Multifaceted role and regulation of aquaporins for efficient stomatal movements.

Stomata are micropores on the leaf epidermis that allow carbon dioxide (CO2) uptake for photosynthesis at the expense of water loss through transpiration. Stomata coordinate the plant gas exchange of carbon and water with the atmosphere through their opening and closing dynamics. In the context of global climate change, it is essential to better understand the mechanism of stomatal movements under different environmental stimuli. Aquaporins (AQPs) are considered important regulators of stomatal movements by contributing to membrane diffusion of water, CO2 and hydrogen peroxide. This review compiles the most recent findings and discusses future directions to update our knowledge of the role of AQPs in stomatal movements. After highlighting the role of subsidiary cells (SCs), which contribute to the high water use efficiency of grass stomata, we explore the expression of AQP genes in guard cells and SCs. We then focus on the cellular regulation of AQP activity at the protein level in stomata. After introducing their post-translational modifications, we detail their trafficking as well as their physical interaction with various partners that regulate AQP subcellular dynamics towards and within specific regions of the cell membranes, such as microdomains and membrane contact sites.

The Glycosaminoglycan Side Chains and Modular Core Proteins of Heparan Sulphate Proteoglycans and the Varied Ways They Provide Tissue Protection by Regulating Physiological Processes and Cellular Behaviour.

This review examines the roles of HS-proteoglycans (HS-PGs) in general, and, in particular, perlecan and syndecan as representative examples and their interactive ligands, which regulate physiological processes and cellular behavior in health and disease. HS-PGs are essential for the functional properties of tissues both in development and in the extracellular matrix (ECM) remodeling that occurs in response to trauma or disease. HS-PGs interact with a biodiverse range of chemokines, chemokine receptors, protease inhibitors, and growth factors in immune regulation, inflammation, ECM stabilization, and tissue protection. Some cell regulatory proteoglycan receptors are dually modified hybrid HS/CS proteoglycans (betaglycan, CD47). Neurexins provide synaptic stabilization, plasticity, and specificity of interaction, promoting neurotransduction, neurogenesis, and differentiation. Ternary complexes of glypican-1 and Robbo-Slit neuroregulatory proteins direct axonogenesis and neural network formation. Specific neurexin-neuroligin complexes stabilize synaptic interactions and neural activity. Disruption in these interactions leads to neurological deficits in disorders of functional cognitive decline. Interactions with HS-PGs also promote or inhibit tumor development. Thus, HS-PGs have complex and diverse regulatory roles in the physiological processes that regulate cellular behavior and the functional properties of normal and pathological tissues. Specialized HS-PGs, such as the neurexins, pikachurin, and Eyes-shut, provide synaptic stabilization and specificity of neural transduction and also stabilize the axenome primary cilium of phototoreceptors and ribbon synapse interactions with bipolar neurons of retinal neural networks, which are essential in ocular vision. Pikachurin and Eyes-Shut interactions with an α-dystroglycan stabilize the photoreceptor synapse. Novel regulatory roles for HS-PGs controlling cell behavior and tissue function are expected to continue to be uncovered in this fascinating class of proteoglycan.

Effects of Dietary Koumine on Growth Performance, Intestinal Morphology, Microbiota, and Intestinal Transcriptional Responses of Cyprinus carpio.

Gelsemium elegans Benth. (GEB) is a traditional medicinal plant in China, and acts as a growth promoter in pigs and goats. Koumine (KM) is the most abundant alkaloid in GEB and produces analgesic, anti-cancer, and immunomodulatory effects. KM can be used as an aquatic immune stimulant, but its growth-promoting effects and transcriptional mechanisms have not been investigated. Diets containing KM at 0, 0.2, 2, and 20 mg/kg were fed to Cyprinus carpio for 71 days to investigate its effects on growth performance, intestinal morphology, microflora, biochemical indicators, and transcriptional mechanisms. Cyprinus carpio fed with KM as the growth promoter, and the number of intestinal crypts and intestinal microbial populations were influenced by KM concentration. KM increased the abundance of colonies of Afipia, Phyllobacterium, Mesorhizobium, and Labrys, which were associated with compound decomposition and proliferation, and decreased the abundance of colonies of pathogenic bacteria Methylobacterium-Methylorubrum. A total of 376 differentially-expressed genes (DEGs) among the four experimental groups were enriched for transforming growth factor-ß1 and small mother against decapentaplegic (TGF-ß1/Smad), mitogen-activated protein kinase (MAPK), and janus kinases and signal transducers and activators of transcription (Jak/Stat) signaling pathways. In particular, tgfbr1, acvr1l, rreb-1, stat5b, smad4, cbp, and c-fos were up-regulated and positively correlated with KM dose. KM had a growth-promoting effect that was related to cell proliferation driven by the TGF-ß1/Smad, MAPK, and Jak/Stat signaling pathways. KM at 0.2 mg/kg optimized the growth performance of C. carpio, while higher concentrations of KM (2 and 20 mg/kg) may induce apoptosis without significantly damaging the fish intestinal structure. Therefore, KM at low concentration has great potential for development as an aquatic growth promotion additive.

