Biology subjects

Johnson, O. T.

Publications and source records attributed to Johnson, O. T..

4 recordsLinked to original sources

bioRxiv2026

A Framework for Benchmarking Pathway Reconstruction Algorithms

Cells coordinate diverse biological processes through interactions among thousands of molecules, but mapping these interactions comprehensively and systematically remains an open problem. Pathway reconstruction algorithms address this problem by linking molecules of interest, identified from high-throughput omics experiments, using prior knowledge encoded as background interaction networks. This process recovers intermediate molecules and interactions that were not directly measured in the experimental data but plausibly connect the observed molecules. It generates testable hypotheses about interactions that drive cell behavior and informs the choice of follow-up experiments. Many algorithms have been created over decades, each optimizing different computational objectives and relying on different assumptions. The resulting heterogeneity has made benchmarking challenging, limiting systematic comparisons. Therefore, selecting an algorithm for a given biological context remains a non-trivial and poorly informed task. This registered report presents a large-scale benchmark of pathway reconstruction algorithms, evaluating 14 algorithms across 822 datasets from four biological settings. To enable this benchmark, we introduce Signaling Pathway Reconstruction Analysis Streamliner (SPRAS), which standardizes algorithm inputs, outputs, and execution into a formal framework, enabling systematic comparison that was previously infeasible. We will assess each algorithm on reconstruction performance against gold standard pathways, algorithm similarity, and computational performance across different biological contexts. Together, these evaluations will provide quantitative evidence for understanding pathway reconstruction algorithm behavior and guiding algorithm selection.

bioinformatics↗

bioRxiv2026

The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

Over the past decades, chikungunya virus (CHIKV), a re-emerging arthropod-borne alphavirus, has caused outbreaks in many (sub)tropical regions, but also in more temperate regions of the world, including Europe. CHIKV poses a significant health burden due to high infection rates during epidemics, symptom progression into chronic arthritic manifestations, and the lack of specific antiviral treatments. Multiple studies have shown that several viruses rely on host molecular chaperones, particularly the central Heat shock protein 70 (Hsp70), for replication. Hsp70s are guided by co-chaperones that drive their functionality and specificity. Here, we used chemical inhibitors of Hsp70-co-chaperone interactions to study the role of this molecular chaperone machinery in CHIKV replication. Our findings revealed that Hsp70 inhibition significantly reduces the CHIKV infectious particle production without affecting host-cell viability. Inhibition of the Hsp70-co-chaperone interaction primarily impedes the post-RNA replication stages of the CHIKV infectious cycle, affecting viral protein expression and reducing both the number and infectivity of released virions. Moreover, Hsp70 inhibition displayed antiviral activity in skin explants. Together, these results suggest that targeting the Hsp70 network could be a viable antiviral strategy against CHIKV infections. Author summaryAs obligatory intracellular parasites, viruses rely entirely on the machinery of the host cell to produce new viral particles. In our study, we investigated whether a specific group of host proteins, known as molecular chaperones, is important for the replication of chikungunya virus (CHIKV), a reemerging mosquito-borne virus that can cause long-lasting joint pains and lacks specific antiviral treatments. We focused on one key family of molecular chaperones, called Heat shock protein 70s (Hsp70s), which supports protein folding and quality control in cells. We used chemical compounds to block Hsp70 function and observed that CHIKV replication was strongly reduced, while host cells remained healthy. We found that Hsp70 is especially important in the later stages of the virus life cycle, where it helps produce viral proteins and new infectious virus particles. When Hsp70 was blocked, fewer and less infectious virus particles were produced. We also showed that this effect holds true in biopsies of mouse skin tissue, which mimics the initial site of infection. These findings identify Hsp70 as an important host factor for CHIKV that may serve as a potential target for new antiviral therapies.

microbiology↗

bioRxiv2023

Conformationally Adaptive Dyes Enable Protein-Adaptive Differential Scanning Fluorometry

Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding. One-Sentence SummaryNext generation protein-adaptive DSF (paDSF) enables rapid and general measurements of protein stability and dynamics.

biochemistry↗

bioRxiv2021

Two distinct classes of co-chaperones compete for the EEVD motif in heat shock protein 70 (Hsp70) to tune its activity

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions (PPIs) with many co-chaperones. One hotspot for co-chaperone binding is the EEVD motif that is found at the extreme C-terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat (TPR) domain co-chaperones, such as the E3 ubiquitin ligase CHIP, and Class B J-domain proteins (JDPs), such as DnaJB4. Although complexes between Hsp70-CHIP and Hsp70-DnaJB4 are both important for chaperone functions, the molecular determinants that dictate the competition between these co-chaperones are not clear. Using a collection of EEVD-derived peptides, we find that DnaJB4 binds to the IEEVD motif of Hsp70s, but not the related MEEVD motif of cytoplasmic Hsp90s. Then, we explored which residues are critical for binding to CHIP and DnaJB4, revealing that they rely on some shared features of the IEEVD motif, such as the C-terminal carboxylate. However, they also had unique preferences, especially at the isoleucine position. Finally, we observed a functionally important role for competition between CHIP and DnaJB4 in vitro, as DnaJB4 can limit the ubiquitination activity of the Hsp70-CHIP complex, while CHIP suppresses the chaperone activities of Hsp70-DnaJB4. Together, these results suggest that the EEVD motif has evolved to support diverse PPIs, such that competition between co-chaperones could help guide whether Hsp70-bound proteins are folded or degraded.

biochemistry↗