Rate Of Proteolysis Research Articles

Leucine Protects Against Skeletal Muscle Atrophy in Lipopolysaccharide-Challenged Rats

Skeletal muscle atrophy is a decrease in muscle mass that occurs when protein degradation exceeds protein synthesis. Leucine (Leu), an essential branched-chain amino acid in animal nutrition, regulates skeletal muscle protein metabolism. Two experiments were conducted to evaluate whether Leu could alleviate lipopolysaccharide (LPS)-induced skeletal muscle wasting by modulating skeletal muscle protein synthesis and degradation. A total of 24 rats were randomly allocated into three groups (n = 8): (1) non-challenged control; (2) LPS-challenged control; and (3) LPS +3.0% Leu. Rats were fed with control or Leu-supplemented (part of the casein was replaced with 3.0% Leu) diets throughout the trial and were injected intraperitoneally with sterile saline or LPS at days 6, 11, 16, and 21. On the morning of day 22, serum samples were collected and rats were then sacrificed for liver and muscle analysis. In vitro protein degradation, nuclear factor-κB (NF-κB) activity, and proteolytic enzyme activities of the muscles from immune-challenged rats were also measured. Our results showed that the LPS challenge resulted in not only enhanced serum interleukin-1 and liver C-reactive protein (CRP) concentrations but also decreased the average daily body weight gain and muscle fiber diameter. However, dietary Leu inclusion attenuated the increase in CRP level and the decrease in muscle fiber diameter. Importantly, the LPS challenge caused a significant elevation in the muscle proteolysis rate, but dietary Leu supplementation significantly blocked the muscle proteolysis. The mRNA expression of NF-κB, muscle atrophy F-box (MAFbx), and muscle ring finger 1 (MuRF1) was upregulated by the LPS challenge in gastrocnemius muscles, but was downregulated by Leu supplementation. Interestingly, when muscles from the LPS-challenged rats were incubated with Leu in vitro, proteasome-, calpain-, and cathepsin-L-dependent muscle proteolysis and NF-κB activity were decreased. Collectively, the data suggest that Leu supplementation could inhibit excessive skeletal muscle degradation, as well as enhance protein synthesis and, thus, attenuate the negative effects caused by the LPS-induced immune challenge.

Acute intermittent hypoxia in rats activates muscle proteolytic pathways through a gluccorticoid-dependent mechanism.

Although it is well known that chronic hypoxia induces muscle wasting, the effects of intermittent hypoxia on skeletal muscle protein metabolism remain unclear. We hypothesized that acute intermittent hypoxia (AIH), a challenge that activates the hypothalamic-pituitary-adrenal axis, would alter muscle protein homeostasis through a glucocorticoid-dependent mechanism. Three-week-old rats were submitted to adrenalectomy (ADX) and exposed to 8 h of AIH (6% O2 for 40 s at 9-min intervals). Animals were euthanized, and the soleus and extensor digitorum longus (EDL) muscles were harvested and incubated in vitro for measurements of protein turnover. AIH increased plasma levels of corticosterone and induced insulin resistance as estimated by the insulin tolerance test and lower rates of muscle glucose oxidation and the HOMA index. In both soleus and EDL muscles, rates of overall proteolysis increased after AIH. This rise was accompanied by an increased proteolytic activities of the ubiquitin(Ub)-proteasome system (UPS) and lysosomal and Ca2+-dependent pathways. Furthermore, AIH increased Ub-protein conjugates and gene expression of atrogin-1 and MuRF-1, two key Ub-protein ligases involved in muscle atrophy. In parallel, AIH increased the mRNA expression of the autophagy-related genes LC3b and GABARAPl1. In vitro rates of protein synthesis in skeletal muscles did not differ between AIH and control rats. ADX completely blocked the insulin resistance in hypoxic rats and the AIH-induced activation of proteolytic pathways and atrogene expression in both soleus and EDL muscles. These results demonstrate that AIH induces insulin resistance in association with activation of the UPS, the autophagic-lysosomal process, and Ca2+-dependent proteolysis through a glucocorticoid-dependent mechanism.NEW & NOTEWORTHY Since hypoxia is a condition in which the body is deprived of adequate oxygen supply and muscle wasting is induced, the present work provides evidence linking hypoxia to proteolysis through a glucocorticoid-dependent mechanism. We show that the activation of proteolytic pathways, atrophy-related genes, and insulin resistance in rats exposed to acute intermittent hypoxia was abolished by surgical removal of adrenal gland. This finding will be helpful for understanding of the muscle wasting in hypoxemic conditions.

