Characterization of opposing responses to phenol by Bacillus subtilis chemoreceptors

Bacillus subtilis employs ten chemoreceptors to move in response to chemicals in its environment. While the sensing mechanisms have been determined for many attractants, little is known about the sensing mechanisms for repellents. In this work, we investigated phenol chemotaxis in B. subtilis . Phenol is an attractant at low, micromolar concentrations, and a repellent at high, millimolar concentrations. McpA was found to be the principal chemoreceptor governing the repellent response to phenol and other related aromatic compounds. In addition, the chemoreceptors McpC and HemAT were found to govern the attractant response to phenol and related compounds. Using chemoreceptor chimeras, McpA was found to sense phenol using its signaling domain rather than its sensing domain. These observations were substantiated in vitro, where direct binding of phenol to the signaling domain of McpA was observed using saturation-transfer difference nuclear magnetic resonance. These results further advance our understanding of B. subtilis chemotaxis and further demonstrate that the signaling domain of B. subtilis chemoreceptors can directly sense chemoeffectors. IMPORTANCE Bacterial chemotaxis is commonly thought to employ a sensing mechanism involving the extracellular sensing domain of chemoreceptors. Some ligands, however, appear to be sensed by the signaling domain. Phenolic compounds, commonly found in soil and root exudates, provide environmental cues for soil microbes like Bacillus subtilis . We show that phenol is sensed both as an attractant and a repellent. While the mechanism for sensing phenol as an attractant is still unknown, we found that phenol is sensed as a repellent by the signaling domain of the chemoreceptor McpA. This study furthers our understanding of the unconventional sensing mechanisms employed by the B. subtilis chemotaxis pathway.

Integrin α6β4 requires plectin and vimentin for adhesion complex distribution and invasive growth

Integrin α6β4 binds plectin to associate with vimentin; however, the biological function remains unclear. Here, we utilized various integrin β4 mutants and CRISPR-Cas9 editing to investigate this association. Upon laminin binding, integrin α6β4 distinctly distributed peripherally, and centrally proximal to the nucleus. Upon fibronectin addition, integrin α6β4 was centrally recruited to large focal adhesions (FAs) and enhanced Fak phosphorylation. Integrin β4 plectin-binding mutants or genetic deletion of plectin inhibited β4 recruitment to FAs and integrin α6β4-enhanced cell spreading, migration and three-dimensional invasive growth. Loss of the β4 signaling domain (but retains plectin binding) blocked migration and invasiveness but not cell spreading, recruitment to FAs or colony growth. Immunostaining revealed that integrin α6β4 redistributed vimentin perinuclearly where it colocalized with plectin and FAs. Depletion of vimentin completely blocked integrin β4-enhanced invasive growth, Fak phosphorylation and proliferation in three-dimensions but not two-dimensions. In summary, we demonstrate the essential roles of plectin and vimentin in promoting an invasive phenotype downstream of integrin α6β4.

Role of ADGRG1/GPR56 in Tumor Progression

Cellular communication plays a critical role in diverse aspects of tumorigenesis including tumor cell growth/death, adhesion/detachment, migration/invasion, angiogenesis, and metastasis. G protein-coupled receptors (GPCRs) which constitute the largest group of cell surface receptors are known to play fundamental roles in all these processes. When considering the importance of GPCRs in tumorigenesis, the adhesion GPCRs (aGPCRs) are unique due to their hybrid structural organization of a long extracellular cell-adhesive domain and a seven-transmembrane signaling domain. Indeed, aGPCRs have been increasingly shown to be associated with tumor development by participating in tumor cell interaction and signaling. ADGRG1/GPR56, a representative tumor-associated aGPCR, is recognized as a potential biomarker/prognostic factor of specific cancer types with both tumor-suppressive and tumor-promoting functions. We summarize herein the latest findings of the role of ADGRG1/GPR56 in tumor progression.

Multi-Omics Analyses Reveal the Regulatory Network and the Function of ZmUGTs in Maize Defense Response

Maize is one of the major crops in the world; however, diseases caused by various pathogens seriously affect its yield and quality. The maize Rp1-D21 mutant (mt) caused by the intragenic recombination between two nucleotide-binding, leucine-rich repeat (NLR) proteins, exhibits autoactive hypersensitive response (HR). In this study, we integrated transcriptomic and metabolomic analyses to identify differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in Rp1-D21 mt compared to the wild type (WT). Genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) were enriched among the DEGs. The salicylic acid (SA) pathway and the phenylpropanoid biosynthesis pathway were induced at both the transcriptional and metabolic levels. The DAMs identified included lipids, flavones, and phenolic acids, including 2,5-DHBA O-hexoside, the production of which is catalyzed by uridinediphosphate (UDP)-dependent glycosyltransferase (UGT). Four maize UGTs (ZmUGTs) homologous genes were among the DEGs. Functional analysis by transient co-expression in Nicotiana benthamiana showed that ZmUGT9250 and ZmUGT5174, but not ZmUGT9256 and ZmUGT8707, partially suppressed the HR triggered by Rp1-D21 or its N-terminal coiled-coil signaling domain (CCD21). None of the four ZmUGTs interacted physically with CCD21 in yeast two-hybrid or co-immunoprecipitation assays. We discuss the possibility that ZmUGTs might be involved in defense response by regulating SA homeostasis.

