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Lipoprotein lipase is nitrated in vivo after lipopolysaccharide challenge
Abstract
Lipopolysaccharide (LPS) administration down-regulates lipoprotein lipase (LPL) activity at the posttranscriptional level. Hypertriglyceridemia is the main metabolic consequence of this fall in LPL activity and is presumably involved in the innate immune response to infection. Nitric oxide (NO) has been implicated in LPS-induced down-regulation of LPL activity, but whether its effects are direct or indirect remains unclear. Here we examined the potential nitration of LPL in vivo in response to LPS challenge in rats. We found hypertriglyceridemia, iNOS expression, NO overproduction, and a generalized decrease in LPL activity in tissues 6 h after LPS administration. LPL sensitivity to nitration was first explored by in vitro exposure of bovine LPL to peroxynitrite, a reactive nitrogen species (RNS). Nitration was confirmed by anti-nitrotyrosine Western blot and subsequent identification of specific nitrotyrosine-containing LPL sequences by tandem mass spectrometry. Further analysis by targeted mass spectrometry revealed three in vivo-nitrated tyrosine residues in heart LPL from LPS-challenged rats. This is the first study to identify nitrated tyrosine residues in LPL, both in vitro and in vivo, and it demonstrates that LPL is a target for RNS in endotoxemia. These results indicate that LPL nitration may be a new mechanism of LPL activity regulation in vivo.
... iNOS seems to affect LPL activity during short-term endotoxemia at the posttranscriptional level. LPL was shown to be nitrated following LPS administration, probably due to iNOS-mediated augmented peroxynitrite levels under these circumstances [130]. Tyrosine nitration of LPL decreases its catalytic efficiency and reduces TG clearance, therefore promoting a hypertriglyceridemic state [130,131]. ...
... LPL was shown to be nitrated following LPS administration, probably due to iNOS-mediated augmented peroxynitrite levels under these circumstances [130]. Tyrosine nitration of LPL decreases its catalytic efficiency and reduces TG clearance, therefore promoting a hypertriglyceridemic state [130,131]. ...
Nitric oxide (NO) is a free radical acting as a cellular signaling molecule in many different biochemical processes. NO is synthesized from l-arginine through the action of the nitric oxide synthase (NOS) family of enzymes, which includes three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). iNOS-derived NO has been associated with the pathogenesis and progression of several diseases, including liver diseases, insulin resistance, obesity and diseases of the cardiovascular system. However, transient NO production can modulate metabolism to survive and cope with stress conditions. Accumulating evidence strongly imply that iNOS-derived NO plays a central role in the regulation of several biochemical pathways and energy metabolism including glucose and lipid metabolism during inflammatory conditions. This review summarizes current evidence for the regulation of glucose and lipid metabolism by iNOS during inflammation, and argues for the role of iNOS as a metabolic enzyme in immune and non-immune cells.
... Thus, we hypothesised that increased expression of Angptl3 promotes fat deposition and adiposity, which in turn leads to the impairment of carcass characteristics and meat quality. Several studies suggest that oxidative stress could stimulate Angptl3 expression and suppress AT-LPL expression (Marcil et al. 2006;Moon et al. 2007;Miida et al. 2008;Casanovas et al. 2009). Therefore, Angptl3 may be a useful target for the improvement of carcass characteristics and meat quality. ...
... A high oxidative stability of muscle-based foods is important for avoiding or delaying development of rancid products or warmed-over flavour (Jiang et al. 2007). Some experiments have shown that oxidative stress may promote the expression of Angptl3 and inhibit the expression of AT-LPL (Moon et al. 2007;Miida et al. 2008;Casanovas et al. 2009). Our results showed that carcass characteristics and meat quality were correlated with the Angptl3 mRNA expression. ...
Angiopoietin-like protein 3 (Angptl3) may promote adipose formation. The present study investigated the beneficial effect of 1,3,5,8-tetrahydroxyxanthone (Xan), a naturally occurring polyphenol agent, on carcass characteristics and meat quality in pigs and the mechanisms involved. Forty-eight Duroc × Landrace × Yorkshire pigs (65.3 ± 7.8 kg) were randomly divided into four groups: control group, untreated high lipid diet (HLD) group and two groups of HLD with Xan (1 or 3%). Forty-two days later, Xan (1 or 3%) treatment significantly increased percentage lean, loin eye area, colour, expression and activity of adipose tissue lipoprotein lipase activity and decreased percentage fat, backfat thickness, total cholesterol concentration, triglyceride concentration, and Angptl3 mRNA expression. The present results suggest that the beneficial effect of Xan on carcass characteristics and meat quality may be related to decreased expression of Angptl3 in pig.
... Despite progress in our understanding of LPL regulation [4], the multiple aspects of LPL biology that remain unclear indicate that we are still left with an incomplete characterization of the enzyme. In recent years, the use of proteomic tools (i.e., two-dimensional electroporesis (2DE) and mass spectrometry (MS)) in LPL research (i) revealed the existence of more than 8 LPL isoforms of the same apparent molecular weight but different pI in the rat heart [10] uncovering a level of diversity previously unknown, (ii) enabled the identification of a novel post-translational modification of the enzyme [11], and (iii) yielded the first (partial) sequence to be described from direct study of rat LPL protein [10]. ...
... However, we demonstrated that phosphorylation does not contribute to LPL pI heterogeneity in the rat [10]. In a recent study, we identified nitrated tyrosine residues in rat LPL in response to LPS challenge [11], demonstrating that LPL can undergo other PTMs in vivo. Hence, tyrosine nitration or other not yet identified PTMs can potentially contribute to LPL pI heterogeneity, although further research will be required to elucidate the molecular differences between LPL isoforms. ...
... Although this can explain the rapidity of the process, how it mediates the process is largely unknown. It has been shown that NO causes nitration of LPL [8], but the significance of nitration in LPL regulation is largely unknown. Intriguingly, a link between NO production and LPL downregulation in WAT has been shown in studies using inflammation models in addition to stress models [9]. ...
... This finding suggests that the hydrolysis of newly absorbed VA, transported as retinyl ester in chylomicra or their remnants, was delayed. Lipoprotein lipase activity is known to be reduced by LPS (34,35). Consistent with the hypothesized delay in chylomicron clearance, liver retinyl esters were lower in the LPS-and VA-supplemented group (23). ...
The association between inflammation and vitamin A (VA) metabolism and status assessment has been documented in multiple studies with animals and humans. The relation between inflammation and carotenoid status is less clear. Nonetheless, it is well known that carotenoids are associated with certain health benefits. Understanding these relations is key to improving health outcomes and mortality risk in infants and young children. Hyporetinolemia, i.e., low serum retinol concentrations, occurs during inflammation, and this can lead to the misdiagnosis of VA deficiency. On the other hand, inflammation causes impaired VA absorption and urinary losses that can precipitate VA deficiency in at-risk groups of children. Many epidemiologic studies have suggested that high dietary carotenoid intake and elevated plasma concentrations are correlated with a decreased risk of several chronic diseases; however, large-scale carotenoid supplementation trials have been unable to confirm the health benefits and in some cases resulted in controversial results. However, it has been documented that dietary carotenoids and retinoids play important roles in innate and acquired immunity and in the body's response to inflammation. Although animal models have been useful in investigating retinoid effects on developmental immunity, it is more challenging to tease out the effects of carotenoids because of differences in the absorption, kinetics, and metabolism between humans and animal models. The current understanding of the relations between inflammation and retinoid and carotenoid metabolism and status are the topics of this review.
... 45 Second, in the circulation, LPS is scavenged by HDL cholesterol, which is often low in conditions with elevated triglycerides and high levels of pro-inflammatory cytokines. 46 Furthermore, LPS downregulates lipoprotein lipase activity, leading to hypertriglyceridemia. 47 Finally, adipocytes express TLR, and binding of LPS may result in increased production of pro-inflammatory cytokines. 23,48 Thus, it is conceivable that the disrupted gut barrier in HIV infection might fuel a possibly interacting effect of LPS and lipids on cardiometabolic risk factors. ...
Objective: Microbial translocation has been suggested to be a driver of immune activation and inflammation. It is hypothesized that microbial translocation may be related to dyslipidemia, insulin resistance, and the risk of coronary heart disease in HIV-infected individuals. Design: Cross-sectional study of 60 HIV-infected patients on combination antiretroviral therapy with viral suppression .2 years and 31 healthy age-matched controls. Methods: Lipopolysaccharide (LPS) was analyzed by limulus amebocyte lysate colorimetric assay. Lipids, including cholesterol, low-density lipoprotein (LDL), and triglycerides, were measured. Glucose metabolism was determined using an oral glucose tolerance test. Body composition was determined using whole-body dual-energy x-ray absorptiometry scans and magnetic resonance imaging. The Framingham risk score was used to assess risk of cardiovascular disease and myocardial infarction.
... In the DM1DL1 patients, we found that bariatric surgery increased iNOS expression, decreased LPL activity, and decreased leptin protein and mRNA, which would confirm the observations of other authors (37). ...
