A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing

The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related.     

Adenosine activates thermogenic adipocytes

Brown or beige fat activation can cause potent anti-obesity and anti-diabetic effects. In a study recently published in Nature, Gnad et al. show that adenosine is a novel activator of brown and beige fat that acts through the A_2A receptor.Brown fat is a thermogenic type of adipose tissue containing abundant mitochondria and multilocular lipid droplets, and is uniquely suited and required for cold tolerance and body weight control¹. Upon brown fat activation, intracellular cyclic AMP (cAMP) levels rise rapidly, leading to lipolysis and upregulation of uncoupling protein 1 (UCP1) which initiates uncoupling of mitochondrial respiration and heat generation^2,3. Additionally, beige or brite fat is a form of white fat that can be “browned” and induced to engage in respiratory uncoupling similarly as brown fat. Activated brown and beige fat consume substantial amounts of metabolic substrate, leading to powerful anti-obesity and anti-diabetic effects in mice. Adult humans possess brown and beige fat, making these tissues potentially important targets for treating obesity and metabolic diseases⁴. Activation of brown and beige fat occurs through several mechanisms including cold, adrenergic signaling, and hormone signaling⁴. However, a critical question remains of which pathway is altered therapeutically, as targeting these known activation pathways may cause discomfort, have undesirable side effects, or be insufficient alone to exert anti-obesity effects in humans. Thus, the need to discover novel pathways for brown and beige fat thermogenic activation persists.Adenosine is a purine nucleoside that alters cAMP signaling in several tissues^5,6. There is a ubiquitous presence of adenosine in the extracellular space, and adenosine levels can be increased extracellularly or intracellularly through conversion of adenine nucleotides. Adenosine binds to four P1 G-protein-coupled receptor subtypes, which include the inhibitory receptors A₁ and A₃ and the stimulatory receptors A_2A and A_2B⁷. Inhibitory receptor activation leads to increased adenylate cyclase activity and decreased cAMP, while stimulatory receptor activation has the opposite effect. The distribution of the adenine receptor subtypes varies widely by tissue and species, and the response is dependent on the level of receptor expression, thus the actions of adenosine can be highly disparate depending on the tissue context⁸.In adipose tissue, adenosine was previously found to inhibit cAMP production in studies performed in hamster and rat, where oxygen consumption and lipolysis also decreased^9,10. In a study recently published in Nature, Gnad et al.¹¹ show that adenosine activates lipolysis and the thermogenic program in brown and white human and murine adipocytes (Figure 1). This phenomenon occurs at significantly lower concentrations of adenosine in brown adipocytes than in white adipocytes. The difference is accounted for by differential expression of the A_2A receptor, which is highly expressed in human and murine brown fat relative to white fat. It is also due to the ratio of expression compared to the inhibitory A₁ receptor, which is expressed at low levels in brown fat compared to white fat. Moreover, the authors show that hamster brown fat expresses almost equal amounts of A₁ and A_2A receptors, explaining the lack of activating effects of adenosine in those cells.Open in a separate windowFigure 1Adenosine activates brown or beige adipocytes through the A_2A receptor. Adenosine activates the A_2A receptor, leading to increased cAMP- and PGC-1α-dependent signaling that enhances thermogenesis and lipolysis in brown and beige adipocytes. Crosstalk occurs with adrenergic signaling, which increases cAMP and adenosine levels. Adenosine is released both through efflux from brown adipocytes and through breakdown of extracellular ATP by the ecto-5′-nucleotidase CD73. AR, adrenergic receptor.The determination of the source of the adenosine driving brown fat activation was also pursued. Adenosine could be derived from breakdown of ATP released from sympathetic neurons and brown adipocytes, or from brown adipocyte efflux of adenosine itself. The authors find that activated brown fat both releases adenosine through efflux and converts extracellular ATP to adenosine, as inhibition of the ecto-5′-nucleotidase CD73, which converts ATP to adenosine, only partially blocks the adenosine increase. Noradrenaline also enhances adenosine release from brown fat without any change in extracellular ATP, suggesting an intracellular crosstalk between catecholamine and adenosine signaling.The author further show that A_2A receptor knockout mice exposed to cold exhibit defective thermogenesis, oxygen consumption, and lipolysis, demonstrating the importance of the A_2A receptor in mediating the thermogenic response. Moreover, pharmacological activation with an A_2A receptor agonist increases oxygen consumption in mice, while inhibition with a specific A_2A antagonist decreases cold-induced oxygen consumption. A_2A receptor agonism acts synergistically with noradrenaline in driving lipolysis, revealing an additive thermogenic effect of adenosine with catecholamine signaling. An A_2A agonist protects against diet-induced obesity in mice, leading to increases in lean mass, oxygen consumption, glucose tolerance, and browning of white fat, which signifies the potential therapeutic importance of adenosine signaling. Importantly, overexpression of the A_2A receptor in inguinal white fat increases its browning, indicating that bolstering of A_2A adenosine receptor expression is sufficient to initiate a thermogenic response in the presence of physiological concentration of adenosine.This study reveals an endogenous purinergic signaling pathway that activates brown and beige fat. The critical signaling effector involved is cAMP, therefore the mechanism of activation is similar to those of other agents that act through the sympathetic nervous system or adrenergic signaling. It is unclear whether adenosine would contribute any additional anti-obesity effects than what has already been achieved with these other agents.While this study demonstrates substantial effects of adenosine on thermogenesis, further studies would better elucidate the specificity toward the A_2A receptor in adipocytes. Treatment of A_2A knockout mice with the receptor agonist would help demonstrate whether there are effects of the agonist independent of the A_2A receptor in vivo. Additionally, adipose tissue-specific A_2A knockout mice would reveal whether there are any phenotypic contributions from knockout in other tissues.Another question is whether A_2A receptor agonism could be used chronically as an anti-obesity therapy in the clinic, since adenosine receptors are expressed in many tissues. This study did not explore the potentially confounding effects of A_2A receptor agonism on tissues such as the brain or heart, including complications from vasodilation or cardiotoxicity. Indeed, A_2A receptor antagonists have been used in clinical trials for Parkinson''s disease, highlighting the possible adverse effects of A_2A receptor agonism on the brain. The effect of adenosine on lipolysis also raises the question of whether this pathway is involved in the lipoatrophy caused by nucleoside analogues used as anti-retroviral drugs. Nevertheless, this work adds important information about a cellular signaling pathway that may be leveraged in finding new therapies for obesity and metabolic diseases.     