Identification of the Regulatory Targets of miR-3687 and miR-4417 in Prostate Cancer Cells Using a Proteomics Approach.

MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter- and Intracellular Regulation.

Despite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter- and intracellular regulation. To achieve external regulation of T-cell/cancer cell interactions, we designed a DNAzyme-based molecular machine with an aptamer and an i-motif, as the MUC-1-selective aptamer allows the specific recognition of cancer cells. The i-motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T-cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme-based molecular machine with mitochondria-targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75-Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects.

The interplay between G protein-coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain.

Formation of biological condensates via phase separation: Characteristics, analytical methods, and physiological implications.

Liquid-liquid phase separation (LLPS) facilitates the formation of condensed biological assemblies with well-delineated physical boundaries, but without lipid membrane barriers. LLPS is increasingly recognized as a common mechanism for cells to organize and maintain different cellular compartments in addition to classical membrane-delimited organelles. Membraneless condensates have many distinct features that are not present in membrane-delimited organelles and that are likely indispensable for the viability and function of living cells. Malformation of membraneless condensates is increasingly linked to human diseases. In this review, we summarize commonly used methods to investigate various forms of LLPS occurring both in 3D aqueous solution and on 2D membrane bilayers, such as LLPS condensates arising from intrinsically disordered proteins or structured modular protein domains. We then discuss, in the context of comparisons with membrane-delimited organelles, the potential functional implications of membraneless condensate formation in cells. We close by highlighting some challenges in the field devoted to studying LLPS-mediated membraneless condensate formation.

Cellular sensing by phase separation: Using the process, not just the products.

Phase separation creates two distinct liquid phases from a single mixed liquid phase, like oil droplets separating from water. Considerable attention has focused on how the products of phase separation-the resulting condensates-might act as biological compartments, bioreactors, filters, and membraneless organelles in cells. Here, we expand this perspective, reviewing recent results showing how cells instead use the process of phase separation to sense intracellular and extracellular changes. We review case studies in phase separation-based sensing and discuss key features, such as extraordinary sensitivity, which make the process of phase separation ideally suited to meet a range of sensory challenges cells encounter.

Catalysis-dependent inactivation of human telomerase and its reactivation by intracellular telomerase-activating factors (iTAFs).

Human telomerase maintains genome stability by adding telomeric repeats to the ends of linear chromosomes. Although previous studies have revealed profound insights into telomerase functions, the low cellular abundance of functional telomerase and the difficulties in quantifying its activity leave its thermodynamic and kinetic properties only partially characterized. Employing a stable cell line overexpressing both the human telomerase RNA component and the N-terminally biotinylated human telomerase reverse transcriptase and using a newly developed method to count individual extension products, we demonstrate here that human telomerase holoenzymes contain fast- and slow-acting catalytic sites. Surprisingly, both active sites became inactive after two consecutive rounds of catalysis, named single-run catalysis. The fast active sites turned off ∼40-fold quicker than the slow ones and exhibited higher affinities to DNA substrates. In a dimeric enzyme, the two active sites work in tandem, with the faster site functioning before the slower one, and in the monomeric enzyme, the active sites also perform single-run catalysis. Interestingly, inactive enzymes could be reactivated by intracellular telomerase-activating factors (iTAFs) from multiple cell types. We conclude that the single-run catalysis and the iTAF-triggered reactivation serve as an unprecedented control circuit for dynamic regulation of telomerase. They endow native telomerase holoenzymes with the ability to match their total number of active sites to the number of telomeres they extend. We propose that the exquisite kinetic control of telomerase activity may play important roles in both cell division and cell aging.