Objective
The possible differences were investigated in 32 morbidly obese patients depending on whether they were “healthy” or had dyslipidemia and/or type 2 diabetes.Methods
Lipid metabolism and insulin resistance were analyzed in subcutaneous (SAT) and visceral adipose tissue (VAT) before and during 6 and 12 months after Roux-en-Y gastric bypass.ResultsSignificant differences have been found in lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) activities in SAT from the different obese group versus normal weight (control) but not between them. The reduced lipase activities in VAT were 43 and 19% smaller (22 and 4% smaller, respectively, vs. control) than the “healthy” obese group for LPL and HSL, respectively, and were accompanied with a reduced expression of these lipases, as well as decreased expression of FAT/CD36, FABP4, and AQ7 in that tissue. In addition, the expression of the other genes measured showed a downregulation not only versus the “healthy” obese but also versus the normal weight group.Conclusions
Being obese is not “healthy,” but it is even less so if morbidly obese patients with diabetes and dyslipidemia were considered. The reduced fat accumulation in these patients may be attributed to the decrease of the expression and activity of the lipases of their adipose tissue.
... 13 The close association between reduced triglyceride levels and reduced plasma LPS could be explained by several contributing mechanisms: First, LPS is cotransported with lipids in chylomicrons from the gut to circulation, 14 where 75%-80% of circulating LPS associates with proatherogenic lipoproteins such as triglyceride-rich very low-density lipoprotein particles, 15 as well as high-density lipoprotein cholesterol, which has antiatherogenic properties and acts as a scavenger of LPS. 16 Furthermore, LPS stimulates triglyceride production in the liver 17 and downregulates lipoprotein lipase activity, leading to reduced elimination of triglycerides, 18 whereas lipase expression is increased in human skeletal muscles after exercise training with weight loss. 19 In conclusion, reduced trunk fat, triglycerides, and improved insulin sensitivity after strength training were associated with reduced LPS levels, possibly mediated through a reduction in triglycerides, although the direction of these associations should be explored in future studies. ...
... First, LPS and triglycerides are cotransported in chylomicrons from the gut to circulation 94 where LPS is scavenged by HDL cholesterol, which is often low in dyslipidemia. 95 Furthermore, LPS down-regulates lipoprotein lipase activity leading to hypertriglyceridemia. 96 Finally, plasma metabolomic analyses showed that certain clusters of lipid abnormalities were associated with LPS and LPS-binding protein in patients receiving protease inhibitors. 97 Insulin resistance and metabolic syndrome Interestingly, increased levels of plasma LPS correlate with lower insulin sensitivity in patients on ART. ...
The widespread access to antiretroviral treatment during the last decades has transformed HIV-infection from a lethal disease to a chronic condition, in which the relative burden of non-AIDS related chronic disorders such as cardiovascular disease, malignancy, renal, liver, and bone disease have increased. The adjusted relative risk for myocardial infarction is reported to be around 2-fold compared to that of the general population, which over time is likely to translate into increased absolute risk in an ageing population. Thus, delineating potentially HIV-specific pathogenetic mechanisms are crucial in order to tailor novel strategies for prophylaxis and treatment. This review will focus on advances in the field that possibly link HIV-induced alterations of the gut mucosa and consequent microbial translocation to cardiometabolic risk factors in HIV-infection. Recent work suggests that markers of microbial translocation are closely associated with several cardiovascular risk factors such as dyslipidemia, insulin resistance, hypertension, coagulation abnormalities, endothelial dysfunction and carotid atherosclerosis. Future studies should investigate whether associations between microbial translocation and cardiovascular risk factors will translate into increased risk of acute events, and whether strategies to target gut microbiota and microbial translocation might reduce such a risk.
... (10) Others. Nitroproteins were discovered in sickle cell disease (Aslan et al., 2003), traumatic brain-injured rats , rat hippocampus after acute inhalation of combustion smoke (Lee et al., 2009a), Eosinophil granule toxins (Ulrich et al., 2008), hypertriglyceridemia (Casanovas et al., 2009), placenta/pre-eclampsia (Webster, Brockman, & Myatt, 2006), human plasma (Hamilton et al., 2008;Hui FIGURE 10. Reaction scheme of the chemical-labeling method as exemplified with an N-terminal nitrotyrosine residue. ...
Oxidative stress plays important roles in a wide range of diseases such as cancer, inflammatory disease, neurodegenerative disorders, etc. Tyrosine nitration in a protein is a chemically stable oxidative modification, and a marker of oxidative injuries. Mass spectrometry (MS) is a key technique to identify nitrotyrosine-containing proteins and nitrotyrosine sites in endogenous and synthetic nitroproteins and nitropeptides. However, in vivo nitrotyrosine-containing proteins occur with extreme low-abundance to severely challenge the use of MS to identify in vivo nitroproteins and nitrotyrosine sites. A preferential enrichment of nitroproteins and/or nitropeptides is necessary before MS analysis. Current enrichment methods include immuno-affinity techniques, chemical derivation of the nitro group plus target isolations, followed with tandem mass spectrometry analysis. This article reviews the MS techniques and pertinent before-MS enrichment techniques for the identification of nitrotyrosine-containing proteins. This article reviews future trends in the field of nitroproteomics, including quantitative nitroproteomics, systems biological networks of nitroproteins, and structural biology study of tyrosine nitration to completely clarify the biological functions of tyrosine nitration. © 2013 Wiley Periodicals, Inc. Mass Spec Rev.
... 45 Second, in the circulation, LPS is scavenged by HDL cholesterol, which is often low in conditions with elevated triglycerides and high levels of pro-inflammatory cytokines. 46 Furthermore, LPS downregulates lipoprotein lipase activity, leading to hypertriglyceridemia. 47 Finally, adipocytes express TLR, and binding of LPS may result in increased production of pro-inflammatory cytokines. 23,48 Thus, it is conceivable that the disrupted gut barrier in HIV infection might fuel a possibly interacting effect of LPS and lipids on cardiometabolic risk factors. ...
Microbial translocation has been suggested to be a driver of immune activation and inflammation. We hypothesized that microbial translocation may be related to dyslipidemia, insulin resistance, and the risk of coronary heart disease in HIV-infected individuals.
Cross sectional study of 60 HIV-infected patients on cART with viral suppression > two years and 31 healthy, age-matched controls.
Lipopolysaccharide (LPS) was analyzed by Limulus Amebocyte Lysate colorimetric assay. Lipids including cholesterol, low-density lipoprotein (LDL), and triglycerides were measured. Glucose metabolism was determined using an oral glucose tolerance test. Body composition was determined using whole body Dual-energy X-ray Absorptiometry (DEXA) scans and Magnetic Resonance Imaging (MRI). The Framingham risk score was used to assess risk of cardiovascular disease (CVD) and myocardial infarction (MI).
HIV-infected patients had higher level of LPS compared to controls (64 pg/mL vs. 50 pg/mL, p=0.002). Likewise, HIV-infected patients had higher triglycerides, LDL and fasting glucose as well as evidence of lower insulin sensitivity compared to controls. Among HIV-infected patients high LPS was associated with a higher level of triglycerides and LDL and with lower insulin sensitivity. Importantly, among HIV-infected patients high LPS was associated and a higher Framingham risk score.
HIV-infected patients with suppressed viral replication had increased level of microbial translocation as measured by LPS. LPS was associated with cardiometabolic risk factors and increased Framingham risk score. Hence, the gastrointestinal mucosal barrier may be a potential therapeutic target to prevent dyslipidemia and future cardiovascular complications in HIV infection.
... The endotoxemia induced the rise of total cholesterol and triglyceride plasma concentration (the highest values at the dose of 5 mg/kg bw). Hyperlipidemia after endotoxin treatment was observed also by other authors [4, 40]. They explained it by inhibition of lipoprotein lipase activity by nitration of tyrosyl residues of this enzyme by nitric oxide. ...
Endotoxin administration is frequently used as a model of systemic inflammatory response which is considered the important pathogenetic factor in muscle wasting development in severe illness, such as sepsis, cancer, injury, AIDS and others. The main purpose of this study was determining the effect of various doses of endotoxin on protein and amino acid metabolism in two types of rat skeletal muscle. Sepsis was induced by intraperitoneal administration of endotoxin in a dose of 1, 3 and 5 mg/kg body weight (bw); control animals received a corresponding volume of the saline solution. After 24 h, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated and used for determination of total and myofibrillar proteolysis, protein synthesis, activity of cathepsins B and L, chymotrypsin-like activity of proteasome and amino acid release. The endotoxemia induced the body weight loss, the rise of total cholesterol and triglyceride plasma concentration and the protein catabolic state in skeletal muscle, which was caused by a higher increase in protein breakdown (due to activation of the proteasome system) than protein synthesis. The more significant effect of endotoxin was seen in EDL than SOL. The dose of 5 mg of endotoxin/kg bw induced the most significant changes in parameters of the protein and amino acid metabolism measured and could be therefore considered appropriate for studies of protein catabolism in young rat skeletal muscle at 24 h after endotoxin treatment.
