Julius Paul Pradeep John

Silk cocoon of Bombyx mori: Proteins and posttranslational modifications – heavy phosphorylation and evidence for lysine-mediated cross links

Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2‐DE was carried out with multi‐enzyme in‐gel digestion followed by MS identification of silk‐peptides. High‐sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys‐>allysine formation have been observed providing evidence for lysine‐mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di‐tyrosine cross links. A high degree of sequence conflicts probably representing single‐nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk.

Hippocampal synapsin isoform levels are linked to spatial memory enhancement by SGS742

Synapsins are essential proteins for synaptic plasticity and there is no information available for their role in cognitive enhancement (CE) of spatial memory formation. It was therefore the aim of the study to link individual synapsin proteins and their isoforms to spatial memory formation enhanced by SGS742 in the mouse. Extracted hippocampal proteins from a cognitive study treating OF1 mice with the cognitive enhancer SGS742 and tested in the Morris water maze, were run on two‐dimensional gel electrophoresis. Subsequently, protein spots were unambiguously identified by qQ‐TOF mass spectrometry. Quantification of proteins from four groups (NaCl‐treated mice, SGS742‐treated mice, SGS742‐treated yoked controls, and NaCl‐treated yoked controls) was carried out according to an in‐gel stable isotope labeling method. A total of 17 protein spots representing synapsin isoforms were identified and quantified. Using quantification of individual synapsin isoforms showed that these can be clearly assigned to CE by the GABAB antagonist SGS742. Quantitative determination of individual synapsin isoform showed an increase in SGS742‐treated mice (mean ± SD) of ratios between light and heavy stable isotope labeled synapsin protein (SGS742 vs. controls: 2.19 ± 0.41 for synapsin Ia, and 1.41 ± 0.81 for synapsin IIa). Synapsins Ib and IIb were not linked to CE. The NaCl‐treated controls and the use of yoked controls that were ruling out swimming‐ and stress‐mediated changes of synapsins, unequivocally allow to propose a role for synapsins Ia and IIa in the mechanism of CE of spatial memory formation.

Strain-dependent expression of signaling proteins in the mouse hippocampus.

Individual mouse strains may differ significantly in terms of behavior and cognitive function. Hippocampal gene expression profiling on several mouse strains has been carried out and points toward substantial strain-specific variation of more than 200 genes including components of major signaling pathways involved in neuronal information storage. Strain-specific hippocampal protein expression, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry has been chosen to address this question by determining strain-dependent expression of signaling proteins in hippocampi of four inbred and one outbred mouse strain. Forty-six spots corresponding to 37 different signaling proteins have been analyzed and quantified. Statistical analysis revealed strain-dependent expression of serine/threonine protein phosphatase 1, serine/threonine protein phosphatase 2A, large GTP binding protein OPA1, guanine nucleotide-binding protein beta, putative GTP-binding protein Ran, receptor of activated protein kinase C1, WASP-family protein member 1, voltage-dependent anion channel 2 and 14-3-3 protein gamma. Differential expression of signaling proteins in the hippocampus may contribute to the molecular understanding of strain-dependent behavioral and cognitive performance. Moreover, these data highlight the importance of the genetic background for the analysis of signaling pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.

Complete sequencing and oxidative modification of manganese superoxide dismutase in medulloblastoma cells

Manganese superoxide dismutase (Mn‐SOD or SOD2) is a key antioxidant enzyme and was assigned several roles in tumor biology. Working on medulloblastoma cell line DAOY, we identified two spots as Mn‐SODs. Because of the proposed pivotal role of this enzyme in oncobiology, we decided to completely sequence the proteins and to determine PTMs. Proteins extracted from DAOY cells were run on 2‐DE, multienzyme digestions were carried out and peptides were analyzed by MALDI‐TOF/TOF, Qq‐TOF and the ion trap using both the CID and ETD principles. Both protein expression forms were completely sequenced and revealed identical protein sequences. Histidines His30 and His31 were oxidized in one protein, whereas tryptophan oxidation (Trp‐186) was observed in both. Histidine oxidation was not only indicated by the mass shift of the peptide but also by specific spectra of 2‐oxo‐histidine and a previously described intermediate (His+14). Complete sequencing of the two Mn‐SOD expression forms unambiguously characterizes this enzyme from a tumor cell line providing evidence that can be used for generation of antibodies and allowing conformational studies. The findings of different PTMs in the same gel represent Mn‐SOD oxidative states, while oxidative modification of His30 and 31 may even reflect decreased Mn‐SOD activity.

Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry

Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC–MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and ModiroTM v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.

Mass spectrometrical verification of stomatin-like protein 2 (SLP-2) primary structure

Stomatin‐like protein 2 (SLP‐2) (syn.: EPB72‐like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP‐2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two‐dimensional gel electrophoresis and SLP‐2 was identified by MALDI‐TOF/TOF. SLP‐2 was digested with trypsin, chymotrypsin, Lys‐C, and de novo sequencing studies as well as Nano‐HPLC‐ESI‐MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N‐terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI‐TOF/TOF, Nano‐HPLC‐ESI‐MS/MS and de novo sequencing completed unambiguous analysis of SLP‐2 primary structure of ≈90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP‐2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance. Proteins 2006.

