An Investigation of the Altered Textural Property in Woody Breast Myopathy Using an Integrative Omics Approach.

Woody breast (WB) is a myopathy observed in broiler Pectoralis major (PM) characterized by its tough and rubbery texture with greater level of calcium content. The objective of this study was to investigate the functionality/integrity of WB sarcoplasmic reticulum (SR), which may contribute to the elevated calcium content observed in WB and other factors that may influence WB texture. Fourteen Ross line broiler PM [7 severe WB and 7 normal (N)] were selected, packaged, and frozen at -20°C at 8 h postmortem from a commercial processing plant. Samples were used to measure pH, sarcomere length, proteolysis, calpain activity, collagenase activity, collagen content, collagen crosslinks density, and connective tissue peak transitional temperature. Exudate was also collected from each sample to evaluate free calcium concentration. The SR fraction of the samples was separated and utilized for proteomic and lipidomic analysis. The WB PM had a higher pH, shorter sarcomeres, lower % of intact troponin-T, more autolyzed µ/m calpain, more activated collagenase, greater collagen content, greater mature collagen crosslinks density, and higher connective tissue peak transitional temperature than the N PM (p ≤ 0.05). Exudate from WB PM had higher levels of free calcium than those from N PM (p < 0.05). Proteomics data revealed an upregulation of calcium transport proteins and a downregulation of proteins responsible for calcium release (p < 0.05) in WB SR. Interestingly, there was an upregulation of phospholipase A2 (PLA2), and cholinesterase exhibited a 7.6-fold increase in WB SR (p < 0.01). Lipidomics data revealed WB SR had less relative % of phosphatidylcholine (PC) and more lysophosphatidylcholine (LPC; p < 0.05). The results indicated that upregulation of calcium transport proteins and downregulation of calcium-release proteins in WB SR may be the muscle's attempt to regulate this proposed excessive signaling of calcium release due to multiple factors, such as upregulation of PLA2 resulting in PC hydrolysis and presence of cholinesterase inhibitors in the system prolonging action potential. In addition, the textural abnormality of WB may be the combined effects of shorter sarcomere length and more collagen with greater crosslink density being deposited in the broiler PM.

ß-carotene oxygenase 2 deficiency-triggered mitochondrial oxidative stress promotes low-grade inflammation and metabolic dysfunction.

Low-grade inflammation is a critical pathological factor contributing to the development of metabolic disorders. ß-carotene oxygenase 2 (BCO2) was initially identified as an enzyme catalyzing carotenoids in the inner mitochondrial membrane. Mutations in BCO2 are associated with inflammation and metabolic disorders in humans, yet the underlying mechanisms remain unknown. Here, we used loss-of-function approaches in mice and cell culture models to investigate the role of BCO2 in inflammation and metabolic dysfunction. We demonstrated decreases in BCO2 mRNA and protein levels and suppression of mitochondrial respiratory complex I proteins and mitochondrial superoxide dismutase levels in the liver of type 2 diabetic human subjects. Deficiency of BCO2 caused disruption of assembly of the mitochondrial respiratory supercomplexes, such as supercomplex III2+IV in mice, and overproduction of superoxide radicals in primary mouse embryonic fibroblasts. Further, deficiency of BCO2 increased protein carbonylation and populations of natural killer cells and M1 macrophages, and decreased populations of T cells, including CD4+ and/or CD8+ in the bone marrow and white adipose tissues. Elevation of plasma inflammatory cytokines and adipose tissue hypertrophy and inflammation were also characterized in BCO2 deficient mice. Moreover, BCO2 deficient mice were more susceptible to high-fat diet-induced obesity and hyperglycemia. Double knockout of BCO2 and leptin receptor genes caused a significantly greater elevation of the fasting blood glucose level in mice at 4 weeks of age, compared to the age- and sex-matched leptin receptor knockout. Finally, administration of Mito-TEMPO, a mitochondrial specific antioxidant attenuated systemic low-grade inflammation induced by BCO2 deficiency. Collectively, these findings suggest that BCO2 is essential for mitochondrial respiration and metabolic homeostasis in mammals. Loss or decreased expression of BCO2 leads to mitochondrial oxidative stress, low-grade inflammation, and the subsequent development of metabolic disorders.

Inhibition of immune pathway-initiating hemolymph protease-14 by Manduca sexta serpin-12, a conserved mechanism for the regulation of melanization and Toll activation in insects.

A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal ß-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr394 and Ile395. Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses.

Proteomic and bioinformatic analyses of putative Mannheimia haemolytica secretome by liquid chromatography and tandem mass spectrometry.

Mannheimia haemolytica is a major bacterial contributor to bovine respiratory disease complex that costs the livestock industry a billion dollars a year in USA. Commercial vaccines are only partially efficacious under field conditions. Earlier studies found that outer membrane protein preparations and culture supernatants can induce immune responses that enhance resistance to challenge by M. haemolytica strains. The objective of this study was to characterize secretome of two M. haemolytica stains grown under two different media. Bacteria-free concentrated supernatants from M. haemolytica culture was subjected to LC-MS/MS. The secretome of M. haemolytica from both strains yielded 923 proteins. Using bioinformatic tools, 283 were identified as secreted proteins. Further breakdown of 283 proteins showed that 114 (40.2%), 184 (65.0%), 138(48.7%), 151 (53.3%) and 172 (60.7%) were characterized as secreted proteins by SignalP 4.1, SecretomeP 2.0, LipoP, Phobius, and PRED-TAT, respectively. A total of 95 (33.56%) proteins were characterized as being secreted via non-classical pathway as opposed to the majority that were secreted in signal peptide dependent pathway. The demonstrated proteins include all previously immunologically characterized M. haemolytica proteins. The potential of using secretome analysis in the design and development of a multivalent vaccine is discussed.

Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass.

Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and composites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitative protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, ß-1,3 glucanase, ß-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed ß-1,3(4) glucanase, ß-1,3 glucanases, ß-glucosidases, ß-mannosidase, ß-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo ß-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. The participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natural biomass fermentation experiments.

Elucidation of the Hsp90 C-terminal inhibitor binding site.

The Hsp90 chaperone machine is required for the folding, activation, and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains; however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein.

Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes.

Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian perilipin A and insect lipid storage proteins (Lsd). Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant Lsd1 and its reconstitution with lipids to form lipoprotein complexes suitable for functional and structural studies. Lsd1 in the lipid bound state is a predominately alpha-helical protein. Using lipoprotein complexes containing triolein it is shown that PKA mediated phosphorylation of Lsd1 promoted a 1.7-fold activation of the main fat body lipase demonstrating the direct link between Lsd1 phosphorylation and activation of lipolysis. Serine 20 was identified as the Lsd1-phosphorylation site triggering this effect.