Effect of Persian and almond gums as fat replacers on the physicochemical, rheological, and microstructural attributes of low-fat Iranian White cheese.

The effect of Persian and almond gums (0, 0.1 and 0.2% (w/w)) as fat replacers and milk fat (0.4, 0.9, and 1.4% (w/w)) on physicochemical and rheological characteristics and microstructure of low‐fat Iranian White cheese was studied. Persian and almond gums both effectively increased moisture‐to‐protein (M:P) ratio of low‐fat cheese samples which in turn led to a significant reduction in the hardness parameters fracture stress and Young's and storage (G’) moduli (p < .05); however, the effect of Persian gum was more pronounced (p < .01). Gum addition promoted cheese yield and proteolysis rate (p < .05). Response surface optimization described that supplementation of cheese milk containing 0.9% fat with 0.2% Persian gum and 0.12% almond gum would result in a low‐fat cheese with textural properties similar to its full‐fat counterpart. Scanning electron microscopy revealed that the fat replacers produced full‐fat‐like structure in the low‐fat Iranian White cheese, when incorporated at the optimum levels.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.

EFEITO DA APLICAÇÃO DE Brevibacterium linens NA MATURAÇÃO DE QUEIJOS TIPO SAINT PAULIN DE CASCA TRATADA

Brevibacterium linens (B. linens) são predominantes na casca de queijos como Gruyère, Limburgo, Reblochon, Port du Salut e Saint Paulin. No entanto, o queijo tipo Saint Paulin fabricado no Brasil, não possui a casca tratada com bactérias e sim, apenas com uma solução salina e corante urucum, o que o deixa com sabor mais suave do que as variedades européias. O objetivo deste trabalho foi avaliar a influência da utilização da Brevibacterium linens na maturação de queijos tipo Saint Paulin. Os queijos foram produzidos utilizando-se três diferentes tratamentos: controle, sem adição de B. linens, com aplicação de B. linens na casca do queijo por esfregaço e com B. linens adicionada ao leite destinado à fabricação dos queijos. Índices de proteólise e pH (próximo a casca e no centro do queijo) foram avaliados durante a maturação, nos tempos 1, 15, 30 e 54 dias, e a composição centesimal foi avaliada no dia 1 de maturação, em três repetições. A análise de pH e de composição centesimal, não indicou diferença significativa (p &gt; 0,05) entre os tratamentos. Os tratamentos com a utilização de B. linens apresentaram maiores índices de proteólise em relação ao tratamento controle. Assim, a utilização desse tipo de cultura adjunta B. linens incentiva a atividade proteolítica no queijo. Além disso, pode ser adicionada tanto no leite como na morge (solução salina) aplicada na casca do queijo.

ITRAQ-based proteomic analysis of myofibrillar contents and relevant synthesis and proteolytic proteins in soleus muscle of hibernating Daurian ground squirrels (Spermophilus dauricus).

