Carol M. Beach

Activation of the MAPK Signal Cascade by the Neural Cell Adhesion Molecule L1 Requires L1 Internalization

L1-mediated axon growth involves intracellular signaling, but the precise mechanisms involved are not yet clear. We report a role for the mitogen-activated protein kinase (MAPK) cascade in L1 signaling. L1 physically associates with the MAPK cascade components Raf-1, ERK2, and the previously identified p90 rsk in brain. In vitro, ERK2 can phosphorylate L1 at Ser1204 and Ser1248 of the L1 cytoplasmic domain. These two serines are conserved in the L1 family of cell adhesion molecules, also being found in neurofascin and NrCAM. The ability of ERK2 to phosphorylate L1 suggests that L1 signaling could directly regulate L1 function by phosphorylation of the L1 cytoplasmic domain. In L1-expressing 3T3 cells, L1 cross-linking can activate ERK2. Remarkably, the activated ERK localizes with endocytosed vesicular L1 rather than cell surface L1, indicating that L1 internalization and signaling are coupled. Inhibition of L1 internalization with dominant-negative dynamin prevents activation of ERK. These results show that L1-generated signals activate the MAPK cascade in a manner most likely to be important in regulating L1 intracellular trafficking.

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.

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.

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 β-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.

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.

Characterization of bison (Bison bison) myoglobin.

Bison is an alternate meat species gaining increased popularity in North America. Although previous investigations reported that bison meat discolors faster than beef, the molecular basis of this observation has not been investigated. Therefore, the objective of the present study was to determine the redox stability, thermostability, and primary structure of bison myoglobin (Mb), in comparison with beef Mb. Purified bison and beef myoglobins were analyzed for autoxidation, lipid oxidation-induced oxidation, and thermostability. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was utilized for determining the exact molecular mass of bison Mb, whereas Edman degradation was employed to determine the amino acid sequence. Bison and beef myoglobins behaved similarly in autoxidation, lipid oxidation-induced oxidation, and thermostability. The observed molecular mass of bison and beef myoglobins was 16,949 Da, and the primary structure of bison Mb shared 100% similarity with beef and yak myoglobins. Noticeably, the amino acid sequence of bison Mb was different from other ruminant myoglobins, such as water-buffalo, sheep, goat, and red-deer. The present study is the first to report the primary structure of bison Mb. Same primary structure and similar biochemical attributes of bison and beef myoglobins suggested that the observed rapid discoloration in bison meat could not be attributed to biochemistry of bison Mb.

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.

Amino acid sequence of myoglobin from emu (Dromaius novaehollandiae) skeletal muscle

The objective of the present study was to characterize the primary structure of emu myoglobin (Mb). Emu Mb was isolated from Iliofibularis muscle employing gel-filtration chromatography. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was employed to determine the exact molecular mass of emu Mb in comparison with horse Mb, and Edman degradation was utilized to characterize the amino acid sequence. The molecular mass of emu Mb was 17,380 Da and was close to those reported for ratite and poultry myoglobins. Similar to myoglobins from meat-producing livestock and birds, emu Mb has 153 amino acids. Emu Mb contains 9 histidines. Proximal and distal histidines, responsible for coordinating oxygen-binding property of Mb, are conserved in emu. Emu Mb shared more than 90% homology with ratite and chicken myoglobins, whereas it demonstrated only less than 70% sequence similarity with ruminant myoglobins.

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.

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.