LTBP-2 acts as a novel marker in human heart failure – a preliminary study

Background: We have observed increased expression of latent TGF-β binding protein (LTBP)-2 mRNA in human failing hearts. This study was aimed to further confirm LTBP-2 act as a novel marker in human acute heart failure.

Methods and results: We demonstrated that median level of LTBP-2 in myocardial samples from heart failure patients was significantly elevated, and TGF-β1 significantly promoted LTBP-2 expression in neonatal rat cardiomyocytes. To investigate the potential of LTBP-2 as a biomarker to diagnose heart failure with reduced ejection fraction (HFREF), another cohort of 133 consecutive patients with dyspnea were enrolled. In receiver operating characteristic (ROC) curve analyses to detect HFREF, LTBP-2 achieved an area under curve (AUC) of 0.67 (95% confidence intervals (CI) 0.58–0.75), comparable to the diagnostic ability of NT-proBNP 0.68 (95% CI 0.59–0.77).

Conclusion: The serum LTBP-2 levels might act as a promising biomarker in HFREF.     

Ferritin acts as a target site for the snowdrop lectin （GNA） in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA （Galanthus nivalis agglutinin） has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor（s） responsible for binding of GNA in the larval midgut of a major lepidopteran pest （the cotton leafworm, Spodoptera littoralis） to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA-binding proteins and BLAST analyses revealed that the N-terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta （76% sequence identity）, Calpodes ethlius （80% sequence identity） and Bombyx mori （61% sequence identity）. Furthermore, the N-terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta （88% sequence identity）.     

Muscle injury-induced thymosin β4 acts as a chemoattractant for myoblasts

Thymosin β4 (Tβ4) is a major intracellular G-actin-sequestering peptide. There is increasing evidence to support important extracellular functions of Tβ4 related to angiogenesis, wound healing and cardiovascular regeneration. We investigated the expression of 'Tβ4' and 'thymosin β10', a closely related peptide, during skeletal muscle regeneration in mice and chemotactic responses of myoblasts to these peptides. The mRNA levels of 'Tβ4' and 'thymosin β10' were up-regulated in the early stage of regenerating muscle fibres and inflammatory haematopoietic cells in the injured skeletal muscles of mice. We found that both Tβ4 and its sulphoxized form significantly accelerated wound closure and increased the chemotaxis of C2C12 myoblastic cells. Furthermore, we showed that primary myoblasts and myocytes derived from muscle satellite cells of adult mice were chemoattracted to sulphoxized form of Tβ4. These data indicate that muscle injury enhances the local production of Tβ4, thereby promoting the migration of myoblasts to facilitate skeletal muscle regeneration.     

Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α

Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is?a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in?vitro as well as in?vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in?vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.     

The G protein Gαs acts as a tumor suppressor in sonic hedgehog signaling-driven tumorigenesis

G protein-coupled receptors (GPCRs) are critical players in tumor growth and progression. The redundant roles of GPCRs in tumor development confound effective treatment; therefore, targeting a single common signaling component downstream of these receptors may be efficacious. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Hyperactive Gα_s signaling can mediate tumor progression in some tissues; however, recent work in medulloblastoma and basal cell carcinoma revealed that Gα_s can also function as a tumor suppressor in neoplasms derived from ectoderm cells including neural and epidermal stem/progenitor cells. In these stem-cell compartments, signaling through Gα_s suppresses self-renewal by inhibiting the Sonic Hedgehog (SHH) and Hippo pathways. The loss of GNAS, which encodes Gα_s, leads to activation of these pathways, over-proliferation of progenitor cells, and tumor formation. Gα_s activates the cAMP-dependent protein kinase A (PKA) signaling pathway and inhibits activation of SHH effectors Smoothened-Gli. In addition, Gα_s-cAMP-PKA activation negatively regulates the Hippo pathway by blocking the NF2-LATS1/2-Yap signaling. In this review, we will address the novel function of the signaling network regulated by Gα_s in suppression of SHH-driven tumorigenesis and the therapeutic approaches that can be envisioned to harness this pathway to inhibit tumor growth and progression.     