... These included the catalytic triad for the active site (Ser159; Asp183; His266); the hydrophobic N-terminus signal peptides (see alsoTable 1) which facilitate enzyme secretion into the circulation system (Jin et al., 2003); disulfide bond forming residues (Cys54/Cys68; Cys243/Cys266; Cys291/Cys302; Cys305/ Cys310; Cys445/Cys466) (the latter disulfide bond is apparently absent in the stickleback LPL sequence); the predictedìid' region (244–265) which covers the active site and participates in lipid substrate binding in analogous lipases (Winkler et al., 1990; Bourne et al., 1994); and a predicted`hingepredicted`hinge' region for vertebrate LPL, containing a proteolytic cleavage site for proprotein convertase (320Arg-321Ala-322Lys-323Arg) (Jin et al., 2003;) (seeFigure 1). Specific tyrosine residues predicted for nitration following lipopolysaccharide (LPS) challenging were identified (tyrosines 121, 127 and 314) which down-regulate LIPL activity and reduce triglyceride clearance from the body (Casanovas et al, 2009). These residues were conserved for all of the vertebrate LPL sequences examined (Figure 1) possibly because of this role in reducing LPL activity following LPS administration, resulting inìipemia' and increased binding of triglyceride-rich lipoproteins with LPS which undergo clearance by the liver (Gouni et al, 1993). ...
Lipoprotein lipase (LIPL or LPL; E.C.3.1.1.34) serves a dual function as a triglyceride lipase of circulating chylomicrons and very-low-density lipoproteins (VLDL) and facilitates receptor-mediated lipoprotein uptake into heart, muscle and adipose tissue. Comparative LPL amino acid sequences and protein structures and LPL gene locations were examined using data from several vertebrate genome projects. Mammalian LPL genes usually contained 9 coding exons on the positive strand. Vertebrate LPL sequences shared 58-99% identity as compared with 33-49% sequence identities with other vascular triglyceride lipases, hepatic lipase (HL) and endothelial lipase (EL). Two human LPL N-glycosylation sites were conserved among seven predicted sites for the vertebrate LPL sequences examined. Sequence alignments, key amino acid residues and conserved predicted secondary and tertiary structures were also studied. A CpG island was identified within the 5'-untranslated region of the human LPL gene which may contribute to the higher than average (×4.5 times) level of expression reported. Phylogenetic analyses examined the relationships and potential evolutionary origins of vertebrate lipase genes, LPL, LIPG (encoding EL) and LIPC (encoding HL) which suggested that these have been derived from gene duplication events of an ancestral neutral lipase gene, prior to the appearance of fish during vertebrate evolution. Comparative divergence rates for these vertebrate sequences indicated that LPL is evolving more slowly (2-3 times) than for LIPC and LIPG genes and proteins.
Context
Humans with obesity and insulin resistance exhibit lipid accumulation in skeletal muscle, but the underlying biological mechanisms responsible for the accumulation of lipid in the muscle of these individuals remain unknown.
Objective
We investigated how plasma insulin modulates the extraction of circulating triglycerides (TGs) and nonesterified fatty acids (NEFAs) from ingested and endogenous origin in the muscle of lean, insulin-sensitive humans (Lean-IS) and contrasted these responses to those in humans with obesity and insulin resistance (Obese-IR).
Methods
The studies were performed in a postprandial state associated with steady-state plasma TG concentrations. The arterio-venous blood sampling technique was employed to determine the extraction of circulating lipids across the forearm muscle before and after insulin infusion. We distinguished the kinetics of TGs and NEFAs from ingested origin from those from endogenous origin across muscle by incorporating stable isotope-labeled triolein in the ingested fat.
Results
Insulin infusion rapidly suppressed the extraction of plasma TGs from endogenous but not ingested origin in the muscle of the Lean-IS, but this response was absent in the muscle of the Obese-IR. Furthermore, in the muscle of the Lean-IS, insulin infusion decreased the extraction of circulating NEFAs from both ingested and endogenous origin; however, this response was absent for NEFAs from ingested origin in the muscle of the Obese-IR subjects.
Conclusion
Partitioning of circulating lipids away from the skeletal muscle when plasma insulin increases during the postprandial period is impaired in humans with obesity and insulin resistance.
The aim of the present study was to investigate retinal microcirculatory and functional metabolic changes in patients after they had recovered from a moderate to severe acute COVID-19 infection. Retinal perfusion was quantified using laser speckle flowgraphy. Oxygen saturation and retinal calibers were assessed with a dynamic vessel analyzer. Arterio-venous ratio (AVR) was calculated based on retinal vessel diameter data. Blood plasma samples underwent mass spectrometry-based multi-omics profiling, including proteomics, metabolomics and eicosadomics. A total of 40 subjects were included in the present study, of which 29 had recovered from moderate to severe COVID-19 within 2 to 23 weeks before inclusion and 11 had never had COVID-19, as confirmed by antibody testing. Perfusion in retinal vessels was significantly lower in patients (60.6 ± 16.0 a.u.) than in control subjects (76.2 ± 12.1 a.u., p = 0.006). Arterio-venous (AV) difference in oxygen saturation and AVR was significantly lower in patients compared to healthy controls (p = 0.021 for AVR and p = 0.023 for AV difference in oxygen saturation). Molecular profiles demonstrated down-regulation of cell adhesion molecules, NOTCH3 and fatty acids, and suggested a bisphasic dysregulation of nitric oxide synthesis after COVID-19 infection. The results of this study imply that retinal perfusion and oxygen metabolism is still significantly altered in patients well beyond the acute phase of COVID-19. This is also reflected in the molecular profiling analysis of blood plasma, indicating a down-regulation of nitric oxide-related endothelial and immunological cell functions.
Trial Registration: ClinicalTrials.gov (https://clinicaltrials.gov) NCT05650905.
Lipoprotein lipase (LPL) is responsible for the intravascular catabolism of triglyceride-rich lipoproteins and plays a central role in whole-body energy balance and lipid homeostasis. As such, LPL is subject to tissue-specific regulation in different physiological conditions, but the mechanisms of this regulation remain incompletely characterized. Previous work revealed that LPL comprises a set of proteoforms with different isoelectric points, but their regulation and functional significance have not been studied thus far. Here we studied the distribution of LPL proteoforms in different rat tissues and their regulation under physiological conditions. First, analysis by two-dimensional electrophoresis and Western blot showed different patterns of LPL proteoforms (i.e., different pI or relative abundance of LPL proteoforms) in different rat tissues under basal conditions, which could be related to the tissue-specific regulation of the enzyme. Next, the comparison of LPL proteoforms from heart and brown adipose tissue between adults and 15-day-old rat pups, two conditions with minimal regulation of LPL in these tissues, yielded virtually the same tissue-specific patterns of LPL proteoforms. In contrast, the pronounced downregulation of LPL activity observed in white adipose tissue during fasting is accompanied by a prominent reconfiguration of the LPL proteoform pattern. Furthermore, refeeding reverts this downregulation of LPL activity and restores the pattern of LPL proteoforms in this tissue. Importantly, this reversible proteoform-specific regulation during fasting and refeeding indicates that LPL proteoforms are functionally diverse. Further investigation of potential differences in the functional properties of LPL proteoforms showed that all proteoforms exhibit lipolytic activity and have similar heparin-binding affinity, although other functional aspects remain to be investigated. Overall, this study demonstrates the ubiquity, differential distribution and specific regulation of LPL proteoforms in rat tissues and underscores the need to consider the existence of LPL proteoforms for a complete understanding of LPL regulation under physiological conditions.
This study aimed to elucidate the underlying mechanisms for the preventive effect of procyanidin B2 (PB2) on a high-fat diet (HFD)-induced dyslipidemia in C57BL/6 mice through a comprehensive analysis of fecal gut microbiome and plasma metabolomics. PB2 significantly reduced the levels of lipid metabolism biomarkers and increased the antioxidant abilities and lipoprotein lipase activity in mice fed with HFD (P < 0.05). Furthermore, the abundance of Blautia genus was increased, and that of Lachnoclostridium, Clostridium, Bilophila, Proteus genera were decreased in the PB2 group of mice compared to the HFD group of mice. Additionally, purine, unsaturated fatty acids, glycerophospholipids, and bile acids were up-regulated in the HFD fed mice plus PB2, and phenylglucuronide was down-regulated. The correlation analysis indicated that the gut microbiome, particularly Bilophila and Proteus genera, were strongly associated with the metabolic pathway modulation, which might have led to the preventive effect of PB2 on dyslipidemia.
Several strategies must be developed and used before the use of mass spectrometry (MS) to identify endogenous nitroproteins and nitrotyrosine sites: different chemical derivation techniques are used to convert the nitro group to an amino group to resolve the varied MS behaviors of a nitro group and different enrichment techniques are used to preferentially enrich endogenous nitropeptides or nitroproteins to overcome the extreme low abundance of endogenous nitropeptides/nitroproteins in a biological system and sensitivity limitations of a mass spectrometer. MS measurement of nitroproteins must finally serve for real application in a biological system. Matrix-assisted laser desorption/ionization (MALDI) ultraviolet (UV)-laser-induced photochemical decomposition significantly consumes the protonated molecule ion of a nitropeptide to impact detection of endogenous low-abundance nitropeptides/nitroproteins. Protein domain/motif analysis, systems pathway analysis, and structural biological analysis of nitroproteins are significantly needed to elucidate the biological roles of tyrosine nitration.
