Molecular handcraft of a well‐folded protein chimera

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Molecular handcraft of a well-folded protein chimera

Discovering new proteins through modular assembly, inspired by both nature's evolution and protein engineering, offers exciting possibilities. By leveraging information on subdomain-sized fragments from the database Fuzzle, we seamlessly integrated a flavodoxin-like fragment into a periplasmic binding protein. The resulting chimera exhibits remarkable folding and stable interfaces, showcasing the adaptability of α/β-proteins. Our work pioneers novel avenues in protein engineering and sheds light on the evolutionary origins of periplasmic binding proteins.


Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous ‘copy-paste’ processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/β-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.

Global transcriptome analysis reveals Salmonella Typhimurium employs nitrate metabolism to combat bile stress

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Global transcriptome analysis reveals Salmonella Typhimurium employs nitrate metabolism to combat bile stress

Our study showed that bile-mediated modulation of bacterial metabolism involves induction of various metabolic processes, e.g., anaerobic respiration dependent on nitrate. We observed that the activation of the nitrate metabolism-related genes fnr and narL is notably higher in the bile-tolerant WT strain compared to the bile-sensitive ΔcspE strain. Consequently, the WT strain displays lower amounts of reactive oxygen species and higher survival compared to the ΔcspE strain during bile stress.


Salmonella Typhimurium is an enteric pathogen that is highly tolerant to bile. Next-generation mRNA sequencing was performed to analyze the adaptive responses to bile in two S. Typhimurium strains: wild type (WT) and a mutant lacking cold shock protein E (ΔcspE). CspE is an RNA chaperone which is crucial for survival of S. Typhimurium during bile stress. This study identifies transcriptional responses in bile-tolerant WT and bile-sensitive ΔcspE. Upregulation of several genes involved in nitrate metabolism was observed, including fnr, a global regulator of nitrate metabolism. Notably, Δfnr was susceptible to bile stress. Also, complementation with fnr lowered reactive oxygen species and enhanced the survival of bile-sensitive ΔcspE. Importantly, intracellular nitrite amounts were highly induced in bile-treated WT compared to ΔcspE. Also, the WT strain pre-treated with nitrate displayed better growth with bile. These results demonstrate that nitrate-dependent metabolism promotes adaptation of S. Typhimurium to bile.

Constraints and solutions for development and uptake of integrated pest management in the UK

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Constraints and solutions for development and uptake of integrated pest management in the UK

Integrated pest management (IPM) yields agricultural improvements that support environmental aims. The IPM innovation system develops exciting products but encounters significant challenges in bringing them to market. This article reports the nine key recommendations emerging from an Association of Applied Biologists conference ‘Bringing Biocontrol and IPM to Market’, at which delegates from four key agricultural industry sectors discussed how to establish an effective IPM innovation system that links multiple stakeholders.


Abstract

Agricultural improvements that reduce conventional pesticide use and support environmental aims are a priority. Current approaches develop promising alternative products but meet significant challenges in bringing them to market. This article reports findings of an Association of Applied Biologists event at which delegates from relevant industry sectors discussed the establishment of an effective integrated pest management innovation system linking multiple stakeholders. Interrelated recommendations were agreed upon, focused on structured gap analysis, co-design processes reflecting the complete innovation system, the approval process, application equipment, enhancing grower confidence, integrating knowledge exchange activities, promulgation of public good information and the need for an overarching national action plan and supporting legislation.

Inflammasomes in epithelial innate immunity: front line warriors

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Inflammasomes in epithelial innate immunity: front line warriors

Strategically placed, our epithelium plays an essential role in maintaining and guarding tissue homeostasis. One key set of defense mechanisms is represented by our inflammasomes, which sense and respond to pathogen attack, through the secretion of pro-inflammatory cytokines. This review will focus on how the inflammasomes, our front-line warriors, maintain our skin, respiratory, and gut epithelial barrier immunity.


Our epithelium represents a battle ground against a variety of insults including pathogens and danger signals. It encodes multiple sensors that detect and respond to such insults, playing an essential role in maintaining and defending tissue homeostasis. One key set of defense mechanisms is our inflammasomes which drive innate immune responses including, sensing and responding to pathogen attack, through the secretion of pro-inflammatory cytokines and cell death. Identification of physiologically relevant triggers for inflammasomes has greatly influenced our ability to decipher the mechanisms behind inflammasome activation. Furthermore, identification of patient mutations within inflammasome components implicates their involvement in a range of epithelial diseases. This review will focus on exploring the roles of inflammasomes in epithelial immunity and cover: the diversity and differential expression of inflammasome sensors amongst our epithelial barriers, their ability to sense local infection and damage and the contribution of the inflammasomes to epithelial homeostasis and disease.

Macromolecular crowding has opposite effects on two critical sub‐steps of transcription initiation

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Macromolecular crowding has opposite effects on two critical sub-steps of transcription initiation

In this work, we study the effect of macromolecular crowding on transcription initiation and report a surprising result: while the first step of promoter opening slows down, the subsequent step of RNA synthesis and promoter escape becomes faster in the presence of crowders. This suggests that the crowded cellular environment significantly impacts gene function, with the effect varying between promoters.


Transcription initiation, the first step in gene expression, has been studied extensively in dilute buffer, a condition which fails to consider the crowded environment in live cells. Recent reports indicate the kinetics of promoter escape is altered in crowded conditions for a consensus bacterial promoter. Here, we use a real-time fluorescence enhancement assay to study the kinetics of unwound bubble formation and promoter escape for three separate promoters. We find that the effect of crowding on transcription initiation is complex, with lower rates of unwound bubble formation, higher rates of promoter escape, and large variations depending on promoter identity. Based on our results, we suggest that altered conditions of crowding inside a live cell can trigger global changes.

Phosphorylation of TG‐interacting factor 1 at carboxyl‐terminal sites in response to insulin regulates adipocyte differentiation

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Phosphorylation of TG-interacting factor 1 at carboxyl-terminal sites in response to insulin regulates adipocyte differentiation

TG-interacting factor 1 (TGIF1) contributes to the differentiation of white preadipocytes; however, its regulation is not well elucidated. We highlight that the insulin-induced ERK activation phosphorylates the T235 or T239 residue of TGIF1, which is crucial for the promotion of mitotic clonal expansion and adipocyte differentiation.


TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27 kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.