Proteome basis of pale, soft, and exudative-like (PSE-like) broiler breast (Pectoralis major) meat.

Pale, Soft, and Exudative (PSE) broiler breast meat has poor protein functionality, which leads to quality problems and economic loss in the poultry industry. Proteomics has been applied to characterize the biochemical mechanisms governing tenderness, color, and water-holding capacity in meat. However, the proteome basis of PSE has not yet been characterized for broiler breast meat. Therefore, this study was conducted to determine the differences in meat quality (cooking loss and shear force), descriptive sensory characteristics, consumer acceptance, and whole muscle proteome between normal and PSE-like broiler breast meat. Male Hubbard × Cobb 500 birds (n = 1,050) were raised in commercial houses. Prior to harvest, a sample of the broilers (n = 900) were subjected to short-term stress (38°C for 2 h), and the remaining broilers (n = 150) were maintained at control conditions (21°C for 2 h). Broiler breast (Pectoralis major) meat was collected and characterized by pH24 and L*24 as normal (pH24 5.8 to 6.2, L*24 45 to 55) or PSE-like (pH24 5.4 to 5.7, L*24 55 to 65) samples. Normal broiler breast meat had lower shear force values than PSE-like meat (P < 0.05). Based on sensory descriptive analysis, normal cooked chicken breast meat was more tender and juicier than PSE-like breast meat (P < 0.05). Consumer sensory analysis results indicated that 81% of consumer panelists liked normal breast meat whereas 62% of the panelists liked PSE-like breast meat. Whole muscle proteome profiling identified fifteen differentially abundant proteins in normal and PSE-like broiler breast samples. Actin alpha, myosin heavy chain, phosphoglycerate kinase, creatine kinase M type, beta-enolase, carbonic anhydrase 2, proteasome subunit alpha, pyruvate kinase, and malate dehydrogenase were over-abundant (P < 0.05) in PSE-like broiler breast whereas phosphoglycerate mutase-1, alpha-enolase, ATP-dependent 6-phosphofructokinase, and fructose 1,6-bisphosphatase were over-abundant (P < 0.05) in normal meat. Thus, results indicated that differences in proteome abundance could be related to the meat quality differences between normal and PSE-like broiler breast meat.

Proteome basis for intramuscular variation in color stability of beef semimembranosus.

The objective of the present study was to characterize the proteome basis for intramuscular color stability variations in beef semimembranosus. Semimembranosus muscles from eight carcasses (n=8) were fabricated into 2.54-cm thick color-labile inside (ISM) and color-stable outside (OSM) steaks. One steak for sarcoplasmic proteome analysis was immediately frozen, whereas other steaks were allotted to retail display under aerobic packaging. Color attributes were evaluated instrumentally and biochemically on 0, 2, and 4days. Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis and tandem mass spectrometry. ISM steaks demonstrated greater (P<0.01) abundance of glycolytic enzymes (fructose-bisphosphate aldolase A, phosphoglycerate mutase 2, and beta-enolase) and phosphatidylethanolamine-binding protein 1 than their OSM counterparts. Possible rapid post-mortem glycolysis in ISM, insinuated by over-abundance of glycolytic enzymes, could lead to rapid pH decline during early post-mortem, which in turn could potentially compromise its color stability. These results indicated that differential abundance of sarcoplasmic proteome contributes to intramuscular variations in beef color stability.

Differential abundance of sarcoplasmic proteome explains animal effect on beef Longissimus lumborum color stability.

The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm steaks. One steak was allotted to retail display, and another was immediately vacuum packaged and frozen at -80°C. Aerobically packaged steaks were stored under display, and color was evaluated on days 0 and 11. The steaks were ranked based on redness and color stability on day 11, and ten color-stable and ten color-labile carcasses were identified. Sarcoplasmic proteome of frozen steaks from the selected carcasses was analyzed. Nine proteins were differentially abundant in color-stable and color-labile steaks. Three glycolytic enzymes (phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2) were over-abundant in color-stable steaks and positively correlated (P<0.05) to redness and color stability. These results indicated that animal variations in proteome contribute to differences in beef color.