ATP-sensitive K+ channel opener acts as a potent Cl− channel inhibitor in vascular smooth muscle cells

We describe the activation of a K⁺ current and inhibition of a Cl^– current by a cyanoguanidine activator of ATP-sensitive K⁺ channels (K_ATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of K_ATP was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nm to 1 m) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC₄(3)], 5 m). The U83757induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlylammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nm to 300 m) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where E _k=–82 mV and E _Cl=0 mV, the reversal potential of the U83757-sensitive current was approximately –70 mV in those experiments where an increase in membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a Cl^– channel blocker as well as a K⁺ channel opener. In experiments in which Cl^– current activation was specifically brought about by cellular swelling and performed in solutions where Cl^– was the major permeant ion, U83757 (300 nm to 300 m) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K⁺-selective current which is sensitive to K_ATP channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K⁺ channel openers in vascular smooth muscle may be due to both the inhibition of Cl^– currents as well as the activation of a K⁺-selective current.This work was supported in part by the following grants: PHS P01 DK44840 and GM36823 (D.J.N.). J.C.M. is an Established Investigator of the American Heart Association.     

Crystallin αB acts as a molecular guard in mouse decidualization: Regulation and function during early pregnancy

Although decidualization is crucial for the establishment of successful pregnancy, the molecular mechanism underlying decidualization remains poorly understood. Crystallin αB (CryAB), a small heat shock protein (sHSP), is up-regulated and phosphorylated in mouse decidua. In mouse primary endometrial stromal cells, CryAB is induced upon progesterone treatment via HIF1α. In addition, CryAB is strongly phosphorylated through the p38-MAPK pathway under stress or during in vitro decidualization. Knockdown of CryAB results in the increase of apoptosis of stromal cells and inhibits decidualization under oxidative or inflammatory stress. Our data indicate that CryAB protects decidualization against stress conditions.     

Balancing acts: the role of TGF-β in the mucosal immune system

The gastrointestinal mucosal immune system faces unique challenges in dealing not only with fed antigens but also both commensal and pathogenic bacteria. It is tasked with digesting, transporting and using nutritional antigens while protecting the host from pathogenic organisms. As such, mechanisms that mediate effective immunity and immune tolerance are active within the gut environment. To accomplish this, the mucosal immune system has evolved sophisticated mechanisms that safeguard the integrity of the mucosal barrier. Transforming growth factor-β (TGF-β) emerges as a key mediator, balancing the tolerogenic and immunogenic forces at play in the gut. In this review, we discuss the role of TGF-β in the generation and functioning of gut lymphocyte populations. We highlight recent findings, summarize controversies, outline remaining questions and provide our personal perspectives.     

Phosphorylation of the p68 subunit of Pol δ acts as a molecular switch to regulate its interaction with PCNA

DNA polymerase delta (Pol δ) is a central enzyme for eukaryotic DNA replication and repair. Pol δ is a complex of four subunits p125, p68, p50, and p12. The functional properties of Pol δ are largely determined by its interaction with its DNA sliding clamp PCNA (proliferating cellular nuclear antigen). The regulatory mechanisms that govern the association of Pol δ with PCNA are largely unknown. In this study, we identified S458, located in the PCNA-interacting protein (PIP-Box) motif of p68, as a phosphorylation site for PKA. Phosphomimetic mutation of S458 resulted in a decrease in p68 affinity for PCNA as well as the processivity of Pol δ. Our results suggest a role of phosphorylation of the PIP-motif of p68 as a molecular switch that dynamically regulates the functional properties of Pol δ.     

PPARα-LXR as a novel metabolostatic signalling axis in skeletal muscle that acts to optimize substrate selection in response to nutrient status

LXR (liver X receptor) and PPARα (peroxisome-proliferator-activated receptor α) are nuclear receptors that control the expression of genes involved in glucose and lipid homoeostasis. Using wild-type and PPARα-null mice fed on an LXR-agonist-supplemented diet, the present study analysed the impact of pharmacological LXR activation on the expression of metabolically important genes in skeletal muscle, testing the hypothesis that LXR activation can modulate PPAR action in skeletal muscle in a manner dependent on nutritional status. In the fed state, LXR activation promoted a gene profile favouring lipid storage and glucose oxidation, increasing SCD1 (stearoyl-CoA desaturase 1) expression and down-regulating PGC-1α (PPARγ co-activator-1α) and PDK4 (pyruvate dehydrogenase kinase 4) expression. PPARα deficiency enhanced LXR stimulation of SCD1 expression, and facilitated elevated SREBP-1 (sterol-regulatory-element-binding protein-1) expression. However, LXR-mediated down-regulation of PGC-1α and PDK4 was opposed and reversed by PPARα deficiency. During fasting, prior LXR activation augmented PPARα signalling to heighten FA (fatty acid) oxidation and decrease glucose oxidation by augmenting fasting-induced up-regulation of PGC-1α and PDK4 expression, effects opposed by PPARα deficiency. Starvation-induced down-regulation of SCD1 expression was opposed by antecedent LXR activation in wild-type mice, an effect enhanced further by PPARα deficiency, which may elicit increased channelling of FA into triacylglycerol to limit lipotoxicity. Our results also identified potential regulatory links between the protein deacetylases SIRT1 (sirtuin 1) and SIRT3 and PDK4 expression in muscle from fasted mice, with a requirement for PPARα. In summary, we therefore propose that a LXR-PPARα signalling axis acts as a metabolostatic regulatory mechanism to optimize substrate selection and disposition in skeletal muscle according to metabolic requirement.     

