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Proteins of the trefoil factor family (TFF) participate in mucosal repair and are formed by single or tandemly repeated trefoil domains. TFFs have been extensively studied in mammals and amphibians, but they have not been functionally characterized in other animals. Here we report the identification of two genes expressed in the hydroid Hydractinia symbiolongicarpus, predicted to encode trefoil domain-containing peptides, one with four trefoil domains in tandem and the other one with a trefoil domain flanked by two ShKT domains. Differential expression analyses by qPCR after an immune challenge and an induced mechanical damage, reveal that the former gene (hysyTFF) had no significant changes in expression after the inductions. However, the latter (hysyTFF-like) was overexpressed after three hours post immune challenge and was downregulated after the first hour post epithelial damage. Immunoblot analyses using specific IgY antibodies revealed that hysyTFF is secreted as a high molecular weight complex. Finally, whole mount immunofluorescence assays showed that hysyTFF was predominantly expressed in the endoderm of stolons and polyps, and sparsely in the ectoderm of both polyps and larvae. Thus, the tissue distribution and expression dynamics of trefoil factor genes in H. symbiolongicarpus suggest that hysyTFF is part of an ancient mechanism of epithelial restitution, and the newly reported hysyTFF-like might act as an immune effector gene, perhaps encoding an antibacterial peptide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Trefoil factor family (TFF) peptides have been examined primarily in the gastrointestinal tract, where they play an important role in the epithelial regeneration. The therapeutic effects of TFFs, particularly the TFF3 protein, have been well studied in humans and in animal models of gastrointestinal injury, whereas little is known about their occurrence and function in the urinary bladder. In this study, we investigated the presence, location, and function of Tff3 in the urinary bladders of wild-type mice (Tff3 WT ) and compared them with Tff3 knockout mice (Tff3 KO ) using molecular and microscopic methods at the light and electron microscopic level. Our results show that Tff3 is expressed in the superficial cells of the urothelium, where it colocalizes with the uroplakin UP1b as one of the fundamental structural components of the apical plasma membrane, which is an important component of the blood-urine permeability barrier. Analysis of the urothelium with experimentally induced injury revealed that injury is more severe in Tff3 KO mice and urothelial regeneration is attenuated compared with Tff3 WT mice, suggesting that Tff3 plays a fine-tuned role in homeostasis and protection of the urothelium. This study provides the first data on the precise location and function of Tff3 in the bladder epithelium.  (J Histochem Cytochem XX. XXX-XXX, XXXX) ., Competing Interests: Competing InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Exocrine-to-endocrine cross talk in the pancreas is crucial to maintain β-cell function. However, the molecular mechanisms underlying this cross talk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of β-cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces β-cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of β-cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared with controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine β-cells in mice., (© 2024 by the American Diabetes Association.)

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl-terminal region of the (internally located) FN of the de novo designed purely-symmetric β-trefoil protein Symfoil-4P. This mutation involves wholesale replacement of a repeating trefoil-fold motif with a "blade" motif from a β-propeller protein, and postulated to trap that region of the Symfoil-4P FN in a nonproductive folding intermediate. The resulting protein (termed "Bladefoil") is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed., (© 2020 The Protein Society.)

Trefoil factor family peptides (TFF1, TFF2, TFF3) are typically co-secreted together with mucins. Tff1 represents a gastric tumor suppressor gene in mice. TFFs are also synthesized in minute amounts in the immune and central nervous systems. In mucous epithelia, they support rapid repair by enhancing cell migration ("restitution") via their weak chemotactic and anti-apoptotic effects. For a long time, as a paradigm, this was considered as their major biological function. Within recent years, the formation of disulfide-linked heterodimers was documented for TFF1 and TFF3, e.g., with gastrokine-2 and IgG Fc binding protein (FCGBP). Furthermore, lectin activities were recognized as enabling binding to a lipopolysaccharide of Helicobacter pylori (TFF1, TFF3) or to a carbohydrate moiety of the mucin MUC6 (TFF2). Only recently, gastric TFF1 was demonstrated to occur predominantly in monomeric forms with an unusual free thiol group. Thus, a new picture emerged, pointing to diverse molecular functions for TFFs. Monomeric TFF1 might protect the gastric mucosa as a scavenger for extracellular reactive oxygen/nitrogen species. Whereas, the TFF2/MUC6 complex stabilizes the inner layer of the gastric mucus. In contrast, the TFF3-FCGBP heterodimer (and also TFF1-FCGBP) are likely part of the innate immune defense of mucous epithelia, preventing the infiltration of microorganisms.

Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain-swapped arrangement at the interface of the N- and C-termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N- and C-termini in the overall architecture; however, the folding requirement of the primordial motif is poorly understood, and tolerance to circular permutation is essentially unknown. The β-trefoil protein fold is a threefold-symmetric architecture where the repeating ~42-mer "trefoil-fold" motif assembles via a domain-swapped arrangement. The trefoil-fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β-trefoil trimeric assembly. The trefoil-fold sequence is not predicted to adopt the trefoil-fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact "blade" motif from the β-propeller architecture. Expression of a trefoil-fold sequence and circular permutants shows that only the wild-type N-terminal motif definition yields an intact β-trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil-fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil-fold structural features, but is more structurally homologous to a β-propeller blade motif., (© 2020 The Protein Society.)

