Unconventional Intestinal Intraepithelial Lymphocytes in Health and Disease.

The intestinal epithelium is constantly exposed to a myriad of antigenic stimuli derived from commensals, food particles and pathogens present in the lumen of the intestines. This complex environment requires a similarly complex immune system capable of preventing exacerbated responses against food particles and commensals, while at the same time eliminating potential pathogens. These functions are accomplished in part by the intraepithelial lymphocyte (IEL) compartment. IELs are a diverse group of immune cells that primarily reside in between intestinal epithelial cells, maintaining an intimate association with these cells. IELs are a diverse population of cells: some of them express a T cell receptor (TCR), while others do not, and within TCR+ and TCR- IELs there are many IEL subpopulations that represent different developmental pathways and functions. In this review, we will focus on "unconventional" T cells present in the intestinal epithelium, in particular TCRγδ+, TCRαß+CD4+CD8αα+, and TCRαß+CD8αα+ IELs. We will discuss their development and potential functions both in humans and in mice.

Osteopontin and iCD8α Cells Promote Intestinal Intraepithelial Lymphocyte Homeostasis.

Intestinal intraepithelial lymphocytes (IEL) comprise a diverse population of cells residing in the epithelium at the interface between the intestinal lumen and the sterile environment of the lamina propria. Because of this anatomical location, IEL are considered critical components of intestinal immune responses. Indeed, IEL are involved in many different immunological processes, ranging from pathogen control to tissue stability. However, despite their critical importance in mucosal immune responses, very little is known about the homeostasis of different IEL subpopulations. The phosphoprotein osteopontin is important for critical physiological processes, including cellular immune responses, such as survival of Th17 cells and homeostasis of NK cells among others. Because of its impact in the immune system, we investigated the role of osteopontin in the homeostasis of IEL. In this study, we report that mice deficient in the expression of osteopontin exhibit reduced numbers of the IEL subpopulations TCRγδ+, TCRß+CD4+, TCRß+CD4+CD8α+, and TCRß+CD8αα+ cells in comparison with wild-type mice. For some IEL subpopulations, the decrease in cell numbers could be attributed to apoptosis and reduced cell division. Moreover, we show in vitro that exogenous osteopontin stimulates the survival of murine IEL subpopulations and unfractionated IEL derived from human intestines, an effect mediated by CD44, a known osteopontin receptor. We also show that iCD8α IEL but not TCRγδ+ IEL, TCRß+ IEL, or intestinal epithelial cells, can promote survival of different IEL populations via osteopontin, indicating an important role for iCD8α cells in the homeostasis of IEL.

Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids.

Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthetic pathway of aromatic amino acids, and a chorismate mutase (CM), functioning at a branch of this pathway leading to the synthesis of tyrosine and phenylalanine. In this study, we characterized this P. nigrescens enzyme and found that its two catalytic activities exhibit substantial hetero-interdependence and that the separation of its two distinct catalytic domains results in a dramatic loss of both DAH7PS and CM activities. The protein displayed a unique dimeric assembly, with dimerization solely via the CM domain. Small angle X-ray scattering (SAXS)-based structural analysis of this protein indicated a DAH7PS-CM hetero-interaction between the DAH7PS and CM domains, unlike the homo-association between DAH7PS domains normally observed for other DAH7PS proteins. This hetero-interaction provides a structural basis for the functional interdependence between the two domains observed here. Moreover, we observed that DAH7PS is allosterically inhibited by prephenate, the product of the CM-catalyzed reaction. This allostery was accompanied by a striking conformational change as observed by SAXS, implying that altering the hetero-domain interaction underpins the allosteric inhibition. We conclude that for this C-terminal CM-linked DAH7PS, catalytic function and allosteric regulation appear to be delivered by a common mechanism, revealing a distinct and efficient evolutionary strategy to utilize the functional advantages of a bifunctional enzyme.

Ion Transport Basis of Diarrhea in a Mouse Model of Adoptive T Cell Transfer Colitis.

