Wei-Qiang Chen

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 protein levels related to spatial memory are different in the Barnes maze and in the multiple T-maze.

The Multiple T-maze (MTM) and the Barnes maze (BM) are land mazes used for the evaluation of spatial memory. The observation that mice are performing differently in individual mazes made us test the hypothesis that differences in cognitive performances in the two land mazes would be accompanied by differences in hippocampal protein levels. C57BL/6J mice were tested in the BM and in the MTM, hippocampi were extirpated 6 h following the probe trials each, and proteins were extracted for gel-based proteomic analysis. Mice learned the task in both paradigms. Levels of hippocampal proteins from several pathways including signaling, chaperone, and metabolic cascades were significantly different between the two spatial memory tasks. Protein levels were linked to spatial memory specifically as yoked controls were used.

Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter IMPLICATIONS FOR ANGELMAN SYNDROME

Background: αCaMKII modulates amphetamine-induced dopamine transporter-mediated substrate efflux. Results: Mice with ablated or blunted αCaMKII function show decreased amphetamine-triggered efflux. Conclusion: Dopamine transporter function is impaired in mice with targeted αCaMKII mutations and in a mouse model of the Angelman syndrome. Significance: Such new insights into dopamine transporter function may further illuminate the complex pathophysiology of the Angelman syndrome. The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP+) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKIIT305D), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP+ efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.

Crystal and solution structure, stability and post‐translational modifications of collapsin response mediator protein 2

The collapsin response mediator protein 2 (CRMP‐2) is a central molecule regulating axonal growth cone guidance. It interacts with the cytoskeleton and mediates signals related to myelin‐induced axonal growth inhibition. CRMP‐2 has also been characterized as a constituent of neurofibrillary tangles in Alzheimer’s disease. CD spectroscopy and thermal stability assays using the Thermofluor method indicated that Ca2+ and Mg2+ affect the stability of CRMP‐2 and prevent the formation of β‐aggregates upon heating. Gel filtration showed that the presence of Ca2+ or Mg2+ promoted the formation of CRMP‐2 homotetramers, and this was further proven by small‐angle X‐ray scattering experiments, where a 3D solution structure for CRMP‐2 was obtained. Previously, we described a crystal structure of human CRMP‐2 complexed with calcium. In the present study, we determined the structure of CRMP‐2 in the absence of calcium at 1.9 Å resolution. When Ca2+ was omitted, crystals could only be grown in the presence of Mg2+ ions. By a proteomic approach, we further identified a number of post‐translational modifications in CRMP‐2 from rat brain hippocampus and mapped them onto the crystal structure.

Protein profiling by the combination of two independent mass spectrometry techniques

Protein profiling in the high-throughput mode is a most useful technique that allows formation of reference databases for cells and tissues and performance of comparative proteomics. In the proposed protocol protein extraction from tissues is followed by 2D gel electrophoresis (2DE) with subsequent in-gel digestion and identification of soluble proteins by two individual mass spectrometric techniques, tandem matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano-liquid chromatography (nano-LC)-MS/MS. The proposed combined use of these two MS approaches leads to a very high identification rate of well-separated protein spots from a gel. In the first step 2DE separates high-abundance proteins (those visualized by nonsensitive Coomassie blue staining) that are subsequently picked, digested and aliquoted for MS applications. Protein samples not identified by MALDI-MS or MS/MS (77% of all spots) are finally unambiguously identified by nano-LC-MS/MS (total identification rate 94%). This protocol can be completed in 6 weeks.

Changes in brain protein expression are linked to magnesium restriction-induced depression-like behavior

There is evidence to suggest that low levels of magnesium (Mg) are associated with affective disorders, however, causality and central neurobiological mechanisms of this link are largely unproven. We have recently shown that mice fed a low Mg-containing diet (10% of daily requirement) display enhanced depression-like behavior sensitive to chronic antidepressant treatment. The aim of the present study was to utilize this model to gain insight into underlying mechanisms by quantifying amygdala/hypothalamus protein expression using gel-based proteomics and correlating changes in protein expression with changes in depression-like behavior. Mice fed Mg-restricted diet displayed reduced brain Mg tissue levels and altered expression of four proteins, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1), manganese-superoxide dismutase (MnSOD), glutamate dehydrogenase 1 (GDH1) and voltage-dependent anion channel 1. The observed alterations in protein expression may indicate increased nitric oxide production, increased anti-oxidant response to increased oxidative stress and potential alteration in energy metabolism. Aberrant expressions of DDAH1, MnSOD and GDH1 were normalized by chronic paroxetine treatment which also normalized the enhanced depression-like behavior, strengthening the link between the changes in these proteins and depression-like behavior. Collectively, these findings provide first evidence of low magnesium-induced alteration in brain protein levels and biochemical pathways, contributing to central dysregulation in affective disorders.