Dietary ractopamine influences sarcoplasmic proteome profile of pork Longissimus thoracis.

Dietary ractopamine improves pork leanness, whereas its effect on sarcoplasmic proteome has not been characterized. Therefore, the influence of ractopamine on sarcoplasmic proteome of post-mortem pork Longissimus thoracis muscle was examined. Longissimus thoracis samples were collected from carcasses (24 h post-mortem) of purebred Berkshire barrows (n=9) managed in mixed-sex pens and fed finishing diets containing ractopamine (RAC; 7.4 mg/kg for 14 days followed by 10.0 mg/kg for 14 days) or without ractopamine for 28 days (CON). Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis and mass spectrometry. Nine protein spots were differentially abundant between RAC and CON groups. Glyceraldehyde-3-phosphate dehydrogenase and phosphoglucomutase-1 were over-abundant in CON, whereas serum albumin, carbonic anhydrase 3, L-lactate dehydrogenase A chain, fructose-bisphosphate aldolase A, and myosin light chain 1/3 were over-abundant in RAC. These results suggest that ractopamine influences the abundance of enzymes involved in glycolytic metabolism, and the differential abundance of glycolytic enzymes could potentially influence the conversion of muscle to meat.

Covalent binding of 4-hydroxy-2-nonenal to lactate dehydrogenase decreases NADH formation and metmyoglobin reducing activity.

Lactate dehydrogenase (LDH) activity can regenerate NADH, which is a critical component in metmyoglobin reduction. However, limited research has determined the effects of lipid oxidation products on LDH activity. The overall objective of this study was to determine the effects of 4-hydroxy-2-nonenal (HNE) on LDH activity. LDH was reacted with HNE at pH 5.6 and 7.4, and LDH activity was measured as NADH formation following the addition of lactate and NAD. The effects of HNE on NADH-dependent metmyoglobin reduction also were analyzed. Mass spectrometric examination revealed that HNE adducts to LDH at both pH 5.6 and 7.4. More specifically, HNE binds with cysteine and histidine residues of LDH at pH 5.6 and 7.4. Covalent binding of HNE decreased NADH formation and metmyoglobin reduction (P < 0.05). These results indicate that secondary lipid oxidation products can inactivate enzymes involved in metmyoglobin reduction and have the potential to increase beef discoloration.

c-Abl and Arg induce cathepsin-mediated lysosomal degradation of the NM23-H1 metastasis suppressor in invasive cancer.

Metastasis suppressors comprise a growing class of genes whose downregulation triggers metastatic progression. In contrast to tumor suppressors, metastasis suppressors are rarely mutated or deleted, and little is known regarding the mechanisms by which their expression is downregulated. Here, we demonstrate that the metastasis suppressor, NM23-H1, is degraded by lysosomal cysteine cathepsins (L,B), which directly cleave NM23-H1. In addition, activation of c-Abl and Arg oncoproteins induces NM23-H1 degradation in invasive cancer cells by increasing cysteine cathepsin transcription and activation. Moreover, c-Abl activates cathepsins by promoting endosome maturation, which facilitates trafficking of NM23-H1 to the lysosome where it is degraded. Importantly, the invasion- and metastasis-promoting activity of c-Abl/Arg is dependent on their ability to induce NM23-H1 degradation, and the pathway is clinically relevant as c-Abl/Arg activity and NM23-H1 expression are inversely correlated in primary breast cancers and melanomas. Thus, we demonstrate a novel mechanism by which cathepsin expression is upregulated in cancer cells (via Abl kinases). We also identify a novel role for intracellular cathepsins in invasion and metastasis (degradation of a metastasis suppressor). Finally, we identify novel crosstalk between oncogenic and metastasis suppressor pathways, thereby providing mechanistic insight into the process of NM23-H1 loss, which may pave the way for new strategies to restore NM23-H1 expression and block metastatic progression.

Effects of pyruvate on lipid oxidation and ground beef color.

