Hypercapnia induces cleavage and nuclear localization of RelB protein, giving insight into CO2 sensing and signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-?B pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-?B family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3ß or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-?B family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg- 1) attenuated protein degradation and increased protein synthesis in skeletal muscle. © 2007 Elsevier Inc. All rights reserved.

Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies in vitro have suggested that activation of the dsRNA-dependent protein kinase (PKR) may be responsible for these changes in protein synthesis and degradation. In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg-1) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2α phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-κB (NF-κB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia. © 2007 Cancer Research UK.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Paternal low protein diet affects adult offspring cardiovascular and metabolic function in mice

Although the association between maternal periconceptional diet and adult offspring health is well characterised, our understanding of the impact of paternal nutrition at the time of conception on offspring phenotype remains poorly defined. Therefore, we determined the effect of a paternal preconception low protein diet (LPD on adult offspring cardiovascular and metabolic health in mice. Male C57BL/6 mice were fed either normal protein diet (NPD; 18% casein or LPD (9% casein for 7 wk before mating. At birth, a reduced male-to-female ratio (P = 0.03 and increased male offspring weight (P = 0.009 were observed in litters from LPD compared with NPD stud males with no differences in mean litter size. LPD offspring were heavier than NPD offspring at 2 and 3 wk of age (P <0.02. However, no subsequent differences in body weight were observed. Adult male offspring derived from LPD studs developed relative hypotension (decreased by 9.2 mmHg and elevated heart rate (P <0.05, whereas both male and female offspring displayed vascular dysfunction and impaired glucose tolerance relative to NPD offspring. At cull (24 wk, LPD males had elevated adiposity (P = 0.04, reduced heart-to-body weight ratio (P = 0.04, and elevated circulating TNF-α levels (P = 0.015 compared with NPD males. Transcript expression in offspring heart and liver tissue was reduced for genes involved in calcium signaling (Adcy, Plcb, Prkcb and metabolism (Fto in LPD offspring (P <0.03. These novel data reveal the impact of suboptimal paternal nutrition on adult offspring cardiovascular and metabolic homeostasis, and provide some insight into the underlying regulatory mechanisms.

Maternal periconceptional and gestational low protein diet affects mouse offspring growth, cardiovascular and adipose phenotype at 1 year of age

Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (Insr) and insulin-like growth factor I receptor (Igf1r) in retroperitoneal fat was significantly elevated in Emb-LPD females (P&0.05), whilst Emb-LPD males displayed significantly decreased expression of the mitochondrial uncoupling protein 1 (Ucp1) gene compared to NPD offspring. LPD females displayed significantly increased expression of Ucp1 in interscapular brown adipose tissue when compared to NPD offspring. Our results demonstrate that aging offspring body weight, cardiovascular and adiposity homeostasis can be programmed by maternal periconceptional nutrition. These adverse outcomes further exemplify the criticality of dietary behaviour around the time of conception on long-term offspring health. © 2011 Watkins et al.

Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss

Atrophy of skeletal muscle is common in patients with cancer and results in increased morbidity and mortality. In order to design effective therapy the mechanism by which this occurs needs to be elucidated. Most studies suggest that the ubiquitin-proteasome proteolytic pathway is most important in intracellular proteolysis, although there have been no reports on the activity of this pathway in patients with different extents of weight loss. In this report the expression of the ubiquitin-proteasome pathway in rectus abdominis muscle has been determined in cancer patients with weight loss of 0-34% using a competitive reverse transcriptase polymerase chain reaction to measure expression of mRNA for proteasome subunits C2 and C5, while protein expression has been determined by western blotting. Overall, both C2 and C5 gene expression was increased by about three-fold in skeletal muscle of cachectic cancer patients (average weight loss 14.5 ± 2.5%), compared with that in patients without weight loss, with or without cancer. The level of gene expression was dependent on the amount of weight loss, increasing maximally for both proteasome subunits in patients with weight loss of 12-19%. Further increases in weight loss reduced expression of mRNA for both proteasome subunits, although it was still elevated in comparison with patients with no weight loss. There was no evidence for an increase in expression at weight losses less than 10%. There was a good correlation between expression of proteasome 20Sα subunits, detected by western blotting, and C2 and C5 mRNA, showing that increased gene expression resulted in increased protein synthesis. Expression of the ubiquitin conjugating enzyme, E214k, with weight loss followed a similar pattern to that of proteasome subunits. These results suggest variations in the expression of key components of the ubiquitin-proteasome pathway with weight loss of cancer patients, and suggest that another mechanism of protein degradation must be operative for patients with weight loss less than 10%. © 2004 Elsevier Ltd. All rights reserved.