BackgroundDaurian ground squirrels (Spermophilus dauricus) deviate from significant increase of protein catabolism and loss of myofibrillar contents during long period of hibernation inactivity.MethodsHere we use iTRAQ based quantitative analysis to examine proteomic changes in the soleus of squirrels in pre-hibernation, hibernation and post-hibernation states. The total proteolysis rate of soleus was measured by the release of the essential amino acid tyrosine from isolated muscles. Immunofluorescent analysis was used to determine muscle fiber cross-sectional area. Western blot was used for the validation of the quantitative proteomic analysis.ResultsThe proteomic responses to hibernation had a 0.4- to 0.8-fold decrease in the myofibrillar contractile protein levels of myosin-3, myosin-13 and actin, but a 2.1-fold increase in myosin-2 compared to pre-hibernation group. Regulatory proteins such as troponin C and tropomodulin-1 were 1.4-fold up-regulated and 0.7-fold down-regulated, respectively, in hibernation compared to pre-hibernation group. Moreover, 10 proteins with proteolytic function in hibernation, which was less than 14 proteins in the post-hibernation group, were up-regulated relative to the pre-hibernation group. The total proteolysis rates of soleus in hibernation and post-hibernation groups were significantly inhibited as compared with pre-hibernation group.ConclusionThese findings suggest that the myofibrillar remodeling and partial suppression of myofibrillar proteolysis were likely responsible for preventing skeletal muscle atrophy during prolonged disuse in hibernation. This is the first study where the myofibrillar contents and relevant synthesis and proteolytic proteins in slow soleus was discussed based on proteomic investigation performed on wild Daurian ground squirrels. Our results lay the foundation for further research in preventing disuse-induced skeletal muscle atrophy in mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0105-x) contains supplementary material, which is available to authorized users.

Ultrasound Pretreatment as an Useful Tool to Enhance Egg White Protein Hydrolysis: Kinetics, Reaction Model, and Thermodinamics.

The impact of ultrasound waves generated by probe-type sonicator and ultrasound cleaning bath on egg white protein susceptibility to hydrolysis by alcalase compared to both thermal pretreatment and conventional enzymatic hydrolysis was quantitatively investigated. A series of hydrolytic reactions was carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations, and temperatures using untreated, and pretreated egg white proteins (EWPs). The kinetic model based on substrate inhibition and second-order enzyme deactivation successfully predicts the experimental behavior providing an effective tool for comparison and optimization. The ultrasound pretreatments appear to greatly improve the enzymatic hydrolysis of EWPs under different conditions when compare to other methods. The apparent reaction rate constants for proteolysis (k2 ) are 0.009, 0.011, 0.053, and 0.045 min-1 for untreated EWPs, and those pretreated with heat, probe-type sonicator, and ultrasound cleaning bath technologies, respectively. The ultrasound pretreatment also decreases hydrolysis activation (Ea ) and enzyme deactivation (Ed ) energy, enthalpy (ΔH), and entropy (ΔS) of activation and for the probe-type sonication this decrease is 61.7%, 61.6%, 63.6%, and 32.2%, respectively, but ultrasound has little change in Gibbs free energy value in the temperature range of 318 to 338 K. The content of sulfhydryl groups and ζ potential show a significant increase (P < 0.05) for both applied ultrasound pretreatments and the reduction of particle size distribution are achieved, providing some evidence that the ultrasound causes EWP structural changes affecting the proteolysis rate.

Dietary Supplementation of Curcumin Alleviates NF-κB-dependent Skeletal Muscle Wasting in Rat.

Activation of the nuclear factor-kappa β (NF-κB) signaling results in releasing of a variety of inflammatory cytokines that can induce skeletal muscle wasting (MW) in mammals. Curcumin is a natural product from spice turmeric that can block biological events in which NF-κB plays a critical role. The present study was conducted to evaluate the beneficial effects of curcumin on the NF-κB-dependent MW in rats. The MW rats were induced by lipopolysaccharide (LPS) injection and rats were fed 15 g curcumin per kg diet for 16 d. LPS not only decreased the growth performance of rat, but significantly elevated its serum IL-1 concentration and muscle proteolysis rate. Interestingly, curcumin significantly prevented the muscle proteolysis rate and down-regulated expression levels of two critical muscle-specific ubiquitin ligases (Atrogin-1 and MuRF-1). When muscles from MW rats were incubated with curcumin in vitro, the calpain-, cathepsin L-, and proteasome-dependent muscle proteolysis were significantly decreased. Moreover, the nuclear NF-κB/p65 protein abundance were also decreased by curcumin. The results not only suggest a molecular mechanism by which curcumin modulates the inflammatory responses, but also offer a feasible avenue to ameliorate the NF-κB-dependent muscle proteolysis.