BACKGROUND: Diarrhea, a major pathological hallmark of inflammatory bowel disease, is characterized by a significant reduction in the expression and function of key intestinal ion transporters. The adoptive naïve CD4+ T cell transfer colitis is an immune-based, chronic colitis mouse model which resembles human Crohn's disease. Although mice with T cell transfer colitis demonstrate diarrhea, the ion transporter basis of this phenotype has not been explored. AIMS/METHODS: In the current studies, we aimed to determine the mRNA and protein levels of the key NaCl transporters DRA and NHE3 along with the mRNA expression of other transporters in the inflamed intestine. RESULTS: Naïve CD4+ T cells, transferred to Rag2 knockout mice, induced severe colonic inflammation characterized by histological damage and increased mRNA levels of cytokines in the colon with no effect in the ileum. Diarrheal phenotype was a key feature of the excised colons of mice where loose stools were evident. Our results demonstrated that the key chloride transporter DRA, mRNA, and protein levels were significantly reduced in the inflamed colon. However, expression of the key sodium hydrogen exchanger NHE3 was unaffected. The mRNA expression of other important transporters was also determined; in this regard, the sodium channel ENACα and the monocarboxylate transporters MCT1 and SMCT1 mRNA levels were also significantly lower compared to control mice. However, CFTR mRNA was not altered in the colon or ileum. CONCLUSIONS: The studies conducted herein for the first time demonstrate the downregulation of important intestinal ion transporters in proximal and distal colon in T cell transfer colitis mouse model, providing valuable evidence for the ion transporter basis of diarrhea in this chronic model of inflammation.

A dimeric catalytic core relates the short and long forms of ATP-phosphoribosyltransferase.

Adenosine triphosphate (ATP) phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of histidine biosynthesis in plants and microorganisms. Two forms of ATP-PRT have been reported, which differ in their molecular architecture and mechanism of allosteric regulation. The short-form ATP-PRT is a hetero-octamer, with four HisG chains that comprise only the catalytic domains and four separate chains of HisZ required for allosteric regulation by histidine. The long-form ATP-PRT is homo-hexameric, with each chain comprising two catalytic domains and a covalently linked regulatory domain that binds histidine as an allosteric inhibitor. Here, we describe a truncated long-form ATP-PRT from Campylobacter jejuni devoid of its regulatory domain (CjeATP-PRTcore). Results showed that CjeATP-PRTcore is dimeric, exhibits attenuated catalytic activity, and is insensitive to histidine, indicating that the covalently linked regulatory domain plays a role in both catalysis and regulation. Crystal structures were obtained for CjeATP-PRTcore in complex with both substrates, and for the first time, the complete product of the reaction. These structures reveal the key features of the active site and provide insights into how substrates move into position during catalysis.

Interdomain Conformational Changes Provide Allosteric Regulation en Route to Chorismate.

Multifunctional proteins play a variety of roles in metabolism. Here, we examine the catalytic function of the combined 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated branch of the pathway for the biosynthesis of amino acids tyrosine and phenylalanine. In line with sequence predictions, the two catalytic functions are located in distinct domains, and these two activities can be separated and retain functionality. For the full-length protein, prephenate, the product of the CM reaction, acts as an allosteric inhibitor for the DAH7PS. The crystal structure of the full-length protein with prephenate bound and the accompanying small angle x-ray scattering data reveal the molecular mechanism of the allostery. Prephenate binding results in the tighter association between the dimeric CM domains and the tetrameric DAH7PS, occluding the active site and therefore disrupting DAH7PS function. Acquisition of a physical gating mechanism to control catalytic function through gene fusion appears to be a general mechanism for providing allostery for this enzyme.

Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both: AN EXAMPLE OF MOLECULAR SYMBIOSIS.

Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex formed by two pathway enzymes: chorismate mutase (CM) and 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Whereas both enzymes are active in isolation, the catalytic activity of both enzymes is enhanced, and in particular that of the much smaller CM is greatly enhanced (by 120-fold), by formation of a hetero-octameric complex between CM and DAH7PS. Moreover, on complex formation M. tuberculosis CM, which has no allosteric response on its own, acquires allosteric behavior to facilitate its own regulatory needs by directly appropriating and partly reconfiguring the allosteric machinery that provides a synergistic allosteric response in DAH7PS. Kinetic and analytical ultracentrifugation experiments demonstrate that allosteric binding of phenylalanine specifically promotes hetero-octameric complex dissociation, with concomitant reduction of CM activity. Together, DAH7PS and CM from M. tuberculosis provide exquisite control of aromatic amino acid biosynthesis, not only controlling flux into the start of the pathway, but also directing the pathway intermediate chorismate into either Phe/Tyr or Trp biosynthesis.