Protein tyrosine nitration is an addition of a nitro group (NO2) to position-3 of the phenolic ring of a tyrosine residue in a protein, is a relatively chemically stable oxidative/nitrative modification, and is a marker of oxidative injuries. A nitro group is an electron-withdrawing group that changes the physical and chemical properties of the phenolic ring of a tyrosine residue. Together with the discovery of an in vivo denitrase, tyrosine nitration also alters the structure and functions of a protein and is involved in multiple pathophysiological processes, including inflammatory disease, cancer, and neurodegenerative disorders. To clarify the biological roles of endogenous tyrosine nitration, it is necessary to identify the nitroprotein and its nitration site with mass spectrometry. However, differential mass spectrometry behaviors of nitropeptides under different mass spectrometry conditions complicate the identification of endogenous nitroproteins. Furthermore, because of the extreme low-abundance of endogenous nitrotyrosine residues and limited detection sensitivity of mass spectrometry, it is essential to preferentially enrich nitroproteins or nitropeptides prior to mass spectrometry analysis with immunoaffinity enrichment and chemical derivation of nitro groups. This chapter reviews tyrosine nitration and addresses mass spectrometry behaviors of tyrosine nitration, global analysis status of tyrosine nitration, enrichment strategies of nitroproteins or nitropeptides, and future trends in the field of tyrosine nitration to clarify biological functions of tyrosine nitration.
Understanding the impact of inflammation on retinoid transport and metabolism is important because it has implications for the use of vitamin A in public health, and other retinoids in clinical practice. This chapter focuses on dietary vitamin A and inflammation as modulators of retinoid transport, turnover and catabolism, considering, firstly, the transport of plasma retinol by its transport protein, retinol-binding protein (RBP), and secondly, the expression of several key genes that regulate the storage and oxidation of retinoids. Since reduced plasma retinol concentrations resulting from inflammation can be a “look-alike” for vitamin A deficiency, inflammation is an important and vexing complication for the interpretation of low vitamin A. At the level of regulation of the genes and enzymes that esterify retinol for storage and oxidize it to retinoic acid to maintain healthful retinoid concentrations, inflammation rapidly and acutely down regulates expression of several such genes.
Abstract Cardiac diseases have been extensively studied following diabetes and altered metabolism has been implicated in its initiation. In this context, there is a shift from glucose utilization to predominantly fatty acid metabolism. We have focused on the micro- and macro-environments that the heart uses to provide fatty acids to the cardiomyocyte. Specifically, we will discuss the cross talk between endothelial cells, smooth muscles and cardiomyocytes, and their respective secretory products that allows for this shift in metabolism. These changes will then be linked to alterations in the cardiovascular system and the augmented heart disease observed during diabetes. Traditionally, the heart was only thought of as an organ that supplies oxygen and nutrients to the body through its function as a pump. However, the heart as an endocrine organ has also been suggested. Secreted products from the cardiomyocytes include the natriuretic peptides atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Both have been shown to have vasodilatory, diuretic and antihypertensive effects. These peptides have been extensively studied and their deficiency is considered to be a major cause for the initiation of cardiovascular and cardiometabolic disorders. Another secretory enzyme, lipoprotein lipase (LPL), has been implicated in diabetic heart disease. LPL is a triglyceride-hydrolyzing enzyme that is synthesized within the cardiomyocyte and secreted towards the lumen under various conditions. For example, moderate or short-term hyperglycemia stimulates the release of LPL from the cardiomyocytes towards the endothelial cells. This process allows LPL to contact lipoprotein triglycerides, initiating their break down, with the product of lipolysis (free fatty acids, FA) translocating towards the cardiomyocytes for energy consumption. This mechanism compensates for the lack of glucose availability following diabetes. Under prolonged, chronic conditions of hyperglycemia, there is a need to inhibit this mechanism to avoid the excess delivery of FA to the cardiomyocytes, an effect that is known to induce cardiac cell death. Thus, LPL inhibition is made possible by a FA-induced activation of PPAR β/δ, which augments angiopoietin-like 4 (Angptl4), an inhibitor of LPL activity. In the current review, we will focus on the mediators and conditions that regulate LPL and Angptl4 secretion from the cardiomyocyte, which are critical for maintaining cardiac metabolic homeostasis.
Angiopoietin-like protein 3 (Angptl3)-lipoprotein lipase (LPL) pathway may be a useful pharmacologic target for hyperlipidemia. The present study was conducted to test the effect of soluble fiber extracted from Undaria pinnatifida (UP), on hyperlipidemia in apolipoprotein E-deficient (ApoE(-/-)) mice. Forty mice were divided into four groups (n = 10): control group (C57BL/6J mice), ApoE(-/-) mice group, and two groups of ApoE(-/-) mice treated with UP fiber (5 or 10 % per day). UP soluble fiber treatment significantly decreased plasma and hepatic total cholesterol, triglycerides levels, plasma low-density lipoprotein cholesterol, and malondialdehyde concentrations and increased plasma high-density lipoprotein cholesterol level and downregulated protein expression of Angptl3 concomitantly with upregulated protein expression of LPL. In addition, T0901317 caused elevated expression of hepatic Angptl3 protein, and the effect of T0901317 was also abrogated by UP soluble fiber in C57BL/6J mice. The present results suggest that the UP soluble fiber regulates Angptl3-LPL pathway to lessen hyperlipidemia in mice.
Ketosis is a metabolic disorder closely associated with both lipid and carbohydrate metabolism. Recent studies show that angiopoietin-like protein 3 (ANGPTL3) contributes to the development of metabolic disorder. The objective of this study was to explore the inhibitory effect of 1,3,5,8-tetrahydroxyxanthone (Xan), a naturally occurring flavonoid compound, on ketosis and the mechanisms involved in this regulation. After 4weeks, Xan (10 or 30mg/kg, intragastrically) treatment decreased plasma total ketone bodies, malondialdehyde, 8-isoprostane, triglyceride, total cholesterol levels, and hepatic ANGPTL3 expression concomitantly with increased plasma glucose concentration and adipose lipoprotein lipase (LPL) expression in ketosis murine. The present results suggest that Xan regulates ANGPTL3-LPL pathway to lessen the ketosis in mice.
Emerging evidence suggests that the lack of PPARα enhances hepatic steatosis and inflammation in Ppara-null mice when fed a high-fat diet (HFD). Thus, the aim of this study was to determine whether Ppara-null mice are more susceptible to nonalcoholic steatohepatitis (NASH) than their wild-type (WT) counterparts following short-term feeding with a HFD. Age-matched male WT and Ppara-null mice were randomly assigned to consume ad libitum a standard Lieber-DeCarli liquid diet (STD) (35% energy from fat) or a HFD (71% energy from fat) for 3 wk. Liver histology, plasma transaminase levels, and indicators of oxidative/nitrosative stress and inflammatory cytokines were evaluated in all groups. Levels of lobular inflammation and the NASH activity score were greater in HFD-exposed Ppara-null mice than in the other 3 groups. Biochemical analysis revealed elevated levels of ethanol-inducible cytochrome P450 2E1 and TNFα accompanied by increased levels of malondialdehyde as well as oxidized and nitrated proteins in Ppara-null mice. Elevated oxidative stress and inflammation were associated with activation of c-Jun-N-terminal kinase and p38 kinase, resulting in increased hepatocyte apoptosis in Ppara-null mice fed a HFD. These results, with increased steatosis, oxidative stress, and inflammation observed in Ppara-null mice fed a HFD, demonstrate that inhibition of PPARα functions may increase susceptibility to high fat-induced NASH.
Lipoprotein lipase (LPL) is the main enzyme responsible for the distribution of circulating triacylglycerides in tissues. Its regulation via release from active sites in the vascular endothelium is poorly understood. In a previous study we reported that in response to acute immobilization (IMMO), LPL activity rapidly increases in plasma and decreases in white adipose tissue (WAT) in rats. In other stress situations IMMO triggers a generalized increase in nitric oxide (NO) production.
Here we demonstrate that in rats: 1) in vivo acute IMMO rapidly increases NO concentrations in plasma 2) during acute IMMO the WAT probably produces NO via the endothelial isoform of nitric oxide synthase (eNOS) from vessels, and 3) epididymal WAT perfused in situ with an NO donor rapidly releases LPL from the endothelium.
We propose the following chain of events: stress stimulus / rapid increase of NO production in WAT (by eNOS) / release of LPL from the endothelium in WAT vessels. This chain of events could be a new mechanism that promotes the rapid decrease of WAT LPL activity in response to a physiological stimulus.
Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.