The priming factor CAPS1 regulates dense-core vesicle acidification by interacting with rabconnectin3ß/WDR7 in neuroendocrine cells.

Vacuolar-type H+-ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways. Dense-core vesicles (DCVs) in neuroendocrine cells are maintained at an acidic pH, which is part of the electrochemical driving force for neurotransmitter loading and is required for hormonal propeptide processing. Genetic loss of CAPS1 (aka calcium-dependent activator protein for secretion, CADPS), a vesicle-bound priming factor required for DCV exocytosis, dissipates the pH gradient across DCV membranes and reduces neurotransmitter loading. However, the basis for CAPS1 binding to DCVs and for its regulation of vesicle pH has not been determined. Here, MS analysis of CAPS1 immunoprecipitates from brain membrane fractions revealed that CAPS1 associates with a rabconnectin3 (Rbcn3) complex comprising Dmx-like 2 (DMXL2) and WD repeat domain 7 (WDR7) proteins. Using immunofluorescence microscopy, we found that Rbcn3α/DMXL2 and Rbcn3ß/WDR7 colocalize with CAPS1 on DCVs in human neuroendocrine (BON) cells. The shRNA-mediated knockdown of Rbcn3ß/WDR7 redistributed CAPS1 from DCVs to the cytosol, indicating that Rbcn3ß/WDR7 is essential for optimal DCV localization of CAPS1. Moreover, cell-free experiments revealed direct binding of CAPS1 to Rbcn3ß/WDR7, and cell assays indicated that Rbcn3ß/WDR7 recruits soluble CAPS1 to membranes. As anticipated by the reported association of Rbcn3 with V-ATPase, we found that knocking down CAPS1, Rbcn3α, or Rbcn3ß in neuroendocrine cells impaired rates of DCV reacidification. These findings reveal a basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity via Rbcn3ß/WDR7 interactions.

The intramembrane protease SPP impacts morphology of the endoplasmic reticulum by triggering degradation of morphogenic proteins.

The endoplasmic reticulum (ER), as a multifunctional organelle, plays crucial roles in lipid biosynthesis and calcium homeostasis as well as the synthesis and folding of secretory and membrane proteins. Therefore, it is of high importance to maintain ER homeostasis and to adapt ER function and morphology to cellular needs. Here, we show that signal peptide peptidase (SPP) modulates the ER shape through degradation of morphogenic proteins. Elevating SPP activity induces rapid rearrangement of the ER and formation of dynamic ER clusters. Inhibition of SPP activity rescues the phenotype without the need for new protein synthesis, and this rescue depends on a pre-existing pool of proteins in the Golgi. With the help of organelle proteomics, we identified certain membrane proteins to be diminished upon SPP expression and further show that the observed morphology changes depend on SPP-mediated cleavage of ER morphogenic proteins, including the SNARE protein syntaxin-18. Thus, we suggest that SPP-mediated protein abundance control by a regulatory branch of ER-associated degradation (ERAD-R) has a role in shaping the early secretory pathway.

A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases.

Accumulating evidence indicates that a wide range of E3 ubiquitin ligases are involved in the development of many human diseases. Searching for small-molecule modulators of these E3 ubiquitin ligases is emerging as a promising drug discovery strategy. Here, we report the development of a cell-based high-throughput screening method to identify modulators of E3 ubiquitin ligases by integrating the ubiquitin-reference technique (URT), based on a fusion protein of ubiquitin located between a protein of interest and a reference protein moiety, with a Dual-Luciferase system. Using this method, we screened for small-molecule modulators of SMAD ubiquitin regulatory factor 1 (SMURF1), which belongs to the NEDD4 family of E3 ubiquitin ligases and is an attractive therapeutic target because of its roles in tumorigenesis. Using RAS homolog family member B (RHOB) as a SMURF1 substrate in this screen, we identified a potent SMURF1 inhibitor and confirmed that it also blocks SMURF1-dependent degradation of SMAD family member 1 (SMAD1) and RHOA. An in vitro auto-ubiquitination assay indicated that this compound inhibits both SMURF1 and SMURF2 activities, indicating that it may be an antagonist of the catalytic activity of the HECT domain in SMURF1/2. Moreover, cell functional assays revealed that this compound effectively inhibits protrusive activity in HEK293T cells and blocks transforming growth factor ß (TGFß)-induced epithelial-mesenchymal transition (EMT) in MDCK cells, similar to the effects on these processes caused by SMURF1 loss. In summary, the screening approach presented here may have great practical potential for identifying modulators of E3 ubiquitin ligases.