Deacetylated αβ-tubulin acts as a positive regulator of Rheb GTPase through increasing its GTP-loading

Ras homolog enriched in brain (Rheb) regulates diverse cellular functions by modulating its nucleotide-bound status. Although Rheb contains a high basal GTP level, the regulatory mechanism of Rheb is not well understood. In this study, we propose soluble αβ-tubulin acts as a constitutively active Rheb activator, which may explain the reason why Rheb has a high basal GTP levels. We found that soluble αβ-tubulin is a direct Rheb-binding protein and that its deacetylated form has a high binding affinity for Rheb. Modulation of both soluble and acetylated αβ-tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated αβ-tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated αβ-tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble αβ-tubulin is a novel type of positive regulator of Rheb and may play a role as a temporal regulator for Rheb during the cell cycle.     

Floral phenotypic plasticity as a buffering mechanism in the globeflower–fly mutualism

A buffering mechanism in co-evolutionary relationships could be to display phenotypic plasticity in response to environmental changes. In the nursery pollination mutualism between the European globeflower and its exclusive fly pollinators, adults feed and mate in flowers, and larvae develop feeding on seeds. Flower number and size influence fitness for both partners, and large flowers attract more flies. We tested floral plasticity in plants from two contrasting environments: a high-altitude heath and low- and intermediate-altitude meadow forests. High-altitude plants have single flowers, while meadow-forest plants sometimes have multiple flowers. Plants were grown for 3 years in a garden and supplied with eight times more nutrients than available in natural soils, given to controls. During the experiment, over 90% of all plants with excess nutrients flowered, while in controls, 40% (high-altitude) to 75–78% (meadow-forest) plants flowered. Excess nutrients stimulated 30% larger flowers, and in meadow-forest plants flower number increased 4.5–5 times. Flower number was only doubled in high-altitude plants. High-altitude plants displayed less plasticity, and possibly, a different genetic strategy involving meristem limitation.     

A specific amino acid residue in the catalytic site of dandelion polyphenol oxidases acts as ‘selector’ for substrate specificity

Key message

Successful site-directed mutagenesis combined with in silico modeling and docking studies for the first time offers experimental proof of the role of the ‘substrate selector’ residue in plant polyphenol oxidases.

Abstract

The plant and fungi enzymes responsible for tissue browning are called polyphenol oxidases (PPOs). In plants, PPOs often occur as families of isoenzymes which are differentially expressed, but little is known about their physiological roles or natural substrates. In a recent study that explored these structure–function relationships, the eleven known dandelion (Taraxacum officinale) PPOs were shown to separate into two different phylogenetic groups differing in catalytic cavity architecture, kinetic parameters, and substrate range. The same study proposed that the PPOs’ substrate specificity is controlled by one specific amino acid residue positioned at the entrance to the catalytic site: whereas group 1 dandelion PPOs possess a hydrophobic isoleucine (I) at position H_B2+1, group 2 PPOs exhibit a larger, positively charged arginine (R). However, this suggestion was only based on bioinformatic analyses, not experiments. To experimentally investigate this hypothesis, we converted group 1 ToPPO-2 and group 2 ToPPO-6 into PPO-2-I₂₄₄R and PPO-6-R₂₅₄I, respectively, and expressed them in E. coli. By performing detailed kinetic characterization and in silico docking studies, we found that replacing this single amino acid significantly changed the PPO’s substrate specificity. Our findings therefore proof the role of the ‘substrate selector’ in plant PPOs.