Oral delivery of insulin for treatment of diabetes: status quo, challenges and opportunities.

Diabetes mellitus is characterised by progressive β-cell destruction and loss of function, or loss of ability of tissues to respond to insulin. Daily subcutaneous insulin injection is standard management for people with diabetes, although patient compliance is hard to achieve due to the inconvenience of injections, so other forms of delivery are being tested, including oral administration. This review summarises the developments in oral insulin administration. The PubMed database was consulted to compile this review comparing conventional subcutaneous injection of insulin to the desired oral delivery. Oral administration of insulin has potential benefits in reducing pain and chances of skin infection, improving the portal levels of insulin and avoiding side effects such as hyperinsulinemia, weight gain and hypoglycaemia. Although oral delivery of insulin is an ideal administration route for patients with diabetes, several physiological barriers have to be overcome. An expected low oral bioavailability can be attributed to its high molecular weight, susceptibility to enzymatic proteolysis and low diffusion rate across the mucin barrier. Strategies for increasing the bioavailability of oral insulin include the use of enzyme inhibitors, absorption enhancers, mucoadhesive polymers and chemical modification for endogenous receptor-mediated absorption. These may help significantly increase patient compliance and disease management.

Ligation of FcγR Alters Phagosomal Processing of Protein via Augmentation of NADPH Oxidase Activity.

Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase (NOX2), and its effect on phagosomal protein degradation. Population-based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor (FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction. Immunoglobulin G (IgG)-stimulated reactive oxygen species (ROS) production and the inhibition of phagosomal proteolysis and disulfide reduction were dependent on NOX2, FcγR and protein kinase C (PKC)/spleen tyrosine kinase (Syk) signaling. In contrast, low levels of ROS production were observed following the phagocytosis of unopsonized beads, which resulted in higher rates of phagosomal proteolysis and disulfide reduction. Phagosomes displayed autonomy with respect to FcγR-mediated differences in NOX2 activation and proteolysis, as phagosomes containing unopsonized cargo retained low NOX2 activation and high proteolysis even in the presence of phagosomes containing IgG-opsonized cargo in the same macrophage. These results show that opsonization of phagocytic cargo results in vastly different phagosomal processing of proteins through the FcγR-triggered, PKC/Syk-dependent local assembly and activation of NOX2.

Bias-free imaging of antigen degradation using bioorthogonal antigens

Abstract Degradation of antigen into short MHC-loadable polypeptides by antigen presenting cells presents a Heisenbergian conundrum: reporter strategies for imaging these processes affect the processes itself to such an extent that the information obtained from reporter antigen strategies may not be representative of the process itself. For example fusion constructs of antigens are only imagable until they are degraded or separated from the antigen, which is essential if a peptide from the antigen is to be loaded on an MHC and reach the surface for sampling by T-cells. This results in an imaging bias to only those parts of the pathway in which reporters remain intact. Further complications can arise from the fact that reporter-constructs can alter the rate of proteolysis and routing by virtue of changing overall physiochemical properties of the antigens. I will present our recent developments of ‘bioorthgonal antigens’, a bias free antigen labelling strategy that allows the imaging of antigen routing with minimal interference in the pathway. These antigens carrying amino acids with tiny chemical modifications (2 or 3 atoms in size) in their side-chains that are stable to proteolysis and other conditions found in the cell, can be selectively visualised using click chemistry. This allows, for example the localization of degrading antigen inside an APC or the determination of the rate of epitope appearance on the cell surface without the need for peptide-MHC-specific antibodies. This offers the first opportunity to have a detectable group that does not affect processing and presentation

