Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND.     

Long-term effect of moderate and profound hypothermia on morphology, neurological, cognitive and behavioural functions in a rat model of perinatal asphyxia

Summary. Background. Perinatal asphyxia is a frequent cause of neurological handicap with no known therapy. However, hypothermic therapy has recently attracted attention owing to its neuroprotective property in brain of immature organisms. Objectives. Hypothermia appears to be promising in reversing the immediate effect of perinatal asphyxia, but data on long-term neuroprotection is still lacking. We therefore intended to test the long-term effect of moderate and profound hypothermia on brain morphology and functions using a well established rat model of perinatal asphyxia. Methods. Rat pups delivered by caesarean section were placed into a water bath, still in patent membranes, at 37 °C and variable hypothermic conditions to induce asphyxia and thereafter given to surrogate mothers. Examinations were performed at the age of three months, consisting of a battery of motor, behavioural, cognition and reflex tests including rota-rod, Morris water maze, multiple T-maze, elevated plus maze and open field studies. Morphological alterations were evaluated by Nissl staining of brain areas known to be hypoxia sensitive. Neurotransmission system markers, including tyrosine hydroxylase, vesicular monoamine transporter, vesicular acetylcholine transporter and excitatory amino acid carrier1 were analyzed by immunohistochemistry. Results. Survival increased with hypothermia. The Nissl stain revealed neuronal loss in hippocampus and hypothalamus of normothermic asphyxiated group (20/37) compared to controls (0/37), but no neuroprotective patterns emerged from hypothermia. An overall inconsistent protection of the neural systems was noted by variable periods of hypothermia. Motor function was significantly impaired in 20/37 as compared to 0/37. In the Morris water maze and multiple T-maze, results were comparable between the groups. In the elevated plus maze, time spent in the closed arm was reduced and in the open field, vertical behaviour was altered in the 20/37 group with horizontal motor behaviour being unaffected. Hypothermia reversed all abnormalities seen in 20/37, with short-term moderate and profound hypothermia being superior to long-term hypothermia. Conclusion. Hypothermia not only significantly increased survival, but also resulted in unimpaired motor as well as improved cognitive functions. Those findings are in contrast to altered brain morphology. As neuronal loss was present in various brain regions, we conclude that deficits may be compensated in the maturing animal. Intrahypoxic hypothermia was able to protect the rat from the devastating effect of perinatal asphyxia not in morphological, but in functional terms. The first and third authors have contributed equally to this work.     

Mass spectrometrical identification of brain proteins including highly insoluble and transmembrane proteins

Conventional two-dimensional electrophoresis (2DE) is the main technique used for protein profiling of tissues and cells, however separation of strongly acidic, basic or highly insoluble proteins is still limited. A series of methods have been proposed to cope with this problem and the use of discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) with subsequent SDS-PAGE followed by mass spectrometry showed that results from 2DE can be complemented by this approach. It was the aim of this study to separate and identify proteins from whole mouse brain that were not demonstrated by 2DE. For this purpose samples were homogenised, soluble proteins were removed by ultracentrifugation and the water-insoluble pellet was resuspended in a mixture containing urea, 16-BAC, glycerol, pyronine Y and dithiothreitol. Electrophoresis was run in the presence of 16-BAC, the strip from the gel containing separated proteins was cut out and was re-run on SDS-PAGE. Protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. One hundred and six individual proteins represented by 187 spots were unambiguously identified consisting of 42 proteins with predicted pI values of pI>8.0, 25 with a 6.0相似文献   

Tuberous sclerosis genes regulate cellular 14-3-3 protein levels

The genes TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for tuberous sclerosis. This autosomal dominant tumor suppressor gene syndrome affects about 1 in 6000 individuals. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. We performed a proteomics approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the cellular levels of four isoforms of the 14-3-3 protein family, 14-3-3 gamma, 14-3-3, 14-3-3 sigma, and 14-3-3 zeta, to be regulated by the two tuberous sclerosis gene products. In the same experiments the protein levels of keratin 7, capZ alpha-1 subunit, ezrin, and nedasin were not affected by ectopic TSC1 or TSC2. Western blot analyses confirmed the deregulation of 14-3-3 proteins upon ectopic overexpression of TSC1 and TSC2. A TSC1 mutant not encoding the transmembrane domain and the tuberin-binding domain but harbouring most of the coiled-coil region and the ERM protein interaction domain of hamartin did not affect 14-3-3 protein levels. The here presented findings suggest that deregulation of 14-3-3 protein amounts might contribute to the development of tumors in tuberous sclerosis patients. These data provide important new insights into the molecular development of this disease especially since both, the TSC genes and the 14-3-3 proteins, are known to be involved in mammalian cell cycle control.     

