Caught in the act - protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease.

Vertebrate proteins that fulfill multiple and seemingly disparate functions are increasingly recognized as vital solutions to maintaining homeostasis in the face of the complex cell and tissue physiology of higher metazoans. However, the molecular adaptations that underpin this increased functionality remain elusive. In this Commentary, we review the PACS proteins - which first appeared in lower metazoans as protein traffic modulators and evolved in vertebrates to integrate cytoplasmic protein traffic and interorganellar communication with nuclear gene expression - as examples of protein adaptation 'caught in the act'. Vertebrate PACS-1 and PACS-2 increased their functional density and roles as metabolic switches by acquiring phosphorylation sites and nuclear trafficking signals within disordered regions of the proteins. These findings illustrate one mechanism by which vertebrates accommodate their complex cell physiology with a limited set of proteins. We will also highlight how pathogenic viruses exploit the PACS sorting pathways as well as recent studies on PACS genes with mutations or altered expression that result in diverse diseases. These discoveries suggest that investigation of the evolving PACS protein family provides a rich opportunity for insight into vertebrate cell and organ homeostasis.

Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors.

ROS1 is the largest receptor tyrosine kinase in the human genome. Rearrangements of the ROS1 gene result in oncogenic ROS1 kinase fusion proteins that are currently the only validated biomarkers for targeted therapy with ROS1 TKIs in patients. While numerous somatic missense mutations in ROS1 exist in the cancer genome, their impact on catalytic activity and pathogenic potential is unknown. We interrogated the AACR Genie database and identified 34 missense mutations in the ROS1 tyrosine kinase domain for further analysis. Our experiments revealed that these mutations have varying effects on ROS1 kinase function, ranging from complete loss to significantly increased catalytic activity. Notably, Asn and Gly substitutions at Asp2113 in the ROS1 kinase domain were found to be TKI-sensitive oncogenic variants in cell-based model systems. In vivo experiments showed that ROS1 D2113N induced tumor formation that was sensitive to crizotinib and lorlatinib, FDA-approved ROS1-TKIs. Collectively, these findings highlight the tumorigenic potential of specific point mutations within the ROS1 kinase domain and their potential as therapeutic targets with FDA-approved ROS1-TKIs.

Resistance Profile and Structural Modeling of Next-Generation ROS1 Tyrosine Kinase Inhibitors.

ROS1 fusion proteins resulting from chromosomal rearrangements of the ROS1 gene are targetable oncogenic drivers in diverse cancers. Acquired resistance to targeted inhibitors curtails clinical benefit and response durability. Entrectinib, a NTRK/ROS1/ALK targeted tyrosine kinase inhibitor (TKI), was approved for the treatment of ROS1 fusion-positive non-small cell lung cancer (NSCLC) in 2019. In addition, lorlatinib and repotrectinib are actively being explored in the setting of treatment-naïve or crizotinib-resistant ROS1 fusion driven NSCLC. Here, we employed an unbiased forward mutagenesis screen in Ba/F3 CD74-ROS1 and EZR-ROS1 cells to identify resistance liabilities to entrectinib, lorlatinib, and repotrectinib. ROS1F2004C emerged as a recurrent entrectinib resistant mutation and ROS1G2032R was discovered in entrectinib and lorlatinib-resistant clones. Cell-based and modeling data show that entrectinib is a dual type I/II mode inhibitor, and thus liable to both types of resistant mutations. Comprehensive profiling of all clinically relevant kinase domain mutations showed that ROS1L2086F is broadly resistant to all type I inhibitors, but remains sensitive to type II inhibitors. ROS1F2004C/I/V are resistant to type I inhibitors, entrectinib and crizotinib, and type II inhibitor, cabozantinib, but retain sensitivity to the type I macrocyclic inhibitors. Development of new, more selective type II ROS1 inhibitor(s) or potentially cycling type I and type II inhibitors may be one way to expand durability of ROS1-targeted agents.

Spectroscopic Analysis of a Library of DNA Tension Probes for Mapping Cellular Forces at Fluid Interfaces.

Oligonucleotide-based probes offer the highest spatial resolution, force sensitivity, and molecular specificity for cellular tension sensing and have been developed to measure a variety of molecular forces mediated by individual receptors in T cells, platelets, fibroblasts, B-cells, and immortalized cancer cell lines. These fluorophore-oligonucleotide conjugate probes are designed with a stem-loop structure that engages cell receptors and reversibly unfolds due to mechanical strain. With the growth of recent work bridging molecular mechanobiology and biomaterials, there is a need for a detailed spectroscopic analysis of DNA tension probes that are used for cellular imaging. In this manuscript, we conducted an analysis of 19 DNA hairpin-based tension probe variants using molecular dynamics simulations, absorption spectroscopy, and fluorescence imaging (epifluorescence and fluorescence lifetime imaging microscopy). We find that tension probes are highly sensitive to their molecular design, including donor and acceptor proximity and pairing, DNA stem-loop structure, and conjugation chemistry. We demonstrate the impact of these design features using a supported lipid bilayer model of podosome-like adhesions. Finally, we discuss the requirements for tension imaging in various biophysical contexts and offer a series of experimental recommendations, thus providing a guide for the design and application of DNA hairpin-based molecular tension probes.

A Broccoli aptamer chimera yields a fluorescent K+ sensor spanning physiological concentrations.

The RNA aptamer Broccoli accepts 2'fluorinated (2'F) pyrimidine nucleotide incorporation without perturbation of structure or fluorescence in the presence of potassium and DFHBI. However, the modification decreases Broccoli's apparent affinity for K+ >30-fold. A chimera of Broccoli RNAs with mixed chemistries displays linear fluorescent gain spanning physiological K+ concentrations, yielding an effective RNA-based fluorescent K+ sensor.

Examining the Protein Interactome and Subcellular Localization of RNase J2 Complexes in Streptococcus mutans.

Regulated RNA turnover is vital for the control of gene expression in all cellular life. In Escherichia coli, this process is largely controlled by a stable degradosome complex containing RNase E and a variety of additional enzymes. In the Firmicutes phylum, species lack RNase E and often encode the paralogous enzymes RNase J1 and RNase J2. Unlike RNase J1, surprisingly little is known about the regulatory function and protein interactions of RNase J2, despite being a central pleiotropic regulator for the streptococci and other closely related organisms. Using crosslink coimmunoprecipitation in Streptococcus mutans, we have identified the major proteins found within RNase J2 protein complexes located in the cytoplasm and at the cell membrane. In both subcellular fractions, RNase J2 exhibited the most robust interactions with RNase J1, while additional transient and/or weaker "degradosome-like" interactions were also detected. In addition, RNase J2 exhibits multiple novel interactions that have not been previously reported for any RNase J proteins, some of which were highly biased for either the cytoplasmic or membrane fractions. We also determined that the RNase J2 C-terminal domain (CTD) encodes a structure that is likely conserved among RNase J enzymes and may have an analogous function to the C-terminal portion of RNase E. While we did observe a number of parallels between the RNase J2 interactome and the E. coli degradosome paradigm, our results suggest that S. mutans degradosomes are either unlikely to exist or are quite distinct from those of E. coli.

A ribose modification of Spinach aptamer accelerates lead(ii) cation association in vitro.

Spinach aptamer fluorescence requires formation of a tripartite complex composed of folded RNA, a GFP-like fluorophore, and selective cation coordination. 2'F pyrimidine modified Spinach has retained fluorescence, increased chemical stability, and accelerated cation association via increased G-quadruplex dynamics, thereby reducing readout time and enhancing Spinach utility for aqueous Pb2+ detection.