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αß T cells) and natural IEL (TCRαßCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαß+CD4-CD8-, TCRαß+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαß+CD4-CD8- and TCRαß+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1ß and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

Ion Transport Basis of Diarrhea, Paneth Cell Metaplasia, and Upregulation of Mechanosensory Pathway in Anti-CD40 Colitis Mice.

BACKGROUND: Anti-Cluster of differentiation (CD)-40-induced colitis, driven by innate inflammatory responses in the intestine, is a potent animal model exhibiting IBD pathophysiology including diarrhea. However, the ion transport basis of diarrhea and some key mucosal pathways (Paneth cells, stem cell niche, and mechanosensory) in this model have not been investigated. METHODS: Mucosal scrapings and intestinal tissue from control and CD40 antibody (150 µg) treated Rag2-/- mice were examined for gut inflammation, Paneth cell numbers, expression of key transporters, tight/adherens junction proteins, stem cell niche, and mechanosensory pathway via hematoxylin and eosin staining, quantitative polymerase chain reaction, and western blotting. RESULTS: Compared with control, anti-CD40 antibody treatment resulted in a significant loss of body weight (P < .05) and diarrhea at day 3 postinjection. Distal colonic tissues of anti-CD40 mice exhibited increased inflammatory infiltrates, higher claudin-2 expression, and appearance of Paneth cell-like structures indicative of Paneth cell metaplasia. Significantly reduced expression (P < .005) of downregulated in adenoma (key Cl- transporter), P-glycoprotein/multidrug resistantance-1 (MDR1, xenobiotic transporter), and adherens junction protein E-cadherin (~2-fold P < .05) was also observed in the colon of anti-CD40 colitis mice. Interestingly, there were also marked alterations in the stem cell markers and upregulation of the mechanosensory YAP-TAZ pathway, suggesting the activation of alternate regeneration pathway post-tissue injury in this model. CONCLUSION: Our data demonstrate that the anti-CD40 colitis model shows key features of IBD observed in the human disease, hence making it a suitable model to investigate the pathophysiology of ulcerative colitis (UC).

Our studies demonstrate the ion transport basis of diarrhea, downregulation of MDR1 and E-cadherin, Paneth cell metaplasia, and induction of claudin-2 and mechanosensory pathway in anti-CD40 colitis (innate immune-based model of IBD), similar to the human disease.

Recombinant expression, detergent solubilisation and purification of insect odorant receptor subunits.

Insect odorant receptors (ORs) are seven transmembrane domain proteins that comprise a novel family of ligand-gated non-selective cation channels. The functional channel is made up of an odour activated ligand-binding OR and the OR co-receptor, Orco. However, the structure, stoichiometry and mechanism of activation of the receptor complex are not well understood. Here we demonstrate that baculovirus-mediated Sf9 cell expression and wheat germ cell-free expression, but not Escherichia coli cell-based or cell-free expression, can be used successfully to over-express a selection of insect ORs. From a panel of 19 detergents, 1%w/v Zwittergent 3-16 was able to solubilise five Drosophila melanogaster ORs produced from both eukaryotic expression systems. A large-scale purification protocol was then developed for DmOrco and the ligand-binding receptor, DmOr22a. The proteins were nickel-affinity purified using a deca-histidine tag in a buffer containing 0.2 mM Zwittergent 3-16, followed by size exclusion chromatography. These purified ORs appear to form similarly sized protein-detergent complexes when isolated from both expression systems. Circular dichroism analysis of both purified proteins suggests they are folded correctly. We also provide evidence that when DmOrco is expressed in Sf9 cells it undergoes post translational modification, probably glycosylation. Finally we show that the recombinant ORs can be incorporated into pre-formed liposomes. The ability to recombinantly express and purify insect ORs to homogeneity on a preparative scale, as well as insert them into liposomes, is a major step forward in enabling future structural and functional studies, as well as their use in OR based biosensors.