The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD(+)

The NAD(+)-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic sensor of the Sirtuin family that mediates homeostatic responses to certain physiological stresses such as nutrient restriction. Previous reports have implicated fluctuations in intracellular NAD(+) concentrations as the principal regulator of SIRT1 activity. However, here we have identified a cAMP-induced phosphorylation of a highly conserved serine (S434) located in the SIRT1 catalytic domain that rapidly enhanced intrinsic deacetylase activity independently of changes in NAD(+) levels. Attenuation of SIRT1 expression or the use of a nonphosphorylatable SIRT1 mutant prevented cAMP-mediated stimulation of fatty acid oxidation and gene expression linked to this pathway. Overexpression of SIRT1 in mice significantly potentiated the increases in fatty acid oxidation and energy expenditure caused by either pharmacological β-adrenergic agonism or cold exposure. These studies support a mechanism of Sirtuin enzymatic control through the cAMP/PKA pathway with important implications for stress responses and maintenance of energy homeostasis.     

Two heteropteran predators in relation to weed management in herbicide-tolerant corn

Deployment of genetically modified (GM), herbicide-tolerant corn may alter weed flora abundance and composition and may affect pests and their natural enemies. Among on-plant predators, Orius spp. are the prevalent group in Spain and were selected to study the impact of glyphosate use on predators. We also studied Nabis sp. which is commonly recorded on corn in the study area. For this, a 4-year study was conducted in NE Spain. Three different herbicide regimes were compared: two glyphosate (a broad-spectrum herbicide) treatments per season, no herbicide treatment, and one pre-emergence conventional treatment with selective herbicides against broadleaf and grassy weeds. Density of main arthropod herbivores and the above two predatory groups was recorded on plants. Differences between herbicide regimes were observed in the two functional groups studied, herbivores and heteropteran predators. The comparison of glyphosate-treated and untreated plots showed significant differences for both functional groups, but the differences between glyphosate-treated and conventionally treated plots for the two functional groups were lower. For Orius spp., annual density per plot was significantly correlated with annual density of leafhoppers and to a lesser extent, with aphids. Nabis sp. densities were never different between glyphosate-treated and conventionally treated plots, and Nabis sp. density showed no relation to any of the herbivores tested. We concluded that no significant changes in heteropteran predator densities may be expected from moderate alterations in weeds arising from the deployment of herbicide-tolerant corn varieties and that leafhoppers are probably the herbivore prey that most influences Orius spp. densities in corn in our study area.     

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB(-/-) mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA(-/-) MEFs, but not FlnB(-/-) MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.     

Molecular mechanisms of disease for mutations at Gly-90 in rhodopsin

Two different mutations at Gly-90 in the second transmembrane helix of the photoreceptor protein rhodopsin have been proposed to lead to different phenotypes. G90D has been classically associated with congenital night blindness, whereas the newly reported G90V substitution was linked to a retinitis pigmentosa phenotype. Here, we used Val/Asp replacements of the native Gly at position 90 to unravel the structure/function divergences caused by these mutations and the potential molecular mechanisms of inherited retinal disease. The G90V and G90D mutants have a similar conformation around the Schiff base linkage region in the dark state and same regeneration kinetics with 11-cis-retinal, but G90V has dramatically reduced thermal stability when compared with the G90D mutant rhodopsin. The G90V mutant also shows, like G90D, an altered photobleaching pattern and capacity to activate Gt in the opsin state. Furthermore, the regeneration of the G90V mutant with 9-cis-retinal was improved, achieving the same A(280)/A(500) as wild type isorhodopsin. Hydroxylamine resistance was also recovered, indicating a compact structure around the Schiff base linkage, and the thermal stability was substantially improved when compared with the 11-cis-regenerated mutant. These results support the role of thermal instability and/or abnormal photoproduct formation in eliciting a retinitis pigmentosa phenotype. The improved stability and more compact structure of the G90V mutant when it was regenerated with 9-cis-retinal brings about the possibility that this isomer or other modified retinoid analogues might be used in potential treatment strategies for mutants showing the same structural features.     