Hyperlipidemia frequently accompanies infectious diseases and may be due to increases in lipoprotein production or decreases in lipoprotein clearance. The administration of endotoxin (LPS) has been used to mimic infection and prior studies demonstrate that LPS produces hypertriglyceridemia. In the present study in rodents, the dose of LPS necessary to induce hyperlipidemia was orders of magnitude less than that necessary to induce shock and death. As little as 10 ng/100 g body weight induced hypertriglyceridemia and this increase in serum triglyceride levels occurred rapidly (78% increase at 2 h). At high doses of LPS (50 micrograms/100 g body weight), the clearance of triglyceride-rich lipoproteins was decreased. At low doses of LPS (100 ng/100 g body weight), triglyceride clearance was not altered but the hepatic secretion of triglyceride was increased. Low dose LPS stimulated hepatic de novo fatty acid synthesis and lipolysis, both of which provided a source of fatty acids for the increase in hepatic triglyceride production. High dose LPS did not increase hepatic fatty acid synthesis or peripheral lipolysis, and hepatic triglyceride secretion was not stimulated. Thus, low dose LPS produces hypertriglyceridemia by increasing hepatic lipoprotein production, while high dose LPS produces hypertriglyceridemia by decreasing lipoprotein catabolism. Administration of anti-tumor necrosis factor (TNF) antibodies or interleukin 1 (IL-1) receptor antagonist did not prevent the increase in serum triglyceride levels induced by LPS. However, anti-TNF antibodies and interleukin 1 receptor antagonist (IL-1ra) blocked the increase in serum triglycerides induced by TNF or IL-1, respectively. These data suggest that neither of these cytokines is absolutely required for the increase in serum triglycerides induced by LPS, raising the possibility that other cytokines, small molecular mediators, or LPS itself may play a crucial role.
Previous studies have demonstrated that endotoxin/lipopolysaccharide treatment causes a decrease in adipose tissue and heart lipoprotein lipase (LPL) activities in rats, producing hypertriglyceridemia in these animals. To examine the mechanisms for this effect of endotoxin, we studied the effects of endotoxin administration on LPL mRNA, and LPL synthetic rates and activity in rat adipose tissue and heart. Endotoxin treatment (i.p., 3 mg/100 g body weight or higher doses) produced a pronounced increase in serum triglycerides associated with a 65% decrease in adipose tissue and heart LPL activities within 7 h. Fast protein liquid chromatography (FPLC), used to separate lipoproteins in rat serum, showed that the increase in triglyceride was all in the very low density lipoprotein fraction which was accompanied by a concomitant decrease in high density lipoprotein. In contrast, there was no change in adipose tissue or heart LPL mRNA up to 24 h after treatment and no change in adipose tissue LPL synthetic rate, as measured by L-[35S]methionine incorporation and immunoprecipitation. Plasma insulin levels remained unchanged. The results indicate that endotoxin-induced hypertriglyceridemia in rats can be attributed to an impaired triglyceride clearance associated with a decrease of LPL activity mediated at a post-transcriptional level.
Reaction of nitric oxide (NO') with superoxide radical generates ~roxynitrite, which can decompose to products that nitrate aromatic amino acids. Such nitro-aromatics may be 'markers' of NW-dependent oxidative damage. Blood serum and synovial fluid from patients with the inffammato~ joint disease rheumatoid arthritis contain 3nitrotyrosine. By contrast, body fluids from normal subjects and patients with osteoarthritis contain no detectable 3-nitrotyrosine; much lower levels were found in serum from patients in the early stages of rheumatoid arthritis. This is evidence that NO plays a role in joint damage in rheumatoid arthritis.
Homogenates from rat spleen and lung could modify nitrotyrosine-containing BSA. With incubation, nitrotyrosine-containing BSA lost its epitope to a monoclonal antibody that selectively recognized nitrotyrosine-containing proteins. In the presence of protease inhibitors, the loss of the nitrotyrosine epitope occurred without protein degradation and hydrolysis. This activity was found in supernatant but not particulate fractions of spleen homogenates. The factor was heat labile, was sensitive to trypsin treatment, and was retained after passage through a membrane with a 10-kDa retention. The activity was time- and protein-concentration dependent. The activity increased about 2-fold in spleen extracts with endotoxin (bacterial lipopolysaccharide) treatment of animals, suggesting that the activity is inducible or regulatable. Other nitrotyrosine-containing proteins also served as substrates, while free nitrotyrosine and some endogenous nitrotyrosine-containing proteins in tissue extracts were poor substrates. Although the product and possible cofactors for this reaction have not yet been identified, this activity may be a "nitrotyrosine denitrase" that reverses protein nitration and, thus, decreases peroxynitrite toxicity. This activity was not observed in homogenates from rat liver or kidney, suggesting that there may also be some tissue specificity for the apparent denitrase activity.
When bacterial lipopolysaccharide (LPS) enters the bloodstream, it is thought to have two general fates. If LPS binds to circulating leukocytes, it triggers innate host defense mechanisms and often elicits toxic reactions. If instead LPS binds to plasma lipoproteins, its bioactivity is largely neutralized. This study shows that lipoproteins can also take up LPS that has first bound to leukocytes. When monocytes were loaded with [(3)H]LPS and then incubated in plasma, they released over 70% of the cell-associated [(3)H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [(3)H]LPS remained tightly associated with the cells. The transfer reaction could be reproduced in the presence of pure native lipoproteins or reconstituted high density lipoprotein. Plasma immunodepletion experiments and experiments using recombinant LPS transfer proteins revealed that soluble CD14 significantly enhances LPS release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell surface can be released. Lipoprotein-mediated LPS release was accompanied by a reduction in several cellular responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo.
Inflammation in asthma, sepsis, transplant rejection, and many neurodegenerative diseases associates an up-regulation of NO synthesis with increased protein nitration at tyrosine. Nitration can cause protein dysfunction and is implicated in pathogenesis, but few proteins that appear nitrated in vivo have been identified. To understand how this modification impacts physiology and disease, we used a proteomic approach toward targets of protein nitration in both in vivo and cell culture inflammatory disease models. This approach identified more than 40 nitrotyrosine-immunopositive proteins, including 30 not previously identified, that became modified as a consequence of the inflammatory response. These targets include proteins involved in oxidative stress, apoptosis, ATP production, and other metabolic functions. Our approach provides a means toward obtaining a comprehensive view of the nitroproteome and promises to broaden understanding of how NO regulates cellular processes.
The nitration of protein tyrosine residues represents an important post-translational modification during development, oxidative stress, and biological aging. To rationalize any physiological changes with such modifications, the actual protein targets of nitration must be identified by proteomic methods. While several studies have used proteomics to screen for 3-nitrotyrosine-containing proteins in vivo, most of these studies have failed to prove nitration unambiguously through the actual localization of 3-nitrotyrosine to specific sequences by mass spectrometry. In this paper we have applied sequential solution isoelectric focusing and SDS-PAGE for the proteomic characterization of specific 3-nitrotyrosine-containing sequences of nitrated target proteins in vivo using nanoelectrospray ionization-tandem mass spectrometry. Specifically, we analyzed proteins from the skeletal muscle of 34-month-old Fisher 344/Brown Norway F1 hybrid rats, a well accepted animal model for biological aging. We identified the 3-nitrotyrosine-containing sequences of 11 proteins, including cytosolic creatine kinase, tropomyosin 1, glyceraldehyde-3-phosphate dehydrogenase, myosin light chain, aldolase A, pyruvate kinase, glycogen phosphorylase, actinin, gamma-actin, ryanodine receptor 3, and neurogenic locus notch homolog. For creatine kinase and neurogenic locus notch homolog, two 3-nitrotyrosine-containing sequences were identified, i.e. at positions 14 and 20 for creatine kinase and at positions 1175 and 1205 for the neurogenic locus notch homolog. The selectivity of the in vivo nitration of creatine kinase at Tyr14 and Tyr20 does not correspond to the product selectivity in vitro, where exclusively Tyr82 was nitrated when creatine kinase was exposed to peroxynitrite. The latter experiments demonstrate that the in vitro exposure of an isolated protein to peroxynitrite may not always be a good model to mimic protein nitration in vivo.
Bacterial endotoxin (i.e., lipopolysaccharide [LPS]) elicits dramatic responses in the host, including elevated plasma lipid
levels due to increased synthesis and secretion of triglyceride-rich lipoproteins by the liver and inhibition of lipoprotein
lipase. This cytokine-induced hyperlipoproteinemia, clinically termed the “lipemia of sepsis,” was customarily thought to
involve the mobilization of lipid stores to fuel the host response to infection. However, because lipoproteins can also bind
and neutralize LPS, we have long postulated that triglyceride-rich lipoproteins (very-low-density lipoproteins and chylomicrons)
are also components of an innate, nonadaptive host immune response to infection. Recent research demonstrates the capacity
of lipoproteins to bind LPS, protect against LPS-induced toxicity, and modulate the overall host response to this bacterial
toxin.
