Adenylate Kinase Isoenzyme Research Articles

Synthesis, kinetic studies, and QSAR of dinucleoside polyphosphate derivatives as human AK1 inhibitors

Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential.In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5′-adenosyl) P5-(5′-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure–activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.

Biomarker discovery and authentication of cold-slaughtered chicken through classical analytical procedures and mass spectrometry based proteomic approaches

ABSTRACT 1. This study evaluated the suitability of routine analytical procedures and used mass spectrometry-based proteomic approaches to distinguish meat from dead chicken/ cold-slaughtered birds (CS), electrically stunned and slaughtered birds, as per standard protocols (ES), and birds slaughtered according to halal guidelines (HS). 2. Meat from CS birds had lower (P < 0.05) pH, water-holding capacity and higher (P < 0.05) lipid oxidation, haem iron content, residual blood and total viable counts relative to ES and HS meat indicating poor quality. 3. The results demonstrated the presence of unique protein bands on SDS-PAGE only in CS meat that can be used for routine screening. 4. Protein analysis using MALDI-TOF mass spectrometry identified haemoglobin subunit alpha-A and alpha-D; Adenylate kinase isoenzyme 1 as reliable and stable marker proteins for authentication of dead chicken meat under raw and cooked conditions and halal slaughtered chicken, respectively. 5. The methods used may be employed by the food safety and regulatory agencies for regular screening of meat quality and to authenticate CS or HS chicken.

Traditional halal meat production without stunning versus commercial slaughter with electrical stunning of slow-growing broiler chicken: impact on meat quality and proteome changes

Impact of traditional halal meat production without stunning (NST) and commercial slaughter with electrical stunning (ST) of 100 slow-growing broiler chicken on blood plasma and different biochemical, enzymatic, hormonal, meat quality, and proteomic changes was evaluated. The results revealed lower (P < 0.05) postmortem pH values and higher redness (a*) scores for ST samples relative to NST group. Myofibrillar fragmentation index and bleeding efficiency (%) were lower (P < 0.05) in ST compared to NST samples. The ST group had higher (P < 0.05) creatinine, total protein, alanine aminotransferase (ALT), and triiodothyronine (T3) than NST group, however, no difference (P > 0.05) in blood glucose, lactate dehydrogenase (LDH), creatine kinase (CK), thyroxine (T4), cortisol, and aspartate aminotransferase (AST) was observed relative to NST samples. The 2-dimensional gel electrophoresis (2-DE) coupled to MALDI-TOF MS of meat samples has identified 14 differentially abundant proteins between 2 groups. Proteins demonstrating positive correlation with stress namely adenylate kinase isoenzyme-1, Rho guanine nucleotide exchange factor (NST), and apolipoprotein A-I (ST) were overabundant. From the current study, it is concluded that electrical stunning of broilers prior to slaughter or traditional halal slaughter without stunning does not adversely affect the meat quality.

Differential plasma protein expression after ingestion of essential amino acid-based dietary supplement verses whey protein in low physical functioning older adults

In a recent randomized, double-blind, placebo-controlled trial, we were able to demonstrate the superiority of a dietary supplement composed of essential amino acids (EAAs) over whey protein, in older adults with low physical function. In this paper, we describe the comparative plasma protein expression in the same subject groups of EAAs vs whey. The plasma proteomics data was generated using SOMA scan assay. A total of twenty proteins were found to be differentially expressed in both groups with a 1.5-fold change. Notably, five proteins showed a significantly higher fold change expression in the EAA group which included adenylate kinase isoenzyme 1, casein kinase II 2-alpha, Nascent polypeptide-associated complex subunit alpha, peroxiredoxin-1, and peroxiredoxin-6. These five proteins might have played a significant role in providing energy for the improved cardiac and muscle strength of older adults with LPF. On the other hand, fifteen proteins showed slightly lower fold change expression in the EAA group. Some of these 15 proteins regulate metabolism and were found to be associated with inflammation or other comorbidities. Gene Ontology (GO) enrichment analysis showed the association of these proteins with several biological processes. Furthermore, protein–protein interaction network analysis also showed distinct networks between upregulated and downregulated proteins. In conclusion, the important biological roles of the upregulated proteins plus better physical function of participants in the EAAs vs whey group demonstrated that EAAs have the potential to improve muscle strength and physical function in older adults. This study was registered with ClinicalTrials.gov: NCT03424265 “Nutritional interventions in heart failure.”