Strain-dependent regulation of neurotransmission and actin-remodelling proteins in the mouse hippocampus

Individual mouse strains differ significantly in terms of behaviour, cognitive function and long‐term potentiation. Hippocampal gene expression profiling of eight different mouse strains points towards strain‐specific regulation of genes involved in neuronal information storage. Protein expression with regard to strain‐ dependent expression of structures related to neuronal information storage has not been investigated yet. Herein, a proteomic approach based on two‐dimensional gel electrophoresis coupled with mass spectrometry (MALDI‐TOF/TOF) has been chosen to address this question by determining strain‐dependent expression of proteins involved in neurotransmission and activity‐induced actin remodelling in hippocampal tissue of five mouse strains. Of 31 spots representing 16 different gene products analysed and quantified, N‐ethylmaleimide‐sensitive fusion protein, N‐ethylmaleimide‐sensitive factor attachment protein‐α, actin‐like protein 3, profilin and cofilin were expressed in a strain‐dependent manner. By treating protein expression as a phenotype, we have shown significant genetic variation in brain protein expression. Further experiments in this direction may provide an indication of the genetic elements that contribute to the phenotypic differences between the selected strains through the expressional level of the translated protein. In view of this, we propose that proteomic analysis enabling to concomitantly survey the expression of a large number of proteins could serve as a valuable tool for genetic and physiological studies of central nervous system function.

Neurotrophin 3/TrkC-regulated proteins in the human medulloblastoma cell line DAOY.

Medulloblastoma (MB) is the most common malignant childhood brain tumor and high neurotrophin (NP) receptor TrkC mRNA expression was identified as a powerful independent predictor of favorable survival outcome. In order to determine downstream effector proteins of TrkC signaling, the MB cell line DAOY was stably transfected with a vector containing the full‐length TrkC cDNA sequence or an empty vector control. A proteomic approach was used to search for expressional changes by two mass spectrometric methods and immunoblotting for validation of significant results. Multiple time points for up to 48 h following NP‐3‐induced TrkC receptor activation were chosen. Thirteen proteins from several pathways (nucleoside diphosphate kinase A, stathmin, valosin‐containing protein, annexin A1, dihydropyrimidinase‐related protein‐3, DJ‐1 protein, glutathione S‐transferase P, lamin A/C, fascin, cofilin, vimentin, vinculin, and moesin) were differentially expressed and most have been shown to play a role in differentiation, migration, invasion, proliferation, apoptosis, drug resistance, or oncogenesis. Knowledge on effectors of TrkC signaling may represent a first useful step for the identification of marker candidates or reflecting probable pharmacological targets for specific treatment of MB.

Identification and characterisation of arsenite (+3 Oxidation State) methyltransferase (AS3MT) in mouse neuroblastoma cell line N1E-115

Summary.Handling and detoxification of metals by enzymes is a major issue that is not in the focus of current biomedical research concepts. The finding of the presence of arsenic (+3 Oxidation State) methyltransferase (AS3MT) in neuroblastoma cells NE-115 as a high abundance protein made us investigate primary structure of AS3MT reflecting an example of metal-handling in eucaryotes. Proteins extracted from NE-115 cells were run on 2-DE followed by two different mass spectrometrical methods. High sequence coverage was obtained by multiple protease digestion and a sequence conflict was solved at arginine 335.These findings are important when future studies on this enzyme are designed at the protein level and in particular, when antibodies against this protein will be generated.

Components of the protein quality control system are expressed in a strain-dependent manner in the mouse hippocampus

Inbred mouse strains are used in forward-genetic experiments, designed to uncover genes contributing to their highly distinct neurophenotypes and multiple reports of variations in mutant phenotypes due to genetic background differences in reverse-genetic approaches have been published. Information on strain-specific protein expression-phenotypes however, is limited and a comprehensive screen of an effect of strain on brain protein levels has not yet been carried out. Herein a proteomic approach, based upon two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF) was used to show significant genetic variation in hippocampal protein levels between five mouse strains. Considering recent evidence for the importance of the intracellular protein quality control system for synaptic plasticity-related mechanism we decided to focus on the analysis of molecular chaperones and components of the ubiquitin-proteasome system. Sixty-six spots, depicting 36 proteins have been unambiguously identified by mass spectrometry. Quantification revealed strain-dependent levels of 18 spots, representing 12 individual gene products. We thus present proteome analysis of hippocampal tissues of several mouse strains as suitable tool to address fundamental questions about genetic control of protein levels and to demonstrate molecular networks of protein metabolism and chaperoning. The findings are useful for designing future studies on these cascades and interpretation of results show that data on brain protein levels cannot be simply extrapolated among different mouse strains.