Nitric oxide (NO) is synthesized from L-arginine by NO synthase in virtually all cell types. Emerging evidence shows that NO regulates the metabolism of glucose, fatty acids and amino acids in mammals. As an oxidant, pathological levels of NO inhibit nearly all enzyme-catalyzed reactions through protein oxidation. However, as a signaling molecule, physiological levels of NO stimulate glucose uptake as well as glucose and fatty acid oxidation in skeletal muscle, heart, liver and adipose tissue; inhibit the synthesis of glucose, glycogen, and fat in target tissues (e.g., liver and adipose); and enhance lipolysis in adipocytes. Thus, an inhibition of NO synthesis causes hyperlipidemia and fat accretion in rats, whereas dietary arginine supplementation reduces fat mass in diabetic fatty rats. The putative underlying mechanisms may involve multiple cyclic guanosine-3',5'-monophosphate-dependent pathways. First, NO stimulates the phosphorylation of adenosine-3',5'-monophosphate-activated protein kinase, resulting in (1) a decreased level of malonyl-CoA via inhibition of acetyl-CoA carboxylase and activation of malonyl-CoA decarboxylase and (2) a decreased expression of genes related to lipogenesis and gluconeogenesis (glycerol-3-phosphate acyltransferase, sterol regulatory element binding protein-1c and phosphoenolpyruvate carboxykinase). Second, NO increases the phosphorylation of hormone-sensitive lipase and perilipins, leading to the translocation of the lipase to the neutral lipid droplets and, hence, the stimulation of lipolysis. Third, NO activates expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, thereby enhancing mitochondrial biogenesis and oxidative phosphorylation. Fourth, NO increases blood flow to insulin-sensitive tissues, promoting substrate uptake and product removal via the circulation. Modulation of the arginine-NO pathway through dietary supplementation with L-arginine or L-citrulline may aid in the prevention and treatment of the metabolic syndrome in obese humans and companion animals, and in reducing unfavorable fat mass in animals of agricultural importance.
Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S-nitrosylation and species-specific peptide identification.
Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the renal medulla may play a key role in the development and progression of hypertension. This may arise through disruption of nitric oxide signalling in the medulla and be accompanied by enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3-nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously hypertensive rats. We have identified several nitrated proteins with both varied subcellular location and functional roles. These proteins are involved in nitric oxide signalling, antioxidant defense and energy metabolism. Moreover, increased nitration was observed in conjunction with enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxidative stress, and that resulting protein modifications may contribute to the progression of hypertension.
A number of recent studies have demonstrated that peroxynitrite (ONOO⁻) can react with a host of biomolecules, particularly those containing aromatic amino acid residues, resulting in a number of modifications which have been found in association with diverse pathological conditions. Electrospray ionisation tandem mass spectrometry with and without liquid chromatographic separation has been used to examine a series of model peptides following their treatment with peroxynitrite at physiological pH. The mass spectra of sequences containing two tyrosine residues showed the formation of both mononitrated and dinitrated species, while those with phenylalanine showed no detectable nitration. Tryptic digests of two of the investigated peptides were also examined by liquid chromatography/electrospray mass spectrometry, which yielded further information on the competition for NO2 by multiple nitration sites within a given sequence. In addition, tryptic digests of the mononitrated component of sequences containing tyrosine and tryptophan indicated that mononitration was limited to tyrosine. Furthermore, some of the presented data indicate that, as well as nitrotyrosine and nitrotryptophan formation, the reaction of ONOO⁻ can result in oxidation as well as the formation of labile adducts with NO2. Copyright © 1999 John Wiley & Sons, Ltd.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
• The effects of sepsis on lipid metabolism have not been clearly defined. This study was designed to observe the changes in adipose tissue lipoprotein lipase (LPL) and fatty acid synthetase (FAS) after administration of Escherichia coli bacteria. Male Lewis rats, weighing 245 to 270 g, were assigned to two groups and fed a powdered chow diet for 14 days. On day 14, one group was inoculated with E coli. Twenty-four hours later, both groups were killed by decapitation. Serum triglyceride levels were significantly elevated in the E coli–treated rats. Adipose tissue LPL and FAS activity was significantly decreased by 50% in E coli–treated rats compared with the control rats. These results suggest that the elevated serum triglyceride levels associated with sepsis may be caused by a decreased rate of clearance of lipids from the blood and an increased rate of hepatic lipid synthesis.(Arch Surg 1982;117:144-147)
Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with leads to formation of peroxynitrite ONOO–, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing.
The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.
The kinetics of decomposition of pernitrite in excess of alkali have been studies at several temperatures. These show that decomposition occurs through the pernitrous acid molecule, HO·ONO, with ΔH‡= 12·5 kcal. mole–1 and ΔS‡= 26 e.u. The value of ΔH‡ is not consistent with the accepted view that pernitrous acid decomposes via homolytic fission of the O–O link. This problem is discussed and other, non-homolytic, mechanisms suggested. An upper limit for the pKA of pernitrous acid at 2° of 8·3 has been calculated. The first isolation of a solid pernitrite is reported. The extinction coefficient of the pernitrite anion at 302 mµ(λmax.) has been redetermined as 1670 ± 50.
A number of recent studies have demonstrated that peroxynitrite (ONOO−) can react with a host of biomolecules, particularly those containing aromatic amino acid residues, resulting in a number of modifications which have been found in association with diverse pathological conditions. Electrospray ionisation tandem mass spectrometry with and without liquid chromatographic separation has been used to examine a series of model peptides following their treatment with peroxynitrite at physiological pH. The mass spectra of sequences containing two tyrosine residues showed the formation of both mononitrated and dinitrated species, while those with phenylalanine showed no detectable nitration. Tryptic digests of two of the investigated peptides were also examined by liquid chromatography/electrospray mass spectrometry, which yielded further information on the competition for NO2 by multiple nitration sites within a given sequence. In addition, tryptic digests of the mononitrated component of sequences containing tyrosine and tryptophan indicated that mononitration was limited to tyrosine. Furthermore, some of the presented data indicate that, as well as nitrotyrosine and nitrotryptophan formation, the reaction of ONOO− can result in oxidation as well as the formation of labile adducts with NO2. Copyright © 1999 John Wiley & Sons, Ltd.
Specific antibodies are essential tools for studying proteins. P66 is a chicken polyclonal antibody raised against bovine lipoprotein lipase (LPL) that has been used in earlier studies. Here, we developed a two-dimensional (2D) Western blot with reducing gels, using commercial bovine LPL (53 kDa) as a standard and P66 for detection. Our results revealed incomplete purification of commercial LPL and nonspecificity of P66, both undetectable in one-dimensional analysis. Antithrombin III (ATIII) was identified as both a major contaminant in commercial LPL and a cross-reacting protein with P66. Although LPL purification methods were presumably designed to eliminate ATIII, here we demonstrate that some procedures fell short of this objective and thus led to the production of a nonspecific antibody. Our results define 2D electrophoresis/Western blot and mass spectrometric protein identification as the most reliable procedure for validating LPL purity and the specificity of antibodies against this enzyme.
Lipoprotein lipase (LPL) plays a pivotal role in lipid metabolism and is implicated in several pathophysiological conditions. A large number of LPL studies have been performed in rat, although the amount of information derived from direct study of the protein in this species is limited. Here we attempted to examine possible modifications of LPL using proteomic tools. By combining high-resolution two-dimensional gel electrophoresis and Western blot with biological mass spectrometry we demonstrate the coexistence of multiple LPL pI isoforms in rat heart. We studied the origin of this pI heterogeneity by: (1) comparison with the 2D pattern of LPL from post-heparin rat plasma (as a source of mature LPL); (2) protein dephosphorylation; (3) protein deglycosylation; and (4) partial sequencing of LPL isoforms. The results reveal that LPL pI heterogeneity does not correspond to different stages of intracellular maturation or protein phosphorylation. It can be partially explained by glycosylation, although other post-translational modifications must also be involved. We also report the first partial sequence to be obtained from direct study of rat LPL protein. These findings should be the basis for further research aimed at identifying the molecular differences between LPL isoforms and exploring their potential functional implications.
Tyrosine nitration is a covalent post-translational protein modification derived from the reaction of proteins with nitrating agents. Tyrosine nitration has been used as a marker of oxidant burden in human diseases. However, it remains unclear whether protein nitration is responsible for alterations in protein function that imparts an increased risk for disease development or unfavorable outcomes. Emerging data implicate tyrosine nitration as a mediator of immune responses suggesting a novel biological function for this protein modification.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.
Lipopolysaccharide (LPS), the active principle of certain endotoxins, protein-free perfused in rat hearts leads in 3 h to a considerable loss of lipoprotein lipase (LPL) activity. In the presence of albumin LPS has virtually no effect. Tumor necrosis factor (TNF) added instead of LPS had no effects on LPL activity during 3 hin vitro perfusion.
LPS injected into rats intravenously leads within 3 h to severe toxic phenomena amongst which increased capillary permeability. This was visualized as increased rate of interstitial fluid formation in Langendorff hearts mounted 3 h after rats had been treated with LPS. LPL activity did not decline in 3 h lasting endotoxemia. Six hours after LPS injection, however, cardiac LPL activity was considerably lowered, although immunoblotting and immunohistochemistry still showed LPL protein to be present. These date indicate the presence of a considerable pool of inactive LPL protein in addition to active LPL, that can be released in the presence of heparin. The LPL activity is lowered by LPS injection after a lag phase of at least 3 h, while capillary endothelial cells are influenced more rapidly. The relatively late expression of TNF toxicity in cardiomyocytes of the intact heart is discussed.