Proteomic Changes in Sarcoplasmic and Myofibrillar Proteins Associated with Color Stability of Ovine Muscle during Post-Mortem Storage.

The objective of this study was to investigate the proteomic characteristics for the sarcoplasmic and myofibrillar proteomes of M. longissimus lumborum (LL) and M. psoasmajor (PM) from Small-tailed Han Sheep. During post-mortem storage periods (1, 3, and 5 days), proteome analysis was applied to elucidate sarcoplasmic and myofibrillar protein changes in skeletal muscles with different color stability. Proteomic results revealed that the identified differentially abundant proteins were glycolytic enzymes, energy metabolism enzymes, chaperone proteins, and structural proteins. Through Pearson’s correlation analysis, a few of those identified proteins (Pyruvate kinase, Adenylate kinase isoenzyme 1, Creatine kinase M-type, and Carbonic anhydrase 3) were closely correlated to representative meat color parameters. Besides, bioinformatics analysis of differentially abundant proteins revealed that the proteins mainly participated in glycolysis and energy metabolism pathways. Some of these proteins may have the potential probability to be predictors of meat discoloration during post-mortem storage. Within the insight of proteomics, these results accumulated some basic theoretical understanding of the molecular mechanisms of meat discoloration.

Differential Association of 4E-BP2-Interacting Proteins Is Related to Selective Delayed Neuronal Death after Ischemia.

Cerebral ischemia induces an inhibition of protein synthesis and causes cell death and neuronal deficits. These deleterious effects do not occur in resilient areas of the brain, where protein synthesis is restored. In cellular stress conditions, as brain ischemia, translational repressors named eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) specifically bind to eIF4E and are critical in the translational control. We previously described that 4E-BP2 protein, highly expressed in brain, can be a molecular target for the control of cell death or survival in the reperfusion after ischemia in an animal model of transient cerebral ischemia. Since these previous studies showed that phosphorylation would not be the regulation that controls the binding of 4E-BP2 to eIF4E under ischemic stress, we decided to investigate the differential detection of 4E-BP2-interacting proteins in two brain regions with different vulnerability to ischemia-reperfusion (IR) in this animal model, to discover new potential 4E-BP2 modulators and biomarkers of cerebral ischemia. For this purpose, 4E-BP2 immunoprecipitates from the resistant cortical region and the vulnerable hippocampal cornu ammonis 1 (CA1) region were analyzed by two-dimensional (2-D) fluorescence difference in gel electrophoresis (DIGE), and after a biological variation analysis, 4E-BP2-interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Interestingly, among the 4E-BP2-interacting proteins identified, heat shock 70 kDa protein-8 (HSC70), dihydropyrimidinase-related protein-2 (DRP2), enolase-1, ubiquitin carboxyl-terminal hydrolase isozyme-L1 (UCHL1), adenylate kinase isoenzyme-1 (ADK1), nucleoside diphosphate kinase-A (NDKA), and Rho GDP-dissociation inhibitor-1 (Rho-GDI), were of notable interest, showing significant differences in their association with 4E-BP2 between resistant and vulnerable regions to ischemic stress. Our data contributes to the first characterization of the 4E-BP2 interactome, increasing the knowledge in the molecular basis of the protection and vulnerability of the ischemic regions and opens the way to detect new biomarkers and therapeutic targets for diagnosis and treatment of cerebral ischemia.

Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies.

Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 104 M−1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.

Novel Proteomic Profiling of Epididymal Extracellular Vesicles in the Domestic Cat Reveals Proteins Related to Sequential Sperm Maturation with Differences Observed between Normospermic and Teratospermic Individuals

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals.

Proteome activity landscapes of tumor cell lines determine drug responses

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work.

Assessing the Global Impact of an Adenylate Kinase Isoenzyme on AMPK and mTOR Signaling Pathways Through Metabolic Profiling in both Brain and Lung Cancer Cell Lines

