directly binds to the folded, ligand-bound GR using three novel interfaces, each of which we demonstrate are critical for FKBP52-dependent potentiation of GR activity in vivo. In addition, we present a 3.23 [A] cryo-EM structure of the GR:Hsp90:FKBP51 complex, which, surprisingly, largely mimics the GR:Hsp90:FKBP52 structure. In both structures, FKBP51 and FKBP52 directly engage the folded GR and unexpectedly facilitate release of p23 through an allosteric mechanism. We also reveal that FKBP52, but not FKBP51, potentiates GR ligand binding in vitro, in a manner dependent on FKBP52-specific interactions. Altogether, we reveal how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation and how FKBP51 and FKBP52 compete for GR:Hsp90 binding, leading to functional antagonism.

biochemistry↗

bioRxiv2022

Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche

Melanoma is an aggressive cancer typically arising from transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. The role of keratinocytes in shaping the melanoma tumor microenvironment remains understudied. We previously showed that temporary loss of the keratinocyte-specific cadherin, Desmoglein 1 (Dsg1) controls paracrine signaling between normal melanocytes and keratinocytes to stimulate the protective tanning response. Here, we provide evidence that melanoma cells hijack this intercellular communication by secreting factors that keep Dsg1 expression low in surrounding keratinocytes, which in turn generate their own paracrine signals that enhance melanoma spread through CXCL1/CXCR2 signaling. Evidence suggests a model whereby paracrine signaling from melanoma cells increases levels of the transcriptional repressor Snai2 (Slug), and consequently decreases the Dsg1 transcriptional activator Grhl1 (Grainyhead like 1), a known promoter of epidermal differentiation and barrier function. Together, these data support the idea that paracrine crosstalk between melanoma cells and keratinocytes resulting in chronic keratinocyte Dsg1 reduction contributes to melanoma cell movement associated with tumor progression.

cancer biology↗

bioRxiv2022

Epinephrine inhibits PI3K alpha via the Hippo kinases.

The phosphoinositide 3-kinase, p110, is an essential mediator of insulin signaling and glucose homeostasis. We systematically interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110 and found that the Hippo kinases phosphorylate and completely inhibit its activity. This inhibitory state corresponds to a conformational change of a membrane binding domain on p110, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases, MST1/2, using forskolin or epinephrine is associated with phosphorylation and inhibition of p110, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules. Our study reveals a novel inhibitory pathway of PI3K signaling and a previously unappreciated link between epinephrine and insulin signaling.

physiology↗