The present studies were designed to delineate changes in heart and adipose tissue lipoprotein lipase (LPL) activity following the administration of E. coli endotoxin. Plasma triglyceride levels were elevated in animals given endotoxin compared to saline-injected controls. Heart LPL activity decreased from 126.4 mumol fatty acid released per gram wet wt per hour in control rats to less than 22.5 mumol . g-1 . h-1 by 7 h following the injection of endotoxin. Although endotoxin was administered in doses producing 0-100% mortalities in a 24-h period, myocardial LPL activity was depressed to the same extent (75-80%) regardless of dose. The response of adipose tissue was less pronounced. Epididymal fat pad LPL activity fell significantly over the 24-h observation period in control and endotoxin-treated rats with the latter group somewhat more depressed 7 h after treatment. The findings are consistent with the suggestion that hypertriglyceridemia often observed during endotoxic shock may be related to depressed LPL activity; the degree of depression is probably tissue dependent.
The effects of sepsis on lipid metabolism have not been clearly defined. This study was designed to observe the changes in adipose tissue lipoprotein lipase (LPL) and fatty acid synthetase (FAS) after administration of Escherichia coli bacteria. Male Lewis rats, weighing 245 to 270 g, were assigned to two groups and fed a powdered chow diet for 14 days. On day 14, one group was inoculated with E coli. Twenty-four hours later, both groups were killed by decapitation. Serum triglyceride levels were significantly elevated in the E coli-treated rats. Adipose tissue LPL and FAS activity was significantly decreased by 50% in E coli-treated rats compared with the control rats. These results suggest that the elevated serum triglyceride levels associated with sepsis maybe caused by a decreased rate of clearance of lipids from the blood and an increased rate of hepatic lipid synthesis.
A new method for the preparation of high concentrations of peroxynitrite (up to 1 M) is described. The synthesis uses a two-phase system and involves a displacement reaction by the hydroperoxide anion (in the aqueous phase) on isoamyl nitrite (in the organic phase). The product peroxynitrite remains in the aqueous phase, whereas isoamyl alcohol forms a new organic phase along with the unreacted isoamyl nitrite. The aqueous phase contains some 0.15 M isoamyl alcohol and the unreacted hydrogen peroxide, but no isoamyl nitrite. Removal of isoamyl alcohol or traces of isoamyl nitrite is accomplished by washing the aqueous phase with dichloromethane, chloroform, or hexane. A near total removal of hydrogen peroxide is then achieved by passing the solutions through a short column of manganese dioxide. The peroxynitrite in these postprocessed solutions has broad absorption spectrum with a maximum around 302 nm, follows a characteristic first-order decomposition at pH 7.2 and 25 degrees C (k = 0.34 +/- 0.1 s-1), and reacts with organic compounds to give either nitrated or one-electron transfer products. When stored frozen at -20 degrees C, these peroxynitrite solutions decompose at a rate of about 1.7 % per day and should be used within 2-4 weeks. For short-term storage of about 1 week or less, these solutions can be stored at refrigerator temperatures (approximately 5 degrees C) where peroxynitrite has a half-life of about 7 days.
Lipoprotein lipase (LPL) activity is known to be synthesized, active and functional in the 1-day-old rat liver: it peaks just at birth triggered by parturition. During suckling LPL mRNA, LPL synthesis and LPL activity are still high at 5 days and then fade reaching adult values at weaning. How LPL expression is gradually extinguished is not known. Therefore we studied the effect of different doses of several hormones on LPL activity released by incubated hepatocytes from 5-day-old rats. In the presence of heparin the release of LPL activity in the medium was linear until 3 h and was always significantly increased vs. without heparin. At 3 h in the presence of heparin the main hormonal effects were: dose-dependent increase (30-60%) with dexamethasone; dose-dependent increase (20-60%) with glucagon; dose-independent decrease (50-60%) with ethinylestradiol, testosterone, progesterone and prolactin; no effect with insulin; 20-40% increase with adrenaline < 1 mM but 40-50% decrease with noradrenaline < 10 microM. Increase of LPL release by glucagon and adrenaline agrees with the increased LPL expression we previously found in an undifferentiated hepatoma cell line when the adenylate cyclase/protein kinase A pathway was activated. The effect of glucagon is concordant with our previous observations that fasting increases liver LPL activity in neonatal rats. The high estradiol levels known to be present in male and female 9-19-day-old rats might contribute to liver LPL extinction during suckling.
We previously reported that tumor necrosis factor-alpha (TNF-alpha)/cachectin suppresses lipoprotein lipase activity and its gene expression in brown adipocytes differentiated in culture. Recent evidence suggests that the effect of TNF-alpha over various cells is related to the enhanced production of nitric oxide (NO). The present study examined whether the suppressive effect of TNF-alpha on lipoprotein lipase activity is mediated by production of NO in the brown adipocytes. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that TNF-alpha caused a concentration- and time-dependent expression of inducible NO synthase in brown adipocytes. Increasing concentrations of TNF-alpha (0.5-50 ng/ml) for 24 h resulted in a concentration-dependent decrease in lipoprotein lipase activity with reciprocal increase in nitrite production in the medium. The suppressive effect of TNF-alpha on lipoprotein lipase activity was significantly prevented by NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, but not by D-NAME, an inactive isomer. Furthermore, 8-bromoguanosine 3',5'-cyclic monophosphate, cell permeant cGMP, suppressed lipoprotein lipase activity and 1 H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor for soluble guanylate cyclase, restored the TNF-alpha-suppressed lipoprotein lipase activity. These results suggest that TNF-alpha stimulates brown adipocytes to express inducible NO synthase, followed by production of NO, which in turn mediates the suppressive effect of TNF-alpha on lipoprotein lipase activity. The effect of NO is mediated, at least partly, through production of cGMP.
Chylomicrons (CM) can bind endotoxin (lipopolysaccharide [LPS]), forming CM-LPS complexes, and protect against endotoxic shock and death in rodent models of gram-negative sepsis. The liver appears to play a central role in this process, as demonstrated by the increased uptake of LPS by this organ. We examined the effect of CM on the uptake and cellular response to injected 125I-LPS by hepatocytes and hepatic nonparenchymal cells. Whereas CM increased the uptake of LPS by both hepatocytes and Kupffer cells, the increase was proportionately greater in hepatocytes than Kupffer cells. Importantly, CM-LPS complexes inhibited inducible nitric oxide synthase (iNOS) mRNA expression and NO production in Kupffer cells and endothelial cells, reducing mRNA levels by 45% to 50% as compared with LPS alone. CM-bound LPS also reduced NO production by hepatocytes in response to cytokine stimulation. Lastly, CM-LPS complexes yielded a concentration-dependent inhibition of LPS-induced tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells in vitro. These data indicate that the mechanism by which CM protect against endotoxicity may involve an increased uptake of LPS by hepatocytes. Moreover, uptake of CM-bound LPS by liver cells attenuates the capacity of these cells to respond to proinflammatory stimulation. These results highlight important anti-inflammatory properties of CM.
Nitric oxide (NO) is a key messenger molecule in several cell types. NO formation is catalyzed by a family of NO synthases (NOS) that use L-arginine as a substrate. Rat adipose tissue expresses the inducible, macrophage-type, nitric oxide (NO) synthase isoform (iNOS). Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) markedly increases the expression and activity of iNOS in both white and brown adipose tissues, as well as in skeletal muscle. iNOS induction can be reproduced in vitro by treatment of cultured white or brown adipocytes or L6 myocytes with LPS and inflammatory cytokines (TNFalpha, IFNgamma). The physiological role of NO in adipose tissues and skeletal muscle is still obscure. Recent evidence suggests that NO may be implicated in the regulation of energy metabolism. Using both pharmacological and genetic models of iNOS invalidation, we have recently begun to uncover a role for NO in the modulation of glucose transport and lipoprotein hydrolysis. These studies support the emerging concept that NO may fulfill the dual role of modulating energy metabolism in both physiological and pathological conditions as well as contributing to local immune defense during inflammatory processes.
Free fatty acids (FAs) derived from adipose tissue (AT) are, quantitatively, the most important fuel in mammals, and provide more than half of the caloric needs during periods when dietary substrates are lacking. Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in the lipolysis of stored triglycerides (TGs) in AT, i.e., the hydrolysis of triacylglycerol to diacylglycerol (for a review on HSL, see
ref.
1). It also catalyzes the subsequent hydrolysis of diglycerides (DGs) and monoglycerides (MGs). A second lipase, mono-glyceride lipase (MGL), is required to obtain complete hydrolysis of MGs. Selective immunoprecipitation of this lipase, from an AT preparation containing both HSL and MGL, leads to marked reduction in the glycerol release and accumulation of MGs (2,3). FAs from the action of HSL and MGL are either released into the circulation or, alternatively, re-esterified directly. In humans, on a typical Western diet, de novo lipogenesis is rarely observed, and storage of TGs is mostly accomplished through re-esterification of preformed FAs derived from the blood. Lipoprotein lipase (LPL), on the endothelial cell, catalyzes the hydrolysis of TGs from chylomicrons and very low density lipoproteins (VLDL, for reviews on LPL, see
refs.