Hippocampal metabolic proteins are modulated in voluntary and treadmill exercise rats

Systematic protein expression studies in the brain of exercising and sedentary animals have not been carried out so far and it was therefore decided to determine differences in metabolic protein levels in rat hippocampus of sedentary, voluntary and involuntary exercising rats by a proteomic approach. Aged, male Sprague-Dawley rats, 23 months old, were used for the study: the first group consisted of sedentary rats, the second of rats with voluntary exercise from five to 23 months and the third group was performing involuntary exercise on a treadmill from five to 23 months. Two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of spots followed by quantification of spots was carried out. Identification of significantly differential proteins was validated by the determination of the corresponding enzyme activity. Five individual metabolic proteins showed differential protein levels in the three groups: mitochondrial precursors of ornithine aminotransferase, isocitrate dehydrogenase [NAD] subunit alpha, malate dehydrogenase, ubiquinol-cytochrome-c reductase complex core protein 1, and ubiquitin carboxyl-terminal hydrolase isozyme L1. The unambiguously identified metabolic proteins were mainly of mitochondrial localization and fit the expectations of altered mitochondrial activity in exercise. Reduced ubiquitin carboxyl-terminal hydrolase isoenzyme L1 levels in treadmill (forced) exercise show the involvement of the proteasomal pathway as a novel finding. These results not only form the basis for functional studies elucidating mechanisms and differences between voluntary and forced exercise in hippocampal metabolism but also highlight the most intriguing aspect that exercise is affecting the brain at the protein level.

Variations of Protein Levels in Human Amniotic Fluid Stem Cells CD117/2 Over Passages 5−25

Stability of cell lines is the prerequisite for all in vitro research, but literature on the stability of protein expression over passages is limited. Determination of specific stability markers, karyotyping, and morphology may not provide full information on this subject. It was the aim of the study to test protein level fluctuations in a human amniotic fluid stem cell line from passages 5, 7, 11, and 25. While karyotype, cell cycle, apoptosis rate, and 10 markers for characterization of the cell line remained unchanged (carried out at passages 5 and 25), cell volume was increased at passage 25. Significant protein fluctuations were observed for signaling, antioxidant, guidance cue, proteasomal, connective tissue, cytoskeleton proteins, chaperones, a chloride channel, and prothymosin at passages 5, 7, 11, and 25. Herein, the use of this gel-based proteomic screen, checking protein stability for the characterization of cell lines in addition to corresponding published markers, is proposed, in particular when experiments are run over several passages.

A gel-based proteomic method reveals several protein pathway abnormalities in fetal Down syndrome brain.

A large series of protein pathway components have been shown to be dysregulated in Down syndrome (DS) brain. No information about pathomechanisms linked to the trisomic state can be obtained from adult DS brain, however, as neurodegeneration occurs from the fourth decade. The aim of the study was to search for protein dysregulation in fetal DS brain before neurodegenerative changes are observed. Proteins were extracted from fetal DS and control frontal cortex, run on 2-DE, followed by quantification of protein spots with subsequent nano-ESI-LC-MS/MS analysis using an ion trap. Aberrant expression of proteins tropomodulin-2, tubulin alpha 1A chain, and alpha-internexin may indicate disturbed synaptic plasticity; fatty acid binding protein 7 suggests impaired maintenance of neuroepithelial cells; and creatine kinase B may reflect defective energy metabolism. RNA binding protein 4B derangement may represent impaired splicing, altered retrotransposon gag domain-containing protein 1 levels may be pointing to altered retrotransposition, and level changes of the potassium-chloride transporter solute carrier family 12 member 7 may lead to impaired ion fluxes with electrophysiological consequences. Taken together, aberrant protein levels from several pathways in fetal DS are challenging as well as fertilizing the area of research and providing the basis for additional neurochemical and functional studies.

Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders

Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk spidroin-1 from Nephila spiders provide the basis for mechanical-elastic property studies of silk for biotechnological and biomedical potential applications. This article is part of a Special Issue entitled: Proteomics of non-model organisms.