In a variety of human cancers, it is well observed that specific altered bioenergetic programs must be sustained in order to maintain the cancer cell phenotype. Well‐described genes play a role in sustaining energy flux for the cancer cell and recent studies add the regulation of AK4 expression, an adenylate kinase localized to the mitochondrial matrix, to the list. Our results demonstrate that in response to altered AK4 expression in cancer cells, intracellular ATP levels drastically increase. Secondly, we demonstrate that in response to perturbed AK4 expression in cancer cells, two critical homeostatic signaling pathways, the 5′ AMP‐activated protein kinase (AMPK) and a mammalian target of rapamycin (mTOR) pathways, are significantly altered. Both mTOR and AMPK proteins govern a metabolic pathway in which they sense cellular energy levels and nutrient status in response to regulate cancer cell growth and/or proliferation. Lastly, AK4 also has an impact on other bioenergetic pathways. For instance, AK4 can catalyze the reaction GTP + AMP ↔ GDP + ADP24 and sustain critical energy flux of nucleotides in the cell. Because AK4 expression appears to regulate cellular metabolic responses at multiple levels, further analyses by obtaining a metabolomic profile is necessary to highlight the global impact AK4 exerts on all the metabolites in the cancer cell. To do this, cell lines will be engineered without the AK4 gene in glioblastomas, lung adenocarcinomas, and HeLa cells then analyzed to identify all known metabolites in the system. It is hypothesized that without the expression of an AK4 protein there will be indirect or direct disruptions in the global nucleotide pools by either increasing nucleotide biosynthesis or decreasing nucleotide consumption. The outcome of this study will add to the small library of research on AK4 as well as provide a comprehensive understanding on the enzymatic activity and interactions where AK4 exerts its biological effects on cancer cell metabolism.Support or Funding InformationFunded by National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) Grant 1R15GM123382‐01.

Endpoint Temperature Influences Sarcoplasmic Proteome Profile of Cooked Beef Longissimus Lumborum

ObjectivesCooking ensures safety and enhances the palatability attributes of meat. Denaturation of myoglobin results in the dull-brown color of cooked meats. The denaturation of sarcoplasmic proteins is influenced by the degree of heat treatment, and their solubility is decreased with an increase in the endpoint cooking temperature. While previous studies examined the relationship between myoglobin denaturation, cooked color, and internal temperature in beef, investigations are yet to be undertaken to characterize the association between endpoint temperature, sarcoplasmic proteome, and color attributes in cooked steaks. Therefore, the objective of the present study was to examine the influence of endpoint cooking temperature (60 and 71°C) on sarcoplasmic proteome and internal color of beef longissimus lumborum (LL) steaks.Materials and MethodsEight (n = 8) beef LL muscles (14 d postmortem; USDA Choice) were obtained from a commercial packing plant. Two 2.5-cm thick steaks were fabricated from the center of the muscles and were cooked to internal endpoint temperature of 60°C (C-60) or 71°C (C-71) in a clam-shell grill. Cooked steaks were immediately cooled in slushed ice, sliced parallel to the grilled surface, and internal redness (a* value) and color stability (R630/580) were evaluated instrumentally. Sarcoplasmic proteome from the interiors of the cooked steaks was analyzed using 2-dimensional electrophoresis, and the gel images were digitally analyzed. The protein spots exhibiting more than 2.5-fold intensity differences (P &lt; 0.05) between C-60 and C-71 were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsThe C-60 steaks demonstrated greater (P &lt; 0.05) a* and R630/580 than their C-71 counterparts. Seven differentially abundant proteins were identified and were over-abundant (P &lt; 0.05) in C-60 compared to C-71. The differentially abundant proteins belong to 6 functional groups, i.e., transport proteins (serum albumin and hemoglobin), energy metabolism (adenylate kinase isoenzyme 1), chaperones (heat shock protein β-1), antioxidant (thioredoxin-dependent peroxide reductase), glycolytic enzymes (fructose-bisphosphate aldolase B), and protease (cytosol aminopeptidase).ConclusionThe findings indicated that the endpoint cooking temperature influences the internal cooked color and the sarcoplasmic proteome profile of beef LL steaks. The overabundant proteins in steaks cooked to 60°C may be utilized as potential biomarkers for undercooked beef, which is a source for foodborne infections.

The Discovery of Proteins Associated with Freshness of Coregonus Peled Muscle During Refrigerated Storage.

The aim of this study is to identify the protein indicator of freshness of Coregonus peled using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) methods. Samples were obtained prior to (control group) and 2, 4, 6, and 8 days after refrigerated storage for quality and proteomics analysis. Three proteins were found to have significant differential abundance in sample groups during the refrigerated storage, including l-lactate dehydrogenase, adenylate kinase isoenzyme 1, and myosin heavy chain, which were associated with freshness changes of C. peled. The freshness of C. peled fish during the refrigerated storage can be differentiated from the comparison of the specific proteins. PRACTICAL APPLICATION: The changes of food quality pose not only the relative economical loses but also the potential implications on consumer's health. Proteomics can represent a powerful tool to explore potential biomarkers that may be related to meat quality defects. The identification of key protein biomarkers linked to freshness of Coregonus peled allows to monitor the response of the food matrix during storage and try to minimizes these defects.