Visual-Tactile Perception of Biobased Composites.

Biobased composites offer unique properties in the context of sustainable material production as well as end-of-life disposal, which places them as viable alternatives to fossil-fuel-based materials. However, the large-scale application of these materials in product design is hindered by their perceptual handicaps and understanding the mechanism of biobased composite perception, and its constituents could pave the way to creating commercially successful biobased composites. This study examines the role of bimodal (visual and tactile) sensory evaluation in the formation of biobased composite perception through the Semantic Differential method. It is observed that the biobased composites could be grouped into different clusters based on the dominance and interplay of various senses in perception forming. Attributes such as Natural, Beautiful, and Valuable are seen to correlate with each other positively and are influenced by both visual and tactile characteristics of the biobased composites. Attributes such as Complex, Interesting, and Unusual are also positively correlated but dominated by visual stimuli. The perceptual relationships and components of beauty, naturality, and value and their constituent attributes are identified, along with the visual and tactile characteristics that influence these assessments. Material design leveraging these biobased composite characteristics could lead to the creation of sustainable materials that would be more attractive to designers and consumers.

The In Vitro Evaluation of Rooster Semen Pellets Frozen with Dimethylacetamide.

Sperm cryopreservation is an effective technique for conserving animal genetic diversity and transmitting superior genetic backgrounds, maintained via a non-invasive sampling and collection of huge quantities of sperm. Nevertheless, cryopreservation in avian species is not commercially viable because of the rooster sperm's susceptibility to damage. This study aims to estimate the impact of dimethylacetamide (DMA) as a cryoprotectant at different levels (3%, 6%, or 9%) on the post-thawed sperm quality, motility, antioxidant-biomarkers, and the expression of anti-freeze related genes. Semen samples were collected twice a week from twelve roosters aged 40 wk, weighing 3400 ± 70 g, and belonging to the Cairo-B2 chicken strain. Fresh semen samples were rapidly appraised, pooled, diluted with two volumes of a basic extender, and divided equally into three groups. The diluted groups were chilled at -20 °C for 7 min, then gently supplemented with 3, 6, or 9% pre-cooled DMA and equilibrated at 5 °C for a further 10 min. Semen pellets were formed by pipetting drops 7 cm above liquid nitrogen (LN2), which were then kept inside cryovials in the LN2. Thawing was performed 2 months later by taking 3-4 pellets of the frozen semen into a glass tube and warming it in a water bath for 8 s at 60 °C. The results showed that 3% DMA increased the proportion of total motile sperm, progressivity, viability, and plasma membrane integrity (%) compared to the 6% and 9% DMA groups. The lipid peroxidation and antioxidant enzyme activity were improved in the 3% group. At the same time, some anti-freeze-related genes' (including ras homolog family member A (RHOA), heat shock protein 70 (HSP70), and small nuclear ribonucleoprotein polypeptide A (SNRPA1)) expressions were upregulated within the 3% DMA group relative to other groups. In conclusion, the 3% DMA group maintained higher post-thawed sperm quality than the other tested groups.

S100P is a novel interaction partner and regulator of IQGAP1.

Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 µM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.

N-terminal acetylation of annexin A2 is required for S100A10 binding.

Annexin A2 (AnxA2), a Ca2+-regulated phospholipid binding protein involved in membrane-cytoskeleton contacts and membrane transport, exists in two physical states, as a monomer or in a heterotetrameric complex mediated by S100A10. Formation of the AnxA2-S100A10 complex is of crucial regulatory importance because only the complex is firmly anchored in the plasma membrane, where it functions in the plasma membrane targeting/recruitment of certain ion channels and receptors. The S100A10 binding motif is located in the first 12 residues of the AnxA2 N-terminal domain, but conflicting reports exist as to the importance of N-terminal AnxA2 acetylation with regard to S100A10 binding. We show here that AnxA2 is subject to N-terminal modification when expressed heterologously in Escherichia coli. Met1 is removed and Ser2 is acetylated, yielding the same modification as the authentic mammalian protein. Bacterially expressed and N-terminally acetylated AnxA2 binds S100A10 with an affinity comparable to AnxA2 from porcine tissue and is capable of forming the AnxA2-S100A10 heterotetramer. Complex formation is competitively inhibited by acetylated but not by non-acetylated peptides covering the N-terminal AnxA2 sequence. These results demonstrate that N-terminal acetylation of AnxA2 is required for S100A10 binding and that this common eukaryotic modification is also obtained upon expression in bacteria.