Epistasis for founder-specific inbreeding depression in rabbits

Inbreeding depression is a topic of main interest in experimental and domestic species, although previous studies simplified this genetically complex effect to the linear (or quadratic) regression coefficient linked to the inbreeding coefficient of each individual or, in more recent studies, to founder-specific inbreeding coefficients. Going beyond generalizing to these traditional scenarios, our research focused on the analysis of gene-by-gene interactions leading to epistasis for inbreeding depression effects. Under a Bayesian context, inbreeding depression effects were evaluated for weaning weight (WW) in a commercial rabbit population founded from 4 bucks and 1 doe (MARET population). Founder-specific inbreeding depression effects for the 4 bucks ranged between -81.1 and 38.3 g for each 1% inbreeding. More interestingly, 2 epistatic interactions between the partial inbreeding coefficient of 2 bucks were also significant and negative, showing a -1.9 and -1.0 g reduction on WW. These results provide the first evidence of epistatic inbreeding depression phenomena in domestic species, emphasizing the complexity of the genetic architecture in mammals.     

Development of memory-like autoregulatory CD8+ T cells is CD4+ T cell dependent

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8(+) cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8(+) T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8(+) T cell memory is CD4(+) T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti-4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4(+) T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4(+) T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2(-/-) mice, but inhibited it in low-affinity TCR-TG NOD.Rag2(-/-) mice. Thus, in chronic autoimmune responses, CD4(+) Th cells contribute to both promoting and suppressing pathogenic autoimmunity.     

High levels of adenosine deaminase on dendritic cells promote autoreactive T cell activation and diabetes in nonobese diabetic mice

Adenosine has been established as an important regulator of immune activation. It signals through P1 adenosine receptors to suppress activation of T cells and professional APCs. Adenosine deaminase (ADA) counters this effect by catabolizing adenosine. This regulatory mechanism has not been tested in a disease model in vivo. Questions also remain as to which cell types are most sensitive to this regulation and whether its dysregulation contributes to any autoimmune conditions. We approached this issue using the NOD model. We report that ADA is upregulated in NOD dendritic cells, which results in their exuberant and spontaneous activation. This, in turn, triggers autoimmune T cell activation. NOD DCs deficient in ADA expression have a greatly reduced capacity to trigger type I diabetes. We also provide evidence that although many cell types, particularly T cells, have been implicated as the suppression targets by adenosine in an in vitro setting, DCs also seem to be affected by this regulatory mechanism. Therefore, this report illustrates a role of ADA in autoimmunity and suggests a potential target for therapeutic intervention.     

A genetic risk score combining ten psoriasis risk loci improves disease prediction

Psoriasis is a chronic, immune-mediated skin disease affecting 2–3% of Caucasians. Recent genetic association studies have identified multiple psoriasis risk loci; however, most of these loci contribute only modestly to disease risk. In this study, we investigated whether a genetic risk score (GRS) combining multiple loci could improve psoriasis prediction. Two approaches were used: a simple risk alleles count (cGRS) and a weighted (wGRS) approach. Ten psoriasis risk SNPs were genotyped in 2815 case-control samples and 858 family samples. We found that the total number of risk alleles in the cases was significantly higher than in controls, mean 13.16 (SD 1.7) versus 12.09 (SD 1.8), p = 4.577×10⁻⁴⁰. The wGRS captured considerably more risk than any SNP considered alone, with a psoriasis OR for high-low wGRS quartiles of 10.55 (95% CI 7.63–14.57), p = 2.010×10⁻⁶⁵. To compare the discriminatory ability of the GRS models, receiver operating characteristic curves were used to calculate the area under the curve (AUC). The AUC for wGRS was significantly greater than for cGRS (72.0% versus 66.5%, p = 2.13×10⁻⁸). Additionally, the AUC for HLA-C alone (rs10484554) was equivalent to the AUC for all nine other risk loci combined (66.2% versus 63.8%, p = 0.18), highlighting the dominance of HLA-C as a risk locus. Logistic regression revealed that the wGRS was significantly associated with two subphenotypes of psoriasis, age of onset (p = 4.91×10⁻⁶) and family history (p = 0.020). Using a liability threshold model, we estimated that the 10 risk loci account for only11.6% of the genetic variance in psoriasis. In summary, we found that a GRS combining 10 psoriasis risk loci captured significantly more risk than any individual SNP and was associated with early onset of disease and a positive family history. Notably, only a small fraction of psoriasis heritability is captured by the common risk variants identified to date.