Endpoint Temperature Influences Sarcoplasmic Proteome Profile of Cooked Beef Longissimus Lumborum

ObjectivesCooking ensures safety and enhances the palatability attributes of meat. Denaturation of myoglobin results in the dull-brown color of cooked meats. The denaturation of sarcoplasmic proteins is influenced by the degree of heat treatment, and their solubility is decreased with an increase in the endpoint cooking temperature. While previous studies examined the relationship between myoglobin denaturation, cooked color, and internal temperature in beef, investigations are yet to be undertaken to characterize the association between endpoint temperature, sarcoplasmic proteome, and color attributes in cooked steaks. Therefore, the objective of the present study was to examine the influence of endpoint cooking temperature (60 and 71°C) on sarcoplasmic proteome and internal color of beef longissimus lumborum (LL) steaks.Materials and MethodsEight (n = 8) beef LL muscles (14 d postmortem; USDA Choice) were obtained from a commercial packing plant. Two 2.5-cm thick steaks were fabricated from the center of the muscles and were cooked to internal endpoint temperature of 60°C (C-60) or 71°C (C-71) in a clam-shell grill. Cooked steaks were immediately cooled in slushed ice, sliced parallel to the grilled surface, and internal redness (a* value) and color stability (R630/580) were evaluated instrumentally. Sarcoplasmic proteome from the interiors of the cooked steaks was analyzed using 2-dimensional electrophoresis, and the gel images were digitally analyzed. The protein spots exhibiting more than 2.5-fold intensity differences (P < 0.05) between C-60 and C-71 were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsThe C-60 steaks demonstrated greater (P < 0.05) a* and R630/580 than their C-71 counterparts. Seven differentially abundant proteins were identified and were over-abundant (P < 0.05) in C-60 compared to C-71. The differentially abundant proteins belong to 6 functional groups, i.e., transport proteins (serum albumin and hemoglobin), energy metabolism (adenylate kinase isoenzyme 1), chaperones (heat shock protein β-1), antioxidant (thioredoxin-dependent peroxide reductase), glycolytic enzymes (fructose-bisphosphate aldolase B), and protease (cytosol aminopeptidase).ConclusionThe findings indicated that the endpoint cooking temperature influences the internal cooked color and the sarcoplasmic proteome profile of beef LL steaks. The overabundant proteins in steaks cooked to 60°C may be utilized as potential biomarkers for undercooked beef, which is a source for foodborne infections.

The effect of sarcoplasmic protein phosphorylation on glycolysis in postmortem ovine muscle

AbstractProtein phosphorylation is a key modulator in glycolysis metabolism and regulates the activity of glycometabolic enzymes. The phosphorylation levels of sarcoplasmic proteins were investigated in relationship to the glycolysis rate in postmortem ovine muscle in the present study. The global phosphorylation levels of sarcoplasmic proteins increased early postmortem and then decreased afterwards in postmortem ovine muscle. Protein bands of different phosphorylation levels were identified as glycometabolism‐related enzymes. The phosphorylation levels of Glycogen phosphorylase and Pyruvate kinase maybe one possible reason for the different glycolytic rates at 0.5 h postmortem. The glycolytic rate attributes were negatively correlated with four bands which were probably phosphofructokinase, enolase and adenylate kinase isoenzyme. In summary, the phosphorylation of sarcoplasmic proteins is related to muscle pH decline early postmortem, influencing the meat rigour mortis and quality development.

Differential Musculoskeletal Adaptations to Exercise of the Soleus and Vastus Lateralis