UNLABELLED: Our overall objective was to better understand the effects of added pyruvate on enhanced beef color stability. The 2 possible mechanisms assessed were the role of pyruvate in lipid oxidation and direct interaction between pyruvate and beef myoglobin. Microsomes were incubated with pyruvate at pH 5.6, 25 °C, and lipid oxidation was measured hourly for 3 h. Bovine oxymyoglobin at pH 5.6 was incubated with pyruvate and used to quantify both redox stability (metmyoglobin formation) and pyruvate-myoglobin adduction using mass spectrometry analysis. Surface color and lipid oxidation were measured on ground beef patties stored for 6 d in polyvinyl chloride over-wrap (PVC) or high oxygen. Addition of pyruvate to microsomes decreased lipid oxidation compared with controls (P < 0.05). Conversely, no effect on myoglobin was observed (no changes in redox stability and no peaks corresponding to pyruvate were observed; P > 0.05). However, pyruvate increased color stability and decreased lipid oxidation of ground beef patties packaged in PVC and high oxygen. Pyruvate decreased nitric oxide metmyoglobin-reducing capacity and oxygen consumption of patties compared with controls (P < 0.05). This research suggests that pyruvate may improve beef color stability primarily through its antioxidant effect on lipids. PRACTICAL APPLICATION: Discoloration of meat often results in significant revenue loss. This study suggests that pyruvate can improve the color stability of patties packaged in high oxygen and PVC primarily through its antioxidant effect on lipids.

Redox instability and hemin loss of mutant sperm whale myoglobins induced by 4-hydroxynonenal in vitro.

The effects of 4-hydroxy-2-nonenal (HNE) on redox stability of Oxy- and Deoxy- wild-type (WT) and recombinant sperm whale myoglobins (P88H/Q152H, L29F, H97A, and H64F) and hemin loss from Met-myoglobin (Mb) were investigated. HNE induced greater redox instability in WT and mutant Mbs compared to controls (p < 0.05). The extent of HNE-induced OxyMb oxidation was lesser in L29F (p < 0.05) and greater in H97A and P88H/Q152H than in WT (p < 0.05). H64F DeoxyMb was more redox stable than WT DeoxyMb in the presence of HNE (p < 0.05). HNE alkylation occurred exclusively on histidine residues, and histidine 48 was alkylated in all sperm whale myoglobins. HNE alkylation accelerated the protoporphyrin moiety loss only in H97A. Met- forms of WT and L29F but not Deoxy- or Oxy- forms released hemin during storage. Primary structure strongly influenced Mb redox stability in the presence of reactive secondary lipid oxidation products.

Proteomics of muscle-specific beef color stability.

The objective of the present study was to differentiate the sarcoplasmic proteome of color-stable (Longissimus lumborum; LL) and color-labile (Psoas major; PM) beef muscles. LL and PM muscles from seven beef carcasses (24 h post-mortem) were fabricated into 2.54 cm steaks, aerobically packaged, and assigned to refrigerated retail display for 9 days. LL steaks demonstrated greater (P < 0.05) color stability and lower (P < 0.05) lipid oxidation than PM steaks. Proteome analyses identified 16 differentially abundant proteins in LL and PM, including antioxidant proteins and chaperones. Proteins demonstrating positive correlation with redness (aldose reductase, creatine kinase, and ß-enolase) and color stability (peroxiredoxin-2, peptide methionine sulfoxide reductase, and heat shock protein-27 kDa) were overabundant in LL, whereas the protein overabundant in PM (mitochondrial aconitase) exhibited negative correlation with redness. The color stability of LL could be attributed to the overabundance of antioxidant proteins and chaperones, and this finding suggests the necessity of developing muscle-specific processing strategies to improve beef color.

The snoRNA MBII-52 (SNORD 115) is processed into smaller RNAs and regulates alternative splicing.

The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader-Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.

SHP-2 is a novel target of Abl kinases during cell proliferation.