RUNX2, a master osteogenic transcript ion factor, is overexpressed in several cancer cells; in melanoma it promotes cells migration and invasion as well as neoangiogenesis. The annual mortality rates related to metastatic melanoma are high and novel agents are needed to improve melanoma patients' survival. It has been shown that lectins specifically target malignant cells since they present the Thomsen-Friedenreich antigen. This disaccharide is hidden in normal cells, while it allows selective lectins binding in transformed cells. Recently, an edible lectin named BEL β-trefoil has been obtained from the wild mushroom Boletus edulis . Our previous study showed BEL β-trefoil effects on transcription factor RUNX2 downregulation as well as on the migration ability in melanoma cells treated in vitro. Therefore, to better understand the role of this lectin, we investigated the BEL β-trefoil effects in a zebrafish in vivo model, transplanted with human melanoma cells expressing RUNX2. Our data showed that BEL β-trefoil is able to spread in the tissues and to reduce the formation of metastases in melanoma xenotransplanted zebrafish. In conclusion, BEL β-trefoil can be considered an effective biomolecule to counteract melanoma disease.

Mucosal healing plays a critical role in combatting the effects of inflammatory bowel disease, fistulae and ulcers. While most treatments for such diseases focus on systemically delivered anti-inflammatory drugs, often leading to detrimental side effects, mucosal healing agents that target the gut epithelium are underexplored. We genetically engineer Escherichia coli Nissle 1917 (EcN) to create fibrous matrices that promote gut epithelial integrity in situ. These matrices consist of curli nanofibers displaying trefoil factors (TFFs), known to promote intestinal barrier function and epithelial restitution. We confirm that engineered EcN can secrete the curli-fused TFFs in vitro and in vivo, and is non-pathogenic. We observe enhanced protective effects of engineered EcN against dextran sodium sulfate-induced colitis in mice, associated with mucosal healing and immunomodulation. This work lays a foundation for the development of a platform in which the in situ production of therapeutic protein matrices from beneficial bacteria can be exploited.

Trefoil factors (TFFs) are regulatory peptides playing critical roles in mucosal repair and protection against a variety of insults within the gastrointestinal tract. This work aimed to explore the effects of deoxynivalenol (DON) on intestinal TFFs expression using in vivo and in vitro models. In an animal trial, twenty-four 28-d-old barrows (Duroc × Landrace × Large White; initial body weight = 7.6 ± 0.7 kg) were randomly divided into three treatments for 28 days, including a control diet (0.61 mg DON/kg feed), and two levels of DON-contaminated diets containing 1.28 and 2.89 mg DON/kg feed, respectively. Piglets exposed to DON had lower mRNA expression of TFF1, TFF2, TFF3, as well as Claudin-4 in the intestine ( P < 0.05). Dietary DON exposure decreased the protein levels of TFF2 and TFF3 in the jejunum as demonstrated by western blot and immunohistochemistry. In intestinal porcine epithelial cells (IPEC-J2), DON depressed the mRNA expression of TFF2, TFF3, and Claudin-4. Overexpression of sterile alpha motif (SAM) pointed domain E26 transformation-specific (ETS) factor (SPDEF) was found to attenuate DON-induced suppression of TFFs in IPEC-J2 cells. Altogether, our work shows, for the first time, that dietary DON exposure depresses the expression of intestinal TFFs in piglets. Given the fundamental role of TFFs in intestinal mucosal homeostasis, our observations indicate that the DON content in animal feed should be strictly controlled based on the existing regulation for DON.

Probiotics are used in the management of some gastrointestinal diseases. However, little is known about their effects on normal gastric epithelial biology. The aim of this study was to explore how the probiotic mixture VSL#3 affects gastric cell lineages in mice with a special focus on protective and aggressive factors. Weight-matching littermate male mice ( n = 14) were divided into treated and control pairs. The treated mice received VSL#3 (5 mg/day/mouse) by gastric gavage for 10 days. Control mice received only the vehicle. Food consumption and bodyweight were monitored. All mice were injected intraperitoneally with bromodeoxyuridine (120 mg/Kg bodyweight) two hours before sacrificed to label S-phase cells. Stomach tissues were processed for lectin- and immunohistochemical examination. ImageJ software was used to quantify immunolabeled gastric epithelial cells. Real-time quantitative polymerase chain reaction was used to provide relative changes in expression of gastric cell lineages specific genes. Results revealed that treated mice acquired (i) increased production of mucus, trefoil factor (TFF) 1 and TFF2, (ii) decreased production of pepsinogen, and (iii) increased ghrelin-secreting cells. No significant changes were observed in bodyweight, food consumption, cell proliferation, or parietal cells. Therefore, VSL#3 administration amplifies specific cell types specialized in the protection of the gastric epithelium.

Trefoil factor family (TFF) peptides are produced rapidly at sites of injury, stimulating epithelial migration, a process involving rapid changes in cell shape and volume, requiring rapid flow of water into and out of the cell. We examined the effect of TFFs on fluidity of cells by measuring their sensitivity to osmotic challenges and cell migration, and determined whether those results were mediated through altering the levels of aquaporins (AQPs), a family of transmembrane water channels involved in cellular water homeostasis. Gastric (AGS) and colonic (Caco-2) cell lines had intrinsic TFF levels determined and the predominant TFF peptide knocked down (RNA interference). Knockdown caused lessened responsiveness to changes in external osmotic challenge (by 51 and 69% in AGS and Caco-2 cells, respectively) and reduced cell migration and transepithelial permeability but did not influence proliferation. Exogenous TFF increased several AQPs, particularly AQP3, and those were reciprocally reduced in knockdown cells. TFF-induced, but not fetal calf serum-induced, cell migration was inhibited by the presence of AQP3 blocker (CuSO 4 ). We summarize that TFF peptides promptly produced at sites of injury increase AQP levels, most notably AQP3, thereby enhancing the cells' ability to rapidly change their shape as part of the restitutive process. TFF peptides also require functioning AQP3 channels to induce cell migration.-Marchbank, T., Playford, R. J. Trefoil factor family peptides enhance cell migration by increasing cellular osmotic permeability and aquaporin 3 levels.