Subcutaneous adipose tissue imaging of human obesity reveals two types of adipocyte membranes: Insulin-responsive and -nonresponsive.

In adipose tissue, resistance to insulin's ability to increase glucose uptake can be induced by multiple factors, including obesity. Impaired insulin action may take place at different spatial loci at the cellular or subcellular level. To begin to understand the spatial response to insulin in human subcutaneous adipose tissue (hSAT), we developed a quantitative imaging method for activation of a major signaling node in the glucoregulatory insulin signaling pathway. After treatment with insulin or control media, biopsied tissues were immunostained for Akt phosphorylation at Thr-308/9 (pAkt) and then imaged by confocal fluorescence microscopy automated to collect a large grid of high resolution fields. In hSAT from 40 men and women with obesity, substantial heterogeneity of pAkt densities in adipocyte membranes were quantified in each image mosaic using a spatial unit of at least twice the size of the point spread function. Statistical analysis of the distribution of pAkt spatial units was best fit as the weighted sum of two separate distributions, corresponding to either a low or high pAkt density. A "high pAkt fraction" metric was calculated from the fraction of high pAkt distributed units over the total units. Importantly, upon insulin stimulation, tissues from the same biopsy showed either a minimal or a substantial change in the high pAkt fraction. Further supporting a two-state response to insulin stimulation, subjects with similar insulin sensitivity indices are also segregated into either of two clusters identified by the amount of membrane-localized pAkt.

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines.

The GalNAc-type O-glycoproteome is orchestrated by a large family of polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with partially overlapping contributions to the O-glycoproteome besides distinct nonredundant functions. Increasing evidence indicates that individual GalNAc-Ts co-regulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of GalNAc-T specificities have mainly been performed with in vitro enzyme assays using short peptide substrates, but recently quantitative differential O-glycoproteomics of isogenic cells with and without GALNT genes has enabled a more unbiased exploration of the nonredundant contributions of individual GalNAc-Ts. Both approaches suggest that fairly small subsets of O-glycosites are nonredundantly regulated by specific GalNAc-Ts, but how these isoenzymes orchestrate regulation among competing redundant substrates is unclear. To explore this, here we developed isogenic cell model systems with Tet-On inducible expression of two GalNAc-T genes, GALNT2 and GALNT11, in a knockout background in HEK293 cells. Using quantitative O-glycoproteomics with tandem-mass-tag (TMT) labeling, we found that isoform-specific glycosites are glycosylated in a dose-dependent manner and that induction of GalNAc-T2 or -T11 produces discrete glycosylation effects without affecting the major part of the O-glycoproteome. These results support previous findings indicating that individual GalNAc-T isoenzymes can serve in fine-tuned regulation of distinct protein functions.

ATP7A and ATP7B copper transporters have distinct functions in the regulation of neuronal dopamine-ß-hydroxylase.

The copper (Cu) transporters ATPase copper-transporting alpha (ATP7A) and ATPase copper-transporting beta (ATP7B) are essential for the normal function of the mammalian central nervous system. Inactivation of ATP7A or ATP7B causes the severe neurological disorders, Menkes disease and Wilson disease, respectively. In both diseases, Cu imbalance is associated with abnormal levels of the catecholamine-type neurotransmitters dopamine and norepinephrine. Dopamine is converted to norepinephrine by dopamine-ß-hydroxylase (DBH), which acquires its essential Cu cofactor from ATP7A. However, the role of ATP7B in catecholamine homeostasis is unclear. Here, using immunostaining of mouse brain sections and cultured cells, we show that DBH-containing neurons express both ATP7A and ATP7B. The two transporters are located in distinct cellular compartments and oppositely regulate the export of soluble DBH from cultured neuronal cells under resting conditions. Down-regulation of ATP7A, overexpression of ATP7B, and pharmacological Cu depletion increased DBH retention in cells. In contrast, ATP7B inactivation elevated extracellular DBH. Proteolytic processing and the specific activity of exported DBH were not affected by changes in ATP7B levels. These results establish distinct regulatory roles for ATP7A and ATP7B in neuronal cells and explain, in part, the lack of functional compensation between these two transporters in human disorders of Cu imbalance.