Effect of high-pressure treatment on hard cheese proteolysis

The application of high hydrostatic pressure (HHP) treatment has been proposed to reduce the ripening time of cheese via modifications in the enzymatic activities or the substrate reactivity. Investigations on the effect of HHP on cheese proteolysis have been undertaken with either encouraging results or little effect according to the treatment conditions and the type of cheese, but information concerning the effect of HHP on the ripening of hard cooked cheese is still lacking. In this report, we describe the effect of HHP treatment on Reggianito cheese proteolysis. For that purpose, 1-d-old miniature cheeses (5.5-cm diameter and 6-cm height) were treated at 100 or 400MPa and 20°C for 5 or 10min, and control cheeses in the trial were not pressurized. All cheeses were ripened at 12°C during 90d. The HHP did not affect gross composition of the cheeses, but microbial load changed, especially because the starter culture count was significantly lower at the beginning of the ripening of the cheeses treated at 400MPa than in controls and cheeses treated at 100MPa. Cheeses treated at 400MPa for 10min had significantly higher plasmin activity than did the others; the residual coagulant activity was not affected by HHP. Proteolysis assessment showed that most severe treatments (400MPa) also resulted in cheeses with increased breakdown of αS1- and β-CN. In addition, nitrogen content in soluble fractions was significantly higher in cheeses treated at 400MPa, as well as soluble peptides and free AA production. Peptide profiles and individual and total content of free AA in 60-d-old treated cheese were as high as in fully ripened control cheeses (90d). Holding time had an effect only on pH-4.6-soluble nitrogen fraction and plasmin activity; cheese treated for 10min showed higher values than those treated for 5min, at both levels of pressure assayed. We concluded that HHP treatments at 400MPa applied 1d after cheesemaking increased the rate of proteolysis, leading to an acceleration of the ripening process in Reggianito Argentino cheese, whereas 100-MPa treatments did not lead to significant changes.

Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

Kinetic stability of cystathionine beta-synthase can be modulated by structural analogs of S-adenosylmethionine: Potential approach to pharmacological chaperone therapy for homocystinuria.

Many pathogenic missense mutations in human cystathionine beta-synthase (CBS) cause misfolding of the mutant enzyme resulting in aggregation or rapid degradation of the protein. Subsequent loss of CBS function leads to CBS-deficient homocystinuria (CBSDH). CBS contains two sets of binding sites for S-adenosylmethionine (SAM) that independently regulate the enzyme activity and kinetically stabilize its regulatory domain. In the present study, we examined the hypothesis that CBS activation may be decoupled from kinetic stabilization and thus CBS regulatory domain can serve as a novel drug target for CBSDH. We determined the effect of SAM and its close structural analogs on CBS activity, their binding to and stabilization of the regulatory domain in the absence and presence of competing SAM. Binding of S-adenosylhomocysteine and sinefungin lead to stabilization of the regulatory domains without activation of CBS. Direct titrations and competition experiments support specific binding of these two SAM analogs to the stabilizing sites. Binding of these two ligands also affects the enzyme proteolysis rate supporting the role of the stabilizing sites in CBS dynamics. Our results indicate that binding of SAM to regulatory and stabilizing sites in CBS may have evolved to display an exquisite thermodynamic and structural specificity towards SAM as well as the ability to transduce the allosteric signal responsible for CBS activation. Thus, ligands may be developed to function as kinetic stabilizers or pharmacological chaperones without interfering with the physiological activation of CBS by SAM.

Antioxidant activity and nutrient release from polyphenol-enriched cheese in a simulated gastrointestinal environment.