RIG-I uses its intrinsically disordered CARDs-Helicase linker in RNA proofreading functions

The innate immune receptor RIG-I provides the first line of defense against viral infections. Viral RNAs are recognized by the C-terminal domain (CTD) of RIG-I, but the RNA must engage the helicase domain to release the signaling domain CARDs from their autoinhibitory CARD2:Hel2i interactions. Because the helicase lacks RNA specificity, there must be mechanisms to proofread RNAs entering the helicase domain. Although such mechanisms are crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely unknown. This study reveals a previously unknown proofreading mechanism that RIG-I uses to ensure the helicase engages RNAs chosen explicitly by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which uses its negatively charged regions to electrostatically antagonize incoming RNAs. In addition to RNA gating, CHL is essential in stabilizing the CARD2:Hel2i interface. The CHL and CARD2:Hel2i interface work together, establishing a tunable gating mechanism that allows CTD-chosen RNAs to bind into the helicase while blocking non-specific RNAs. With its critical regulatory functions, CHL represents a novel target for RIG-I-based therapeutics.

Characterization of opposing responses to phenol by Bacillus subtilis chemoreceptors

ABSTRACTBacillus subtilis employs ten chemoreceptors to move in response to chemicals in its environment. While the sensing mechanisms have been determined for many attractants, little is known about the sensing mechanisms for repellents. In this work, we investigated phenol chemotaxis in B. subtilis. Phenol is an attractant at low, micromolar concentrations, and a repellent at high, millimolar concentrations. McpA was found to be the principal chemoreceptor governing the repellent response to phenol and other related aromatic compounds. In addition, the chemoreceptors McpC and HemAT were found to the govern the attractant response to phenol and related compounds. Using receptor chimeras, McpA was found to sense phenol using its signaling domain rather than its sensing domain. These observations were substantiated in vitro, where direct binding of phenol to the signaling domain of McpA was observed using saturation-transfer difference nuclear magnetic resonance. These results further advance our understanding of B. subtilis chemotaxis and demonstrate that the signaling domain of B. subtilis chemoreceptors can directly sense chemoeffectors.IMPORTANCEBacterial chemotaxis is commonly thought to employ a sensing mechanism involving the extracellular sensing domain of chemoreceptors. Some ligands, however, appear to be sensed by the signaling domain. Phenolic compounds, commonly found in soil and root exudates, provide environmental cues for soil microbes like Bacillus subtilis. We show that phenol is sensed both as an attractant and a repellent. While mechanism for sensing phenol as an attractant is still unknown, we found that phenol is sensed as a repellent by the signaling domain of the chemoreceptor McpA. This study furthers our understanding of the unconventional sensing mechanisms employed by the B. subtilis chemotaxis pathway.

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

Chimeric antigen receptors (CARs) consist of an extracellular antigen-binding region fused to intracellular signaling domains, thus enabling customized T cell responses against target cells. Due to the low-throughput process of systematically designing and functionally testing CARs, only a small set of immune signaling domains have been thoroughly explored, despite their major role in T cell activation, effector function and persistence. Here, we present speedingCARs, an integrated method for engineering CAR T cells by signaling domain shuffling and functional screening by single-cell sequencing. Leveraging the inherent modularity of natural signaling domains, we generated a diverse library of 180 unique CAR variants, which were genomically integrated into primary human T cells by CRISPR-Cas9. Functional and pooled screening of the CAR T cell library was performed by co-culture with tumor cells, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), thus enabling high-throughput profiling of multi-dimensional cellular responses. This led to the discovery of several CAR variants that retained the ability to kill tumor cells, while also displaying diverse transcriptional signatures and T cell phenotypes. In summary, speedingCARs substantially expands and characterizes the signaling domain combinations suited for CAR design and supports the engineering of next-generation T cell therapies.

CARPOOL: A library-based platform to rapidly identify next generation chimeric antigen receptors

CD19-targeted CAR therapies have successfully treated B cell leukemias and lymphomas, but many responders later relapse or experience toxicities. CAR intracellular domains (ICDs) are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3𝛇, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domain combinations that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.

The Molecular Mechanism of Fludioxonil Action Is Different to Osmotic Stress Sensing

The group III two-component hybrid histidine kinase MoHik1p in the filamentous fungus Magnaporthe oryzae is known to be a sensor for external osmotic stress and essential for the fungicidal activity of the phenylpyrrole fludioxonil. The mode of action of fludioxonil has not yet been completely clarified but rather assumed to hyperactivate the high osmolarity glycerol (HOG) signaling pathway. To date, not much is known about the detailed molecular mechanism of how osmotic stress is detected or fungicidal activity is initiated within the HOG pathway. The molecular mechanism of signaling was studied using a mutant strain in which the HisKA signaling domain was modified by an amino acid change of histidine H736 to alanine A736. We found that MoHik1pH736A is as resistant to fludioxonil but not as sensitive to osmotic stress as the null mutant ∆Mohik1. H736 is required for fludioxonil action but is not essential for sensing sorbitol stress. Consequently, this report provides evidence of the difference in the molecular mechanism of fludioxonil action and the perception of osmotic stress. This is an excellent basis to understand the successful phenylpyrrole-fungicides’ mode of action better and will give new ideas to decipher cellular signaling mechanisms.