High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli

Background

The realization of hydrogenase-based technologies for renewable H₂ production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.

Principal Findings

In this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H₂ evolution with rates comparable to those of enzymes isolated from their respective native organisms.

Significance

The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H₂-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.     

Tyrosine,Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation

Background

[FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H₂. These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated.

Principal Findings

We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation.

Significance

The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.     

Development of an in vitro compartmentalization screen for high-throughput directed evolution of [FeFe] hydrogenases

Background

[FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible.

Methodology/Principal Findings

Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library.

Conclusions/Significance

[FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy.     

Floral thermogenesis of three species of Hydnora (Hydnoraceae) in Africa

Background and Aims

Floral thermogenesis occurs in at least 12 families of ancient seed plants. Some species show very high rates of respiration through the alternative pathway, and some are thermoregulatory, with increasing respiration at decreasing ambient temperature. This study assesses the intensity and regulation of respiration in three species of African Hydnora that represent the Hydnoraceae, an unusual family of holoparasitic plants from arid environments.

Methods

Long-term respirometry (CO₂ production) and thermometry were carried out on intact flowers of H. africana, H. abyssinica and H. esculenta in the field, and short-term measurements were made on floral parts during the protogynous flowering sequence.

Key Results

For H. africana, there was no temperature elevation in either the osmophores or the gynoecial chamber in any phase, and mass-specific respiration rates of the flower parts were low (maximum 8·3 nmol CO₂ g⁻¹ s⁻¹ in osmophore tissue). Respiration tracked ambient and floral temperatures, eliminating the possibility of the inverse relationship expected in thermoregulatory flowers. Hydnora abyssinica flowers had higher respiration (maximum 27·5 nmol g⁻¹ s⁻¹ in the osmophores) and a slight elevation of osmophore temperature (maximum 2·8 °C) in the female stage. Respiration by gynoecial tissue was similar to that of osmophores in both species, but there was no measurable elevation of gynoecial chamber temperature. Gynoecial chamber temperature of H. esculenta could reach 3·8 °C above ambient, but there are no respiration data available. Antheral tissue respiration was maximal in the male phase (4·8 nmol g⁻¹ s⁻¹ in H. africana and 10·3 nmol g⁻¹ s⁻¹ in H. abyssinica), but it did not raise the antheral ring temperature, which showed that thermogenesis is not a by-product of pollen maturation or release.

Conclusions

The exceptionally low thermogenesis in Hydnora appears to be associated with scent production and possibly gynoecial development, but has little direct benefit to beetle pollinators.Key words: Pollination biology, Hydnora, thermogenesis, respiration rate, temperature, flowers, insects     

Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells

Background & Aims

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs).

Methods & Results

Ldlr^−/− mice were transplanted with wild-type (Wt), Cd36^−/− or Msr1^−/− bone marrow and fed a Western diet for 3months. Cd36^−/−- and Msr1^−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36^−/−- and Msr1^−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation.

Conclusion

CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.     

The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients

Background

The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown.

Methods

We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion.

Results

Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca²⁺-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl⁻conductance.

Conclusions

We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.     

Synthetic biology for improved hydrogen production in Chlamydomonas reinhardtii

Hydrogen is a clean alternative to fossil fuels. It has applications for electricity generation and transportation and is used for the manufacturing of ammonia and steel. However, today, H₂ is almost exclusively produced from coal and natural gas. As such, methods to produce H₂ that do not use fossil fuels need to be developed and adopted. The biological manufacturing of H₂ may be one promising solution as this process is clean and renewable. Hydrogen is produced biologically via enzymes called hydrogenases. There are three classes of hydrogenases namely [FeFe], [NiFe] and [Fe] hydrogenases. The [FeFe] hydrogenase HydA1 from the model unicellular algae Chlamydomonas reinhardtii has been studied extensively and belongs to the A1 subclass of [FeFe] hydrogenases that have the highest turnover frequencies amongst hydrogenases (21,000 ± 12,000 H₂ s⁻¹ for CaHydA from Clostridium acetobutyliticum). Yet to date, limitations in C. reinhardtii H₂ production pathways have hampered commercial scale implementation, in part due to O₂ sensitivity of hydrogenases and competing metabolic pathways, resulting in low H₂ production efficiency. Here, we describe key processes in the biogenesis of HydA1 and H₂ production pathways in C. reinhardtii. We also summarize recent advancements of algal H₂ production using synthetic biology and describe valuable tools such as high-throughput screening (HTS) assays to accelerate the process of engineering algae for commercial biological H₂ production.     

Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]?hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H₂ production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.     

RLIP76, a glutathione-conjugate transporter, plays a major role in the pathogenesis of metabolic syndrome

Purpose

Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76^−/− mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76^−/− mice.

Research Design and Methods

Blood glucose (BG) and lipid measurements were performed in RLIP76^+/+ and RLIP76^−/− mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining.

Results

The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76^−/− mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK.

Conclusions/Significance

All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76^−/− mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome.     

LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease

Background & Aims

Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr^−/− mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time.

Methods

APOE2ki and Ldlr^−/− mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control.

Results

Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr^−/− mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr^−/− mice. Finally, bone-marrow-derived-macrophages of Ldlr^−/− mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice.

Conclusion

Ldlr^−/− mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr^−/− mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis.     

Cathepsin S deficiency results in abnormal accumulation of autophagosomes in macrophages and enhances Ang II-induced cardiac inflammation

Background

Cathepsin S (Cat S) is overexpressed in human atherosclerotic and aneurysmal tissues and may contributes to degradation of extracellular matrix, especially elastin, in inflammatory diseases. We aimed to define the role of Cat S in cardiac inflammation and fibrosis induced by angiotensin II (Ang II) in mice.

Methods and Results

Cat S-knockout (Cat S^−/−) and littermate wild-type (WT) C57BL/6J mice were infused continuously with Ang II (750 ng/kg/min) or saline for 7 days. Cat S^−/− mice showed severe cardiac fibrosis, including elevated expression of collagen I and α-smooth muscle actin (α-SMA), as compared with WT mice. Moreover, macrophage infiltration and expression of inflammatory cytokines (tumor necrosis factor α, transforming growth factor β and interleukin 1β) were significantly greater in Cat S^−/− than WT hearts. These Ang II-induced effects in Cat S^−/− mouse hearts was associated with abnormal accumulation of autophagosomes and reduced clearance of damaged mitochondria, which led to increased levels of reactive oxygen species (ROS) and activation of nuclear factor-kappa B (NF-κB) in macrophages.

Conclusion

Cat S in lysosomes is essential for mitophagy processing in macrophages, deficiency in Cat S can increase damaged mitochondria and elevate ROS levels and NF-κB activity in hypertensive mice, so it regulates cardiac inflammation and fibrosis.     

Deletion of the Innate Immune NLRP3 Receptor Abolishes Cardiac Ischemic Preconditioning and Is Associated with Decreased Il-6/STAT3 Signaling

Objective

Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart.

Principal Findings

Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC^−/− and NLRP3^−/− mice. Deletion of NLRP3 inflammasome components ASC^−/− or NLRP3^−/− did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC^−/− hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3^−/− hearts against I/R injury. NLRP3^−/− hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1β levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1β signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3^−/− hearts when compared to WT hearts.

Conclusions

The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.     

Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.     

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis

Background & Aims

CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.

Methods

Acute inflammation and recovery in wild-type (WT) and CCR9^−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.

Results

CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9^−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9^−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9^−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.

Conclusions

Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.     

CO2 efflux from cleared mangrove peat

Background

CO₂ emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils.

Methodology/Principal Findings

We measured CO₂ efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO₂ efflux. CO₂ efflux from soils declines from time of clearing from ∼10 600 tonnes km⁻² year⁻¹ in the first year to 3000 tonnes km² year⁻¹ after 20 years since clearing. Disturbing peat leads to short term increases in CO₂ efflux (27 umol m⁻² s⁻¹), but this had returned to baseline levels within 2 days.

Conclusions/Significance

Deforesting mangroves that grow on peat soils results in CO₂ emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks.     

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.     

eNOS protects from atherosclerosis despite relevant superoxide production by the enzyme in apoE mice

Background

All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme.

Principal Findings

Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE^−/−/eNOS^−/−), while P/E-interactions did not differ, compared to apoE^−/−. eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE^−/− vessels.

Conclusion

Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE^−/−/eNOS^−/− vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE^−/− atherosclerosis but does not negate the enzyme''s strong protective effects.     

CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies

Background

Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L – an established marker and mediator of cardiovascular disease – induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo.

Methodology/Principal Findings

WT or CD40L^−/− mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L^−/− mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L^−/− mice. However, CD40L^−/− mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L^−/− mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels.

Conclusion

We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.