Previously, we showed that Abl family tyrosine kinases are activated by growth factors, and Abl is required for transition from G1 to S phase during PDGF-mediated proliferation. Here, we show that the SHP-2 tyrosine phosphatase, which acts to promote proliferation in response to cytokines and growth factors, is a novel substrate of endogenous Abl kinases during growth factor-mediated cellular proliferation. Using a pharmacological inhibitor and RNAi, we show that endogenous Abl kinases phosphorylate SHP-2 on Y580, and induce sustained activation of ERK kinases in response to growth factor stimulation in fibroblasts. Consistent with these data, SHP-2 is required for Abl-dependent PDGF-mediated proliferation since expression of an activated form of SHP-2 rescues the ability of Abl-Arg null fibroblasts to transit from G1 to S phase, whereas inhibition of SHP-2 signaling reduces the ability of Abl kinases to rescue the proliferation defect. Abl kinases also indirectly mediate phosphorylation of SHP-2 on Y63 and Y279, which are frequent sites of germline mutation in two cancer susceptibility syndromes. Significantly, we demonstrate that phosphorylation of SHP-2 on Y279 downregulates growth factor-induced sustained ERK activation and proliferation, supporting a role for Abl kinases not only in potentiating growth factor-mediated SHP-2 signaling, but also in negative-feedback regulation.

Changing transcriptional initiation sites and alternative 5'- and 3'-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyltransferase2 variants to different subcellular compartments.

Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT) genes encoding enzymes (EC 2.1.1.77) capable of converting uncoded l-isoaspartyl residues, arising spontaneously at l-asparaginyl and l-aspartyl sites in proteins, to l-aspartate. PIMT2 produces at least eight transcripts by using four transcriptional initiation sites (TIS; resulting in three different initiating methionines) and both 5'- and 3'-alternative splice site selection of the first intron. The transcripts produce mature proteins capable of converting l-isoaspartate to l-aspartate in small peptide substrates. PIMT:GFP fusion proteins generated a detectable signal in the nucleus. However, whether the protein was also detectable in the cytoplasm, endo-membrane system, chloroplasts, and/or mitochondria, depended on the transcript from which it was produced. On-blot-methylation of proteins, prior to the completion of germination, indicated that cruciferin subunits contain isoaspartate. The implications of using transcriptional mechanisms to expand a single gene's repertoire to protein variants capable of entry into the cell's various compartments are discussed in light of PIMT's presumed role in repairing the proteome.

Cerebrospinal fluid proteomics and human immunodeficiency virus dementia: preliminary observations.

Protein profiling using mass spectrometry may be useful in identifying previously unknown protein markers in human immunodeficiency virus (HIV) dementia and provide insight into disease pathogenesis. Six samples of matched cerebrospinal (CSF) and blood serum from patients with no, mild, and moderate dementia were prepped for biomarker screening by the Ciphergen system. Chips were analyzed in the matrix-assisted laser desorption/ionization (MALDI) mass spectrometer at low mass (700 to 20,000 Da) and at higher mass (5000 to 100,000 Da). In both serum and CSF samples, differences in protein intensity appeared to correlate with degree of dementia. This preliminary study suggests that protein markers of HIV dementia may be detected by MALDI mass spectrometry.

L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1.

Dynamic regulation of the cell surface expression of adhesion molecules is an important mechanism for controlling neuronal growth cone motility and guidance. Clathrin-mediated vesicular internalization of L1 via the tyrosine-based endocytosis motif YRSL regulates adhesion and signaling by this Ig superfamily molecule. Here, we present evidence that tyrosine-1176 (Y1176) of the YRSL motif is phosphorylated in vivo. The nonreceptor tyrosine kinase (p60src) is implicated in L1-mediated neurite outgrowth, and we find that p60src phosphorylates Y1176 in vitro. Phosphorylation of Y1176 prevents L1 binding to AP-2, an adaptor required for clathrin-mediated internalization of L1. mAb 74-5H7 recognizes the sequence immediately NH2-terminal to the tyrosine-based motif and binds L1 only when Y1176 is dephosphorylated. 74-5H7 identifies a subset of L1 present at points of cell-cell contact and in vesicle-like structures that colocalize with an endocytosis marker. L1-L1 binding or L1 cross-linking induces a rapid increase in 74-5H7 immunoreactivity. Our data suggest a model in which homophilic binding or L1 cross-linking triggers transient dephosphorylation of the YRSL motif that makes L1 available for endocytosis. Thus, the regulation of L1 endocytosis through dephosphorylation of Y1176 is a critical regulatory point of L1-mediated adhesion and signaling.