Swine unconventional T cells.

Pigs are important domestic livestock and a comprehensive understanding of their immune system is critical to improve swine vaccine efficacy. Pig models represent an excellent animal model for immunological studies because of their anatomical and physiological similarities to humans. A significant portion of pig immunological studies focused on characterizing the conventional T cell (Tconv) immune responses. These cells recognize peptides presented by major histocompatibility complex (MHC) proteins. In contrast, unconventional T cells are non-MHC-restricted and profoundly regulate conventional T cells. Key subsets of unconventional T cells reviewed here include natural killer T (NKT) cells, γδ T cells, mucosal-associated invariant T (MAIT) cells, intraepithelial lymphocytes (IELs), and two potential unconventional T cell subsets expressing NKp46 or CD11b. Unlike Tconvs, most of these cells recognize lipids, small molecule metabolites, or modified peptides, and they generally show simplified patterns of T cell receptor (TCR) expression and rapid effector responses. Here, we review that unconventional T cells are an abundant and critical component of the porcine immune system, summarize the current understanding of these cells, and highlight some of the key differences among mouse, human, and porcine unconventional T cells.

Effect of Dimethylacetamide Concentration on Motility, Quality, Antioxidant Biomarkers, Anti-Freeze Gene Expression, and Fertilizing Ability of Frozen/Thawed Rooster Sperm.

Sperm cryopreservation is of great importance for the poultry industry but still needs to be optimized. The high susceptibility of poultry sperm to cryodamage leads to low fertility rates after cryopreservation. Therefore, the present study aimed at evaluating the effect of including a cryoprotectant, dimethylacetamide (DMA), in the chicken semen freezing extenders at a final concentration of 3%, 6%, or 9% on the post-thawed sperm motility, quality, antioxidant biomarkers, anti-freeze gene expression, and fertilizing ability. Results showed that the total motile sperm, progressivity, and viability were quadratically increased (p < 0.05) in the 6% DMA group. The antioxidant enzyme activity and lipid peroxidation were negatively (p < 0.05) affected by the increase in DMA concentration. Furthermore, some anti-freeze-associated genes such as heat shock protein 70 (HSP70) and ras homolog family member A (RHOA) were linearly and quadratically down-regulated (p < 0.05) with the high concentration of DMA. Finally, the fertility and hatchability rates did not indicate statistical differences between DMA groups. It can be concluded that using the low concentration of 3−6% DMA in the freezing semen extender is preferable to obtain acceptable results in the post-thawed sperm quality and fertility.

Granzyme B prevents aberrant IL-17 production and intestinal pathogenicity in CD4+ T cells.

CD4+ T cell activation and differentiation are important events that set the stage for proper immune responses. Many factors are involved in the activation and differentiation of T cells, and these events are tightly controlled to prevent unwanted and/or exacerbated immune responses that may harm the host. It has been well-documented that granzyme B, a potent serine protease involved in cell-mediated cytotoxicity, is readily expressed by certain CD4+ T cells, such as regulatory T cells and CD4+CD8αα+ intestinal intraepithelial lymphocytes, both of which display cytotoxicity associated with granzyme B. However, because not all CD4+ T cells expressing granzyme B are cytotoxic, additional roles for this protease in CD4+ T cell biology remain unknown. Here, using a combination of in vivo and in vitro approaches, we report that granzyme B-deficient CD4+ T cells display increased IL-17 production. In the adoptive transfer model of intestinal inflammation, granzyme B-deficient CD4+ T cells triggered a more rapid disease onset than their WT counterparts, and presented a differential transcription profile. Similar results were also observed in granzyme B-deficient mice infected with Citrobacter rodentium. Our results suggest that granzyme B modulates CD4+ T cell differentiation, providing a new perspective into the biology of this enzyme.