Green tea polyphenols are recognized for their antioxidant properties and their effects on lipid digestion kinetics. Polyphenols are sensitive to degradation in the intestinal environment. Interactions with dairy proteins could modulate the stability and biological activity of polyphenols during digestion. The objective of this study was to evaluate the release of nutrients (polyphenols, fatty acids and peptides) and the antioxidant activity in polyphenol-enriched cheese containing different levels of calcium in a simulated gastrointestinal environment. The relationship between cheese matrix texture, matrix degradation and nutrient release during digestion was also studied. Green tea extract was added to milk at 0% or 0.1%, and cheeses were produced on a laboratory scale. The level of available calcium was adjusted to low (Ca(low)), regular (Ca(reg)) or high (Ca(high)) during the salting step of the cheese-making process. Cheeses were subjected to simulated digestion. The rate and extent of fatty acid release were 21% lower for Ca(low) cheese than for Ca(reg) and Ca(high) cheeses. The greater adhesiveness of Ca(low) cheese, which resulted in lower rates of matrix degradation and proteolysis, contributed to the reduced rate of lipolysis. The presence of green tea extract in cheese reduced the release of free fatty acids at the end of digestion by 7%. The addition of green tea extract increased cheese hardness but did not influence matrix degradation or proteolysis profiles. The formation of complexes between tea polyphenols and proteins within the cheese matrix resulted in a more than twofold increase in polyphenol recovery in the intestinal phase compared with the control (tea polyphenol extract incubated with polyphenol-free cheese). Antioxidant activity was 14% higher in the digest from polyphenol-enriched cheese than in the control. These results suggest that cheese is an effective matrix for the controlled release of nutrients and for the protection of green tea polyphenol integrity and biological activity in the gastrointestinal environment.

Physicochemical, Biochemical and Instrumental Attributes and Consumer Acceptability of Dry-Fermented Sausages Elaborated with Combined Partial Substitutions of Sodium Chloride and Pork Backfat

We performed 6 fabrications of dry-fermented sausages to investigate, at laboratory scale, the effects of combined partial substitutions of sodium chloride by potassium chloride and pork backfat by sunflower oil on key physical-chemical and biochemical parameters, instrumental colour and texture measurements, and the consumer acceptability of the end-products. Regarding the physical-chemical parameters, statistical analysis of results showed that final product weight loss was impacted by fat content and use of sunflower oil; final mean water activity value was only affected by salt level; and animal fat content impacted pH values, only at the end of drying. Regarding the biochemical parameters investigated, we statistically found a marked impact of partial substitution of NaCl by KCl on the proteolysis evolution, of fat level and sodium content on the end-product lipolysis, and finally, a significant effect of animal fat level and incorporation of sunflower oil on both protein and lipid oxidations. However, the new product formulations combining salt and fat substitutions lead to globally acceptable water loss and water activity values and similar rates of proteolysis, lipolysis and lipid oxidation, but less protein oxidation. From a practical point of view, the results clearly showed that sodium and animal fat contents in dryfermented sausages can be drastically reduced with no too marked adverse effect on colour, final textural properties or consumer acceptability. On the basis of these laboratory results, new healthier dry-fermented products can be manufactured by an industrial company in the near future.

Endogenous and exogenous pathways maintain the reductive capacity of the phagosome.

Although endosomes, lysosomes, and phagosomes require a reductive environment for the optimal activity of disulfide reductases and other thiol-dependent enzymes, how these reductive environments are established and maintained remain unknown. Our goal in this study was to begin to elucidate the redox control systems responsible for maintaining redox-sensitive enzymatic activities in the phagolysosome of murine macrophages. Through the use of specific inhibitors and genetic knockdown of known redox enzymes, we identified redox pathways that influence phagosomal disulfide reduction. In particular, known inhibitors of the NADPH-dependent selenoprotein, thioredoxin reductase, were shown to inhibit phagosomal disulfide reduction and phagosomal proteolysis. This was supported by the observation that conditional deletion of the selenocysteine tRNA in macrophages decreased phagosomal disulfide reduction capacity. In addition, pharmacologic inhibition of the pentose phosphate pathway decreased rates of disulfide reduction and proteolysis in the phagosome, implicating NADPH as a source of phagosomal reductive energy. Finally, by analyzing the effect of extracellular redox couples, such as cysteine:cystine on thiol-dependent phagosomal processes, we demonstrated that the extracellular space can additionally supply the phagosome with reductive energy. Collectively, these data demonstrate that defined cytosolic reductive pathways act in concert with the uptake of cysteine from the extracellular space to support thiol-dependent chemistries in the phagosome.