4–7). The released fatty acids are either transported into AT for re-esterification and storage or, alternatively, returned into the circulation. MGs, particularly, 2-monoacylglycerols from the action of LPL, are believed to be hydrolyzed by MGL after their diffusion into the adipocytes.
Hypertriglyceridemia (hyperTG) is a common characteristic of endotoxemia. At high doses of endotoxin, hyperTG is caused by a reduction in the activity of lipoprotein lipase (LPL), the enzyme responsible for TG intravascular clearance. The present studies were aimed at evaluating the role of nitric oxide (NO), overproduced in endotoxemia through the activation of inducible NO synthase (iNOS), in the decrease in tissue LPL activity brought about by lipopolysaccharide (LPS, 15 mg/kg). In rats, the specific iNOS inhibitor aminoguanidine (AGN, 100 mg/kg) prevented the LPS-induced threefold elevation in plasma TG concentration, through actions that were not related to changes in hepatic TG secretion. AGN administration abrogated the LPS- induced reduction in LPL activity in skeletal muscle but not that in white adipose tissue (WAT). Quantification of LPL mRNA revealed that AGN prevented LPS-mediated alterations in muscle LPL activity through posttranscriptional mechanisms. The above findings were confirmed in iNOS (-/-) knockout mice injected with endotoxin. LPS increased plasma TG concentration in wild-type mice by 75%, whereas hyperTG did not occur in knockout mice. Skeletal muscle LPL activity decreased by 40% in wild-type mice but remained unchanged in knockout mice 6 h after LPS injection, whereas WAT LPL activity decreased to one-third of control levels in both genotypes. These findings demonstrate that, after LPS administration, reduced muscle LPL activity and the resulting hyperTG are caused by the overproduction of NO via the induction of iNOS. The findings further provide evidence for a central role of muscle LPL in the response of TG metabolism to acute endotoxemia.
Nitric oxide (NO) is a signal molecule with functions such as neurotransmission, local vascular relaxation, and anti-inflammation in many physiological and pathological processes. Various factors regulate its intracellular lifetime. Due to its high reactivity in biological systems, it is transformed in the bloodstream into nitrates (NO(-)(3)) by oxyhemoglobin. The Griess reaction is a technically simple method (spectrophotometric, 540 nm) for the analysis of nitrites (NO(-)(2)) in aqueous solutions. We studied the interference of common anticoagulants in the quantification of nitrate and nitrite in plasma samples by the Griess method. We obtained rat plasma using heparin or sodium EDTA as anticoagulants, then added, or otherwise, known NO(-)(3) amounts in order to calculate their recovery. We also studied the effect of ultra-filtration performed before Griess reaction on plasma and aqueous solutions of various anticoagulants (heparin, EDTA, and also sodium citrate) to compare the recoveries of added NO(-)(3) or NO(-)(2). We used standards of NO(-)(3) or NO(-)(2) for quantification. We conclude that: (i) The bacterial nitrate reductase used to reduce NO(-)(3) to NO(-)(2) is unstable in certain storage conditions and interferes with different volumes of plasma used. (ii) The ultrafiltration (which is sometimes performed before the Griess reaction) of plasma obtained with EDTA or citrate is not recommended because it leads to overestimation of NO(minus sign)(3). In contrast, ultrafiltration is necessary when heparin is used. (iii) The absorbance at 540 nm attributed to plasma itself (basal value or background) interferes in final quantification, especially when ultrafiltration is not performed. For the quantification of plasma NO(-)(3) we recommend: sodium EDTA as anticoagulant, no ultrafiltration of plasma, and measurement of the absorbance background of each sample.
The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.
Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.
The role of nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) is very complex. Induction of iNOS expression and hence NO production has been described to have beneficial antiviral, antiparasital, microbicidal, immunomodulatory, and antitumoral effects. However, induced at the wrong place or at the wrong time, iNOS has detrimental consequences and seems to be involved in the pathophysiology of different human diseases. The pathways regulating iNOS expression seem to vary in different cells or different species. In general, activation of the transcription factors nuclear factor (NF)-kappaB and signal transducer and activator of transcription (STAT)-1alpha and thereby activation of the iNOS promoter seems to be an essential step in the regulation of iNOS expression in most cells. Also, post-transcriptional mechanisms are critically involved in the regulation of iNOS expression.
S-nitrosylation, the covalent attachment of a nitrogen monoxide group to the thiol side chain of cysteine, has emerged as an important mechanism for dynamic, post-translational regulation of most or all main classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of nitric oxide (NO) on cellular signal transduction, and provides a mechanism for redox-based physiological regulation.
Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O2- leads to formation of peroxynitrite ONOO-, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.
In the Griess reaction, first reported by Johann Peter Griess in 1879 as a method of analysis of nitrite (NO(2)(-)), nitrite reacts under acidic conditions with sulfanilic acid (HO(3)SC(6)H(4)NH(2)) to form a diazonium cation (HO(3)SC(6)H(4)-N[triple bond]N(+)) which subsequently couples to the aromatic amine 1-naphthylamine (C(10)H(7)NH(2)) to produce a red-violet coloured (lambda(max) approximately 540 nm), water-soluble azo dye (HO(3)SC(6)H(4)-NN-C(10)H(6)NH(2)). The identification of nitrite in saliva has been the first analytical application of this diazotization reaction in 1879. For a century, the Griess reaction has been exclusively used to identify analytically bacterial infection in the urogenital tract, i.e. to identify nitrite produced by bacterial reduction of nitrate (NO(3)(-)), the major nitrogen oxide anion in human urine. Since the discovery of the l-arginine/nitric oxide (l-Arg/NO) pathway in 1987, however, the Griess reaction is the most frequently used analytical approach to quantitate the major metabolites of NO, i.e. nitrite and nitrate, in a variety of biological fluids, notably blood and urine. The Griess reaction is specific for nitrite. Analysis of nitrate by this reaction requires chemical or enzymatic reduction of nitrate to nitrite prior to the diazotization reaction. The simplicity of the Griess reaction and its easy and inexpensive analytical feasibility has attracted the attention of scientists from wide a spectrum of disciplines dedicated to the complex and challenging L-Arg/NO pathway. Today, we know dozens of assays based on the Griess reaction. In principle, every laboratory in this area uses its own Griess assay. The simplest Griess assay is performed in batch commonly as originally reported by Griess. Because of the recognition of numerous interferences in the analysis of nitrite and nitrate in biological fluids and of the desire to analyze these anions simultaneously, the Griess reaction has been repeatedly modified and automated. In recent years, the Griess reaction has been coupled to HPLC, i.e. is used for post-column derivatization of chromatographically separated nitrite and nitrate. Such a HPLC-Griess system is even commercially available. The present article gives an overview of the currently available assays of nitrite and nitrate in biological fluids based on the Griess reaction. Special emphasis is given to human plasma and urine, to quantitative aspects, as well as to particular analytical and pre-analytical factors and problems that may be associated with and affect the quantitative analysis of nitrite and nitrate in these matrices by assays based on the Griess reaction. The significance of the Griess reaction in the L-Arg/NO pathway is appraised.
Nitric oxide-derived metabolites have been demonstrated to covalently modify cellular protein repertoire, thus affecting specific enzymatic functions. Among the various redox posttranslational modifications, protein nitration has been broadly recognized by immunological, spectroscopical, and chromatographic methods as a widespread reaction regulating essential phatophysiological processes. With the introduction of matrix-assisted laser desorption and electrospray as soft ionization methods for mass spectrometry of biomolecules, nitration has been investigated directly at the protein level, assigning polypeptide modification sites. Peptide mass fingerprinting and fragment fingerprinting upon collisional fragmentation approaches have been widely used to this purpose. This chapter describes how minimal levels of nitration present on a model protein, namely bovine serum albumin, generated in vitro by ONOO(-) treatment, were ascertained by integrated mass spectrometry approaches, identifying sites of modification. Critical considerations on the limits of each mass spectrometric ionization methods are also provided.
Protein 3-nitrotyrosine is a posttranslational modification found in many pathological conditions from acute to chronic diseases. Could 3-nitrotyrosine formation participate on the basis of these diseases or is it just a marker connected with the associated nitroxidative stress? In vitro and in vivo data, including proteomic research, show that protein tyrosine nitration is a selective process where only a small amount of proteins is found nitrated and one or a few tyrosine residues are modified in each. Accumulating data suggest a strong link between protein 3-nitrotyrosine and the mechanism involved in disease development. In this review, we analyze the factors determining protein 3-nitrotyrosine formation, the functional and biological outcome associated with protein tyrosine nitration, and the fate of the nitrated proteins.
Peptide nitration by peroxynitrite: characterisation of the nitration sites by liquid chromatography/tandem mass spectrometry
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