The vastus lateralis (VL) and soleus (SOL) muscles show vigorous changes when exposed to unloading conditions. There is evidence that the SOL muscle shows an increased sensitivity to loading yet has been shown to be resistant to exercise-induced adaptations. PURPOSE: To utilize high-resolution two-dimensional gel electrophoresis combined with mass spectrometry to identify anomalous biomarkers of the SOL muscle. METHODS: Biopsy samples of the VL and SOL muscles were obtained from three healthy, inactive individuals (1 male, 21 yrs, 92.5 kg, 167.5 cm; 2 females, 18 and 19 yrs, 66.6 and 71.7 kg, 153.3 and 161.9 cm respectively). Muscle tissue was homogenized in a bead homogenizer and protein quantified with a DC protein assay. Two-dimensional gel electrophoresis was performed and differences in spot abundance between the two muscles were used to select spots of interest. Proteins of the 24 selected spots were subsequently identified by MALDI-TOF MS/MS scanning and Mascot database searching against Swiss-Prot human protein database. RESULTS: Results from the 2D gel electrophoresis varied across the three subjects. Proteins identified from spots of greater intensity in the VL were myosin light chain isoforms, actin, adenylate kinase isoenzyme, alpha-crystallin B, ankyrin repeat domain-containing protein, plasminogen receptor, G-protein coupled receptor, and troponin I. The main proteins identified in the SOL were related to myoglobin with a protein phosphatase protein also being identified. Western blotting will be conducted to verify the identified proteins. CONCLUSION: This preliminary work has identified differential proteins between the SOL and VL relating to oxygen transport, cytoskeletal components, and energy regulation. Future work should examine changes to the proteome between these two muscle with exercise and unloading. Supported by NIH Grants UL1GM118979, TL4GM118980, and RL5GM118978.

Comparative proteomic analysis reveals that juvenile hormone binding protein and adenylate kinase may be involved in the molting process of silkworm, Bombyx mori.

The molting is an essential part of the silkworm metamorphosis development. Although previous studies have demonstrated that molting in silkworm is associated with prothoracicotropic hormone (PTTH), molting hormone (MH), and juvenile hormone (JH), the changes of proteins and genes during silkworm molting, as well as the molecular mechanism about its generating and maintaining remains unclear. In this paper, the proteomic approaches were employed to investigate this issue. Totally, 35 different proteins were successfully identified through mass spectrometry and database searches, among which 42 % proteins were involved in cell structure and 16 % proteins belonged to the metabolism group. Meanwhile, vacuolar ATP synthase, juvenile hormone binding protein precursor and adenylate kinase isoenzyme were found to be down-regulated at early, mid-molt stages, which were further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Taken together, our data suggests that juvenile hormone binding protein (JHBP) and adenylate kinase (AK) play a critical role in the process of silkworm molting, which may participate in the regulation of silkworm molting.

Proteomic Biomarkers for Color in Beef Longissimus Lumborum Aged for 21 Days

ObjectivesMeat color is highly critical to fresh beef marketability as it influences consumer purchase decisions at the point-of-sale. Longissimus lumborum (LL) is an economically important and color-stable muscle in beef hindquarter which has been extensively studied with respect to color biochemistry. Previous research indicated that sarcoplasmic proteome influences fresh beef color. Post-mortem aging employed to improve beef tenderness and palatability can influence color as well as the sarcoplasmic proteome. Our objective was to examine color attributes and sarcoplasmic proteome profile of beef LL during wet-aging, and to identify potential biomarkers for beef color attributes.Materials and MethodsLL muscles were obtained from both sides of eight (n = 8) beef carcasses (USDA Choice, 24 h post-mortem). LL from each side was further divided into two equal-length sections and vacuum-packaged. The vacuum-packaged muscle sections were randomly assigned to aging at 2°C for either 0 (LL0), 7 (LL7), 14 (LL14), or 21 (LL21) days. On each aging period, muscle sections were fabricated into 2.5-cm thick steaks. Samples for proteome analysis obtained during fabrication were frozen at –80°C. After respective aging, the steaks were allowed to bloom for 2 h, and lightness (L*), redness (a*), yellowness (b*), hue (trueness of red), chroma (saturation index), pH, and metmyoglobin reducing activity (MRA) were evaluated. The color data, pH, and MRA were analyzed using MIXED procedure in SAS (SAS Inst. Inc., Cary, NC). Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis (pH 5 to 8; 13.5% acrylamide gels). The images of Coomassie Blue-stained gels were obtained using VersaDoc and were analyzed by PDQuest software. The influence of aging on proteome profile was examined by comparing LL0 against rest of the aging days. Protein spots exhibiting 1.5–fold intensity difference and associated with P 0.05) on all aging days. Proteome analyses revealed that adenylate kinase isoenzyme 1 was more abundant in LL7, whereas creatine kinase M-type, β-enolase, phosphoglucomutase-1, heat shock cognate 71 kDa protein, and alanine aminotransferase 1 were more abundant in LL14. Comparison of LL0 and LL21 proteomes revealed greater abundance of ɑ-enolase in LL21, whereas malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were more abundant in LL0.ConclusionOur results indicated that beef LL demonstrates lower surface redness, lightness, yellowness, hue, and chroma on d 0 of aging compared to the other aging periods. Majority of the differentially abundant proteins observed in LL during aging are chaperones and enzymes associated with energy metabolism. Chaperones have been reported to prevent protein denaturation and aggregation in biological systems, whereas metabolic enzymes are well-known to play a critical role on beef color biochemistry. Therefore, these findings indicated that differentially abundant sarcoplasmic proteins could be utilized as potential biomarkers for color attributes in fresh beef LL.