In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids

It is currently admitted that food structure can facilitate or delay the release of nutrients during digestion and their absorption by the human body. The aim of this study is to propose an in vitro method able to assess the behavior of lipo-proteinic matrices with different structures during digestion. Two model matrices of exactly the same compositions (10% oil, 15% whey proteins, w/w) were designed: i) A liquid emulsion (LE) made of small fat droplets (1μm) dispersed in a liquid continuous phase containing native whey proteins, ii) a solid emulsion (SE) made of a continuous whey protein gel entrapping large oil droplets (20μm). The two matrices were digested through an in vitro gastro-intestinal protocol based on the INFOGEST guidelines using pH-stat to monitor the enzymatic hydrolyses of both proteins and lipids. By further digesting lipid-free matrices in the same conditions, the contributions of the proteolytic and lipolytic reactions were evaluated. Significant differences were observed between matrices at both short and long digestion times. The initial rates of both proteolysis and lipolysis were slower for SE than LE because of the gel state of the continuous phase. At the end of the experiments, SE led to a smaller extent of lipolysis (DHlip_SE=51%<DHlip_LE=81%) but a greater extent of proteolysis (DHprot_SE=80%>DHprot_LE=52%) because of the higher sensitivity to digestion of denatured whey proteins. These results highlight the impact of matrix structure on enzyme accessibility, and show that the proposed method is suitable to monitor the digestion of complex food matrices.

Anti-DNA antibody mediated catalysis is isotype dependent.

Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies.

Gender-specific effects of intrauterine growth restriction on the adipose tissue of adult rats: a proteomic approach.

BackgroundIntrauterine growth restriction (IUGR) may program metabolic alterations affecting physiological functions and lead to diseases in later life. The adipose tissue is an important organ influencing energy homeostasis. The present study was aimed at exploring the consequences of IUGR on the retroperitoneal adipose tissue of adult male and female rats, using a proteomic approach.Methods and ResultsPregnant Wistar rats were fed with balanced chow, either ad libitum (control group) or restricted to 50 % of control intake (restricted group) during the whole gestation. The offspring were weaned to ad libitum chow and studied at 4 months of age. Retroperitoneal fat was analyzed by two-dimensional gel electrophoresis followed by mass spectrometry.Both male and female restricted groups had low body weight at birth and at weaning but normal body weight at adulthood. The restricted males had normal fat pads weight and serum glucose levels, with a trend to hyperinsulinemia. The restricted females had increased fat pads weight with normal glucose and insulin levels.The restricted males showed up-regulated levels of proteasome subunit α type 3, branched-chain-amino-acid aminotransferase, elongation 1- alpha 1, fatty acid synthase levels, cytosolic malate dehydrogenase and ATP synthase subunit alpha. These alterations point to increased proteolysis and lipogenesis rates and favoring of ATP generation.The restricted females showed down-regulated levels of L-lactate dehydrogenase perilipin-1, mitochondrial branched-chain alpha-keto acid dehydrogenase E1, and transketolase. These findings suggest impairment of glycemic control, stimulation of lipolysis and inhibition of proteolysis, pentose phosphate pathway and lipogenesis rates.In both genders, several proteins involved in oxidative stress and inflammation were affected, in a pattern compatible with impairment of these responses.ConclusionsThe proteomic analysis of adipose tissue showed that, although IUGR affected pathways of substrate and energy metabolism in both males and females, important gender differences were evident. While IUGR males displayed alterations pointing to a predisposition to later development of obesity, the alterations observed in IUGR females pointed to a metabolic status of established obesity, in agreement with their increased fat pads mass.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0088-z) contains supplementary material, which is available to authorized users.