Hippocampal AMPA-type receptor complexes containing GluR3 and GluR4 are paralleling training in the Multiple T-Maze

Although it is well-known that AMPA receptors are involved in spatial learning and memory, published data on GluR3 and GluR4 are limited. Moreover, there is no information about GluR3 and GluR4 receptor complex levels in spatial memory training. It was therefore the aim of the study to determine the above-mentioned receptor levels following training in the Multiple T-Maze (MTM). Results from the MTM and hippocampal membrane proteins from C57BL/6J mice were taken from an own previous study and GluR3 and GluR4 receptor complexes were run on blue native gel electrophoresis followed by immunoblotting and quantification of bands. Subsequently, GluR3 and GluR4 were identified under denaturing conditions from two-dimensional gels by mass spectrometry (nano-LC-ESI-MS/MS). Hippocampal levels of GluR3 containing complexes (apparent molecular weight between 480 and 720) were decreased while GluR4 containing complexes (apparent molecular weight between 480 and 720) were increased. GluR4 complex levels in trained mice were correlating with latency and speed. Mass spectrometry unambiguously identified the two receptor subunits. The findings show that GluR3 and GluR4 may have different functions in the processes of spatial memory training in the MTM and indeed, different neurobiological functions of the two receptor subunits have been already reported. GluR3 and GluR4 receptor complex rather than subunit levels are paralleling training in the MTM and GluR4 complex levels were even linked to memory training, which may be of relevance for understanding molecular memory processes, interpretation of previous work or for design of future AMPA receptor studies.     

Intraperitoneal injection of saline modulates hippocampal brain receptor complex levels but does not impair performance in the Morris Water Maze

The involvement of the hippocampus in pain has been demonstrated but key players, i.e. the major brain receptors have not been shown to be modulated by pain. It was therefore the aim of the study to show the concerted action and pattern of brain receptor complex levels in a non-invasive model of moderate pain. C57BL/6J mice were divided into four groups of 14 animals each: trained injected, trained non-injected, yoked injected and yoked non-injected. Animals were tested in the open field and the elevated plus maze for behavioural evaluation and cognitive functions were tested using the Morris Water Maze. Hippocampi were taken 6 h following sacrification. Membrane proteins were prepared by ultracentrifugation and run on blue native gels to keep the native state, blotted to membranes and western blotting was carried out using the primary antibodies against serotonin receptor 5HT1A, muscarinic acetylcholine receptor M1 (mAChR-M1), nicotinic acetylcholine receptor alpha7 (nAChR-alpha7), glutamate (AMPA) receptor (GluR1) and neurokinin receptor 1 (NK-1). There was no difference between performance in behaviour or in the MWM between groups. Brain receptor level changes involved all receptors given above. Pain affected mAChR-M1, GluR1 and NK-1 complex levels when yoked-injected were compared with yoked non-injected animals. Memory mechanisms affected mAChR-M1 complex levels when trained non-injected animals were compared with yoked non-injected controls. Taken together, the neurochemical basis for testing receptor agonists/antagonists on the role of pain and the hippocampus was generated that may be useful for interpretations of the role of this complex area in moderate pain.     

Modafinil improves performance in the multiple T-Maze and modifies GluR1, GluR2, D2 and NR1 receptor complex levels in the C57BL/6J mouse

Modafinil has been shown to modify behavioural and cognitive functions and to effect several brain receptors. Effects, however, were not observed at the receptor protein complex level and it was therefore the aim of the study to train mice in the multiple T-Maze (MTM) as a paradigm for spatial memory and to determine paralleling brain receptor complex levels. Sixty C57BL/6J mice were used in the study and divided into four groups (trained drug injected; trained vehicle injected; yoked drug injected; yoked vehicle injected). Animals obtained training for 4?days and were killed 6?h following the last training session on day 4. Hippocampi were dissected from the brain, membrane fractions were prepared by ultracentrifugation and were run on blue-native gels and immunoblotted with antibodies against major brain receptors. Modafinil treatment led to decreased latency and increased average speed, but not to changes in pathlength and number of correct decisions in the MTM. Drug effects were modifying receptor complexes of GluR1, GluR2, D2 and NR1. Training effects on receptor complex levels were observed for GluR3, D1 and nicotinic acetylcholine receptor alpha 7 (Nic7). GluR1 levels were correlating with GluR2 and D1 levels were correlating with D2 and NR1. Involvement of the glutamatergic, NMDA, dopaminergic and nicotinergic system in modafinil and memory training were herein described for the first time. A brain receptor complex pattern was revealed showing the concerted action following modafinil treatment.     

Mass spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2A-D and five novel phosphorylation sites on NR2A and NR2B

The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.     

Proteomic analysis of human hippocampus shows differential protein expression in the different hippocampal subfields

In the current investigation, we aimed to characterize the differential protein expression in each of the hippocampal subregions in healthy control samples (n = 20). We used laser-assisted microdissection and difference in-gel electrophoresis to enrich for these tissues and to compare protein profiles. Image analysis was carried out using Progenesis SameSpots. Samples with a false discovery rate smaller than 5%, a p-value of < 0.01, and an expression of at least ± 1.2 were considered significant. Proteins were identified using LC-ESI-MS/MS. The raw mass spectral data were analyzed using DataAnalysis software. Data were searched against the Swissprot database using MASCOT. Samples were grouped according to the different subregions and we found 182 spots to be differentially expressed between the different hippocampal subregions. These have been made available as part of the UCD-2DPAGE database at http://proteomics-portal.ucd.ie:8082. The associated MS data have been submitted to PRIDE (Accession numbers 21593-21745). This baseline data will be helpful in helping us to understand the central role of the hippocampus in health and the evidence that particular hippocampal subregions are differentially affected in disease.     

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.