Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based directed evolution platform in plants to ...evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for resistance to splicing inhibitors. SF3B1 mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of resistance to splicing inhibitors. Studies of the structural basis of the splicing inhibitor binding to SGRs corroborate the resistance phenotype. This directed evolution platform can be used to interrogate and evolve the molecular functions of key biomolecules and to engineer crop traits for improved performance and adaptation under climate change conditions.
Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic ...platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.
Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this “background” level of receptor phosphorylation is insufficient to effect a downstream ...response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling.
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► Grb2 is constitutively bound to FGFR2 prior to growth factor binding ► Grb2 binds FGFR2 as a dimer bringing two receptor molecules together ► The preformed FGFR2 dimer is active but incapable of effecting signal transduction ► Phosphorylation of Grb2 by FGFR2 releases it from the receptor and promotes signaling
Grb2 dimers prevent ligand-independent signaling downstream of the FGF receptor by occluding binding sites for signaling proteins. FGF binding leads to phosphorylation of Grb2, triggering its release from the receptor and initiation of signaling.
Solving the phase problem in protein X‐ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not ...uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co‐purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR‐based screening of crystallographic data against currently 62 known contaminants. The web‐based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for `crystallization and preliminary X‐ray analysis' publications. Thus, in addition to potentially saving X‐ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data.
A webserver, titled ContaMiner, has been established, which allows fast molecular‐replacement‐based screening of crystallographic data against a database (ContaBase) of currently 62 potential contaminants. ContaMiner enables systematic screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for `crystallization and preliminary X‐ray analysis' publications.
► Intramolecular and intermolecular interactions of FAK enable environment-specific functions. ► FAK is a scaffolding protein, with a phosphorylation switch between actions. ► Localization controls ...FAK enrichment, which controls FAK-dependent phosphorylation. ► Serine or tyrosine phosphorylations on FAK trigger different functions.
Focal adhesion kinase (FAK) has an astonishing number of ligands and functions, which enable it to contribute to embryonic development and human health. FAK can promote different effects in similar cellular environments or similar effects in different cellular environments. Recent advances in structural and cellular analysis of FAK are starting to reveal the interrelationships between the conformations, localizations, interactions, and functions of FAK. This review focuses on our emerging understanding of how the structural framework of FAK mechanistically allows it to integrate manifold stimuli into environment-specific functions.
The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for rapid and sensitive protein detection and quantification in simple and robust formats for widespread point-of-care ...applications. Here, we report on nanobody-functionalized organic electrochemical transistors with a modular architecture for the rapid quantification of single-molecule-to-nanomolar levels of specific antigens in complex bodily fluids. The sensors combine a solution-processable conjugated polymer in the transistor channel and high-density and orientation-controlled bioconjugation of nanobody-SpyCatcher fusion proteins on disposable gate electrodes. The devices provide results after 10 min of exposure to 5 μl of unprocessed samples, maintain high specificity and single-molecule sensitivity in human saliva and serum, and can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available. We used the sensors to detect green fluorescent protein, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) spike proteins, and for the COVID-19 screening of unprocessed clinical nasopharyngeal swab and saliva samples with a wide range of viral loads.
We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition ...to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A. DNA copy number changes resulted in recurrent 1q gain, MYCN amplification, LIN28B gain, and MIRLET7A loss. Unexpected germline variants involved PALB2 and CHEK2. Integrated analyses support two major classes of genetic changes that preserve the progenitor state and/or interrupt normal development.
Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a ...permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which—RAC1, PPP6C, and STK19—harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.
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► Landscape of driver mutations by exon sequencing of 121 melanoma tumor/normal pairs ► Method for detecting genes with driver mutations in high-mutation-rate setting ► PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2 are significantly mutated melanoma genes ► Signature spectrum of UV mutagenesis accounts for 46% of driver mutations found
A statistical approach for analyzing exome sequencing data differentiates between driver mutations and the abundant passenger mutations found in melanoma due to UV light exposure. Analysis of whole-exome sequence data from 121 tumors identifies six new melanoma genes and defines a landscape of driver mutations in this challenging malignancy.
Focal adhesion kinase (FAK) and its close paralogue, proline-rich tyrosine kinase 2 (PYK2), are key regulators of aggressive spreading and metastasis of cancer cells. While targeted small-molecule ...inhibitors of FAK and PYK2 have been found to have promising antitumor activity, their clinical long-term efficacy may be undermined by the strong capacity of cancer cells to evade anti-kinase drugs. In healthy cells, the expression and/or function of FAK and PYK2 is tightly controlled via modulation of gene expression, competing alternatively spliced forms, non-coding RNAs, and proteins that directly or indirectly affect kinase activation or protein stability. The molecular factors involved in this control are frequently deregulated in cancer cells. Here, we review the endogenous mechanisms controlling FAK and PYK2, and with particular focus on how these mechanisms could inspire or improve anticancer therapies.
Interleukin 17-producing helper T cells (TH 17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. ...We found that interleukin 26 (IL-26), a human TH 17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, TH 17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of TH 17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.
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