Synthesis, self-assembly and (absence of) protein interactions of poly(glycerol methacrylate)-silicone macro-amphiphiles

The present study focuses on the synthesis of amphiphilic block copolymers containing poly(glycerol monomethacrylate) (PGMMA), showing the advantages of a protection/deprotection strategy based on silyl groups. PGMMA blocks were synthesized via ATRP started by a double functional poly(dimethyl siloxane) (PDMS) macroinitiator of molecular weight ≈7000 g mol-1. The resulting triblock copolymers were characterized by low polydispersity (generally ≤1.1) and their aggregation concentration in water was essentially dominated by the PDMS block length (critical aggregation concentration substantially invariant for GMMA degree of polymerization ≥30). For GMMA blocks with DP > 50, the self-assembly in water produced 35-50 nm spherical micelles, while shorter hydrophilic chains produced larger aggregates apparently displaying worm-like morphologies. Block copolymers with long GMMA chains (DP ≈ 200) produced particularly stable micellar aggregates, which were then selected for a preliminary assessment of the possibility of adsorption of plasma proteins (albumin and fibrinogen); using diffusion NMR as an analytical technique, no significant adsorption was recorded both on micelles and on soluble PGMMA employed as a control, indicating the possibility of a "stealth" behaviour. This journal is © 2013 The Royal Society of Chemistry.

The application of counter immunoelectrophoresis (CIE) in ocular protein studies. Part II:kinin activity in the lens wearing eye

The kinin family are a group of bioactive peptides that are closely involved in the modulation of vascular inflammation and local injury. We have demonstrated here, for the first time, a link between kinin activity and contact lens wear. Protein extracts from daily and extended wear etafilcon A, Group IV, Acuvue lenses (Vistakon), were analysed by counter immunoelectrophoresis. In this way, kinin activity associated with contact lens wear was detected. High molecular weight kininogen was used as the marker protein. In contrast, no kinin activity was detected in the non-lens wearing normal eye. © 2002 British Contact Lens Association. Published by Elsevier Science Ltd. All rights reserved.

Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo's potential, affecting adult health

Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences.

Association behavior of the synthetic protein-lipid complex analogues

Hypercoiling poly(styrene-ALT-maleic anhydride) (PSMA) is known to undergo conformational transition in response to environmental stimuli. This behavior allows it to associate with the phospholipid, 2-dilauryl-SN-glycero-3- phosphocholine (DLPC) to produce nanostructures analogous to lipoproteins. The complex represents a new bio-mimetic delivery vehicle with applications in the cosmetic and pharmaceutical industries. This study investigates, for the first time, the association behavior of PSMA and DLPC through the combination of different analytical techniques. The results indicate that the association is primarily driven by hydrophobic interactions and depends on various factors including the polymer/lipid ratio, the polymer molecular weight and the pH of the aqueous environment. The conformational transition of PSMA leads to the formation of discrete micellar complexes involving anisotropic-to-isotropic lipid phase transformation. As the number of hydrophobic moieties in the polymer is increased, the pH-dependent conformational transition of the polymer plays less important part in achieving this phase transition of the lipid. © (2012) Trans Tech Publications.

Membrane protein extraction and purification using styrene-maleic acid (SMA) co-polymer:effect of variations in polymer structure

The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene to maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA) which vary in size and shape were used. Our results show that several polymers can be used to extract membrane proteins comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular weight (7.5-10 kDa) is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification SMA 2000 was found to be the best choice for yield, purity and function. However the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.