Generation and characterization of a novel, permanently active S100P mutant.

S100 proteins function as Ca2+ signal transducers by regulating cellular targets in their Ca2+ bound conformation. S100P is a member of the S100 protein family that can activate the membrane and F-actin binding protein ezrin in a Ca2+ dependent manner at least in vitro. Here we generated a novel tool to elucidate directly the S100P-ezrin interaction in vivo. This was achieved by constructing a S100P derivative that contained mutations in the two EF hand loops predicted to lock the protein in a permanently active state. The resulting S100P mutant, termed here S100P pa, could be purified as a soluble protein and showed biochemical properties displayed by wild-type S100P only in the presence of Ca2+. Importantly, S100P pa bound to the N-terminal domain of ezrin in the absence of Ca2+ showing an affinity only slightly reduced as compared to that of Ca2+-bound WT S100P. In line with this permanent complex formation, S100P pa colocalized with ezrin to plasma membrane protrusions of epithelial cells even in the absence of intracellular Ca2+ transients. Thus, S100P pa is a novel type of S100 protein mutant locked in a permanently active state that shows an unregulated complex formation with its cellular target ezrin.

Manipulating intradiol dioxygenases by C-terminus truncation.

Intradiol dioxygenases (EC 1.13.11.1) are bacterial enzymes that catalyze the ring cleavage of catechols which is a central step in the aerobic degradation of aromatic compounds. Some members of this enzyme group have a C-terminus which is 4-5% longer (an additional 13-18 amino acids) compared to the majority of known sequences. The longer C-terminus itself is not highly conserved and appears to be poorly integrated in the protein structural models developed for representative intradiol dioxygenases. Using a protein engineering approach variant intradiol dioxygenases were produced by truncating the C-terminus to a size comparable to the shorter versions of the enzyme. Three enzymes were selected and were originally described from the model organisms; Burkholderia xenovorans LB400, Pseudomonas putida KT2440 and Acinetobacter baylyi ADP1. The activity of the truncated enzymes were compared to the unmodified enzymes which revealed that truncation of the C-terminus could alter the enzyme activity; increasing the LB400 enzyme activity by as much as five fold, but reducing the activity of the intradiol dioxygenases from KT2440 and ADP1. The difference in effect is explained by the presence of a greater number of amino acid residues that can contribute to forming stable protein structures in the KT2440 and ADP1 enzymes. It is hypothesized that C-terminal truncation could in some cases provide a useful strategy for increasing intradiol dioxygenase activity for biotechnological production of muconic and adipic acids.

Innate CD8αα+ cells promote ILC1-like intraepithelial lymphocyte homeostasis and intestinal inflammation.

Innate CD8αα+ cells, also referred to as iCD8α cells, are TCR-negative intraepithelial lymphocytes (IEL) possessing cytokine and chemokine profiles and functions related to innate immune cells. iCD8α cells constitute an important source of osteopontin in the intestinal epithelium. Osteopontin is a pleiotropic cytokine with diverse roles in bone and tissue remodeling, but also has relevant functions in the homeostasis of immune cells. In this report, we present evidence for the role of iCD8α cells in the homeostasis of TCR-negative NKp46+NK1.1+ IEL (ILC1-like). We also show that the effect of iCD8α cells on ILC1-like IEL is enhanced in vitro by osteopontin. We show that in the absence of iCD8α cells, the number of NKp46+NK1.1+ IEL is significantly reduced. These ILC1-like cells are involved in intestinal pathogenesis in the anti-CD40 mouse model of intestinal inflammation. Reduced iCD8α cell numbers results in a milder form of intestinal inflammation in this disease model, whereas treatment with osteopontin increases disease severity. Collectively, our results suggest that iCD8α cells promote survival of NKp46+NK1.1+ IEL, which significantly impacts the development of intestinal inflammation.