Proteomic Analysis of Protein Expression Throughout Disease Progression in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. Mice in the transgenic AβPPswe/PS1dE9 mouse line express a chimeric mouse/human amyloid-β protein precursor (Mo/HuAβPP695swe) and mutant human presenilin 1 (PS1-dE9) associated with early-onset AD. Knowing the protein expression in these mice may offer better understanding of the pathological changes in AD. In this study, we used two-dimensional gel electrophoresis combined with mass spectrometry techniques to compare protein expression in AβPPswe/PS1dE9 mice with age-matched wild-type mice throughout the disease progression. We identified 15 proteins that were significantly different between the AβPPswe/PS1dE9 mice and age-matched controls and also changed with disease development. Among those, the expression levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times higher than those in normal mice: DCC-interacting protein 13-beta, serum albumin, creatine kinase B-type, heat shock 70 kDa protein 1A, T-complex protein 1 subunit beta, adenylate kinase isoenzyme 1, pyruvate dehydrogenase E1 component subunit beta mitochondrial, and V-type proton ATPase catalytic subunit A. Levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times lower than those in normal mice: dihydropyrimidinase-related protein 2, actin cytoplasmic 2, isoform 1 of V-type proton ATPase catalytic subunit, tubulin alpha-1C chain, F-actin-capping protein subunit alpha-2, ubiquitin carboxyl-terminal hydrolase isozyme L1, and actin cytoplasmic 1. These proteins are involved in regulating various cellular functions, including cytoskeletal structure, energy metabolism, synaptic components, and protein degradation. These findings indicate altered protein expression in the pathogenesis of AD and illuminate novel therapeutic avenues for treatment in AD.

Differential proteomic analysis of the anti-depressive effects of oleamide in a rat chronic mild stress model of depression

Depression is a complex psychiatric disorder, and its etiology and pathophysiology are not completely understood. Depression involves changes in many biogenic amine, neuropeptide, and oxidative systems, as well as alterations in neuroendocrine function and immune-inflammatory pathways. Oleamide is a fatty amide which exhibits pharmacological effects leading to hypnosis, sedation, and anti-anxiety effects. In the present study, the chronic mild stress (CMS) model was used to investigate the antidepressant-like activity of oleamide. Rats were exposed to 10weeks of CMS or control conditions and were then subsequently treated with 2weeks of daily oleamide (5mg/kg, i.p.), fluoxetine (10mg/kg, i.p.), or vehicle. Protein extracts from the hippocampus were then collected, and hippocampal maps were generated by way of two-dimensional gel electrophoresis (2-DE). Altered proteins induced by CMS and oleamide were identified through mass spectrometry and database searches. Compared to the control group, the CMS rats exhibited significantly less body weight gain and decreased sucrose consumption. Treatment with oleamide caused a reversal of the CMS-induced deficit in sucrose consumption. In the proteomic analysis, 12 protein spots were selected and identified. CMS increased the levels of adenylate kinase isoenzyme 1 (AK1), nucleoside diphosphate kinase B (NDKB), histidine triad nucleotide-binding protein 1 (HINT1), acyl-protein thioesterase 2 (APT-2), and glutathione S-transferase A4 (GSTA4). Compared to the CMS samples, seven spots changed significantly following treatment with oleamide, including GSTA4, glutathione S-transferase A6 (GSTA6), GTP-binding nuclear protein Ran (Ran-GTP), ATP synthase subunit d, transgelin-3, small ubiquitin-related modifier 2 (SUMO2), and eukaryotic translation initiation factor 5A-1 (eIF5A1). Of these seven proteins, the level of eIF5A1 was up-regulated, whereas the remaining proteins were down-regulated. In conclusion, oleamide has antidepressant-like properties in the CMS rat model. The identification of proteins altered by CMS and oleamide treatment provides support for targeting these proteins in the development of novel therapies for depression.

Proteomic analysis indicates that overexpression and nuclear translocation of lactoylglutathione lyase (GLO1) is associated with tumor progression in murine fibrosarcoma

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.