Regulation of articular chondrocyte catabolic genes by growth factor interaction.

Osteoarthritis is characterized by a loss of articular cartilage homeostasis in which degradation exceeds formation. Several growth factors have been shown to promote cartilage formation by augmenting articular chondrocyte anabolic activity. This study tests the hypothesis that such growth factors also play an anticatabolic role. We transferred individual or combinations of the genes encoding insulin-like growth factor-I, bone morphogenetic protein-2, bone morphogenetic protein-7, transforming growth factor-ß1, and fibroblast growth factor-2, into adult bovine articular chondrocytes and measured the expression of catabolic marker genes encoding A disintegrin and metalloproteinase with thrombospondin motifs-4 and -5, matrix metalloproteinases-3 and -13, and interleukin-6. When delivered individually, or in combination, these growth factor transgenes differentially regulated the direction, magnitude, and time course of expression of the catabolic marker genes. In concert, the growth factor transgenes regulated the marker genes in an interactive fashion that ranged from synergistic inhibition to synergistic stimulation. Synergistic stimulation prevailed over synergistic inhibition, reaching maxima of 15.2- and 2.7-fold, respectively. Neither the magnitude nor the time course of the effect of the transgene combinations could be predicted on the basis of the individual transgene effects. With few exceptions, the data contradict our hypothesis. The results demonstrate that growth factors that are traditionally viewed as chondrogenic tend also to promote catabolic gene expression. The competing actions of these potential therapeutic agents add an additional level of complexity to the selection of regulatory factors for restoring articular cartilage homeostasis or promoting repair.

Methionine in Proteins: It's Not Just for Protein Initiation Anymore.

Methionine in proteins is often thought to be a generic hydrophobic residue, functionally replaceable with another hydrophobic residue such as valine or leucine. This is not the case, and the reason is that methionine contains sulfur that confers special properties on methionine. The sulfur can be oxidized, converting methionine to methionine sulfoxide, and ubiquitous methionine sulfoxide reductases can reduce the sulfoxide back to methionine. This redox cycle enables methionine residues to provide a catalytically efficient antioxidant defense by reacting with oxidizing species. The cycle also constitutes a reversible post-translational covalent modification analogous to phosphorylation. As with phosphorylation, enzymatically-mediated oxidation and reduction of specific methionine residues functions as a regulatory process in the cell. Methionine residues also form bonds with aromatic residues that contribute significantly to protein stability. Given these important functions, alteration of the methionine-methionine sulfoxide balance in proteins has been correlated with disease processes, including cardiovascular and neurodegenerative diseases. Methionine isn't just for protein initiation.

HNF4α and CDX2 Regulate Intestinal YAP1 Promoter Activity.

The Hippo pathway is important for tissue homeostasis, regulation of organ size andgrowth in most tissues. The co-transcription factor yes-associated protein 1 (YAP1) serves as a maindownstream effector of the Hippo pathway and its dysregulation increases cancer development andblocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation ofYAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene andtranscription factors important for intestinal expression. Bioinformatic analysis of caudal typehomeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitatedDNA from differentiated Caco-2 cells revealed potential intragenic enhancers in the YAP1 gene.Transfection of luciferase-expressing YAP1 promoter-reporter constructs containing the potentialenhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco-2 and T84cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silicotranscription factor binding site analysis and protein-DNA binding was confirmed in vitro usingelectrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experimentsthat CDX2 and HNF4α bind to the YAP1 enhancer in Caco-2 cells. These results reveal a previouslyunknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for highexpression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding areimportant for the YAP1 enhancer activity in intestinal epithelial cells.

Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation.

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.