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.     

Differential expression of synaptic proteins in unilateral 6‐OHDA lesioned rat model—A comparative proteomics approach

Parkinson's disease (PD) is characterized as a movement disorder due to lesions in the basal ganglia. As the major input region of the basal ganglia, striatum plays a vital role in coordinating movements. It receives afferents from the cerebral cortex and projects afferents to the internal segment of the globus pallidus and substantia nigra pars reticulate. Additionally, accumulating evidences support a role for synaptic dysfunction in PD. Therefore, the present study explores the changes in protein abundance involved in synaptic disorders in unilateral lesioned 6‐OHDA rat model. Based on ¹⁸O/¹⁶O‐labeling technique, striatal proteins were separated using online 2D‐LC, and identified by nano‐ESI‐quadrupole‐TOF. A total of 370 proteins were identified, including 76 significantly differentially expressed proteins. Twenty‐two downregulated proteins were found in composition of vesicle, ten of which were involved in neuronal transmission and recycling across synapses. These include N‐ethylmaleimide‐sensitive fusion protein attachment receptor proteins (SNAP‐25, syntaxin‐1A, syntaxin‐1B, VAMP2), synapsin‐1, septin‐5, clathrin heavy chain 1, AP‐2 complex subunit beta, dynamin‐1, and endophilin‐A1. Moreover, MS result for syntaxin‐1A was confirmed by Western blot analysis. Overall, these synaptic changes induced by neurotoxin may serve as a reference for understanding the functional mechanism of striatum in PD.     

Effects of simulated microgravity on the expression of presynaptic proteins distorting the GABA/glutamate equilibrium – A proteomics approach

Microgravity may cause cognition‐related changes in the animal nervous system due to the resulting uneven flow of fluids in the body. These changes may restrict the long‐term stay of humans in space for various purposes. In this study, a rat tail suspension model (30^o) was used to explore the effects of 21 days of prolonged simulated microgravity (SM) on the expression of proteins involved in cognitive functions in the rat hippocampus. SM decreased the content of γ‐aminobutyric acid (GABA) and increased the content of glutamate (Glu) in the rat hippocampus. A comparative ¹⁸O‐labeled quantitative proteomics strategy was applied to detect the differential expression of synaptic proteins under SM. Fifty‐three proteins were found to be differentially expressed under SM. Microgravity induces difficulty in the formation of the SNARE complex due to the down‐regulation of vesicle‐associated membrane protein 3(VAMP3) and syntaxin‐1A. Synaptic vesicle recycling may also be affected due to the dysregulation of syntaxin‐binding protein 5 (tomosyn), rab3A and its effector rim2. Both processes are disturbed, indicating that presynaptic proteins mediate a GABA/Glu imbalance under SM. These findings provide clues for understanding the mechanism of the GABA/Glu equilibrium in the hippocampus induced by microgravity in space and represent steps toward safe space travel.     

Identification of common and differential mechanisms of glomerulus and tubule senescence in 24‐month‐old rats by quantitative LC–MS/MS

Kidney aging together with related renal disease had become a major clinical problem. Understanding the mechanisms of aging was important for suspending senescence and decreasing the incidence of aging‐related diseases. In the present work, 24‐month‐old F344 rats were used as aging rats and 3‐month‐old rats were used as young controls. Senescence‐associated‐β‐galactosidase staining results showed that the degree of senescence in renal tubules was more severe than that in glomeruli. We performed quantitative LC–MS to assess the differential protein expression profiles of senescent glomeruli and tubules. Bioinformatics analysis showed that aging, response to oxidative stress, nucleotide metabolism, amine acid metabolism, and inflammatory response were common mechanisms of glomerulus and tubule senescence. Differentially expressed proteins network mediated Golgi vesicle transport, actin filament based process, and regulation of cell death were associated with tubule senescence. More importantly, we found that the changes of four and a half LIM protein 2 (FHL2) were opposite in senescent glomeruli and tubules, and FHL2 could regulate p16 by suppressing T‐box 3, which was involved in regulation of senescence in glomeruli and tubules. In conclusion, we assessed the mechanisms of senescence in aging glomeruli and tubules, and the results yielded new insight into kidney senescence.     

Quantitative proteomic profiling identifies new renal targets of copper(II)‐selective chelation in the reversal of diabetic nephropathy in rats

This study aimed to identify new diabetic nephropathy (DN)‐related proteins and renal targets of the copper(II)‐selective chelator, triethylenetetramine (TETA) in streptozotocin‐diabetic rats. We used the recently developed iTRAQ? technology to compare renal protein profiles among non‐diabetic, diabetic, and TETA‐treated diabetic rats. In diabetic kidneys, tubulointerstitial nephritis antigen (TINag), voltage‐dependent anion‐selective channel (VDAC) 1, and VDAC2 were up‐regulated in parallel with alterations in expression of proteins with functions in oxidative stress and oxidative phosphorylation (OxPhos) pathways. By contrast, mitochondrial HSP 60, Cu/Zn‐superoxide dismutase, glutathione S‐transferase α3 and aquaporin‐1 were down‐regulated in diabetic kidneys. Following TETA treatment, levels of D ‐amino acid oxidase‐1, epoxide hydrolase‐1, aquaporin‐1, and a number of mitochondrial proteins were normalized, with concomitant amelioration of albuminuria. Changes in levels of TINag, collagen VIα1, actinin 4α, apoptosis‐inducing factor 1, cytochrome C, histone H3, VDAC1, and aquaporin‐1 were confirmed by Western blotting or immunohistochemistry. Changes in expression of proteins related to tubulointerstitial function, podocyte structure, and mitochondrial apoptosis are implicated in the mechanism of DN and their reversal by TETA. These findings are consistent with the hypothesis that this new experimental therapy may be useful for treatment of DN.     

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.     

Role of JAK2–STAT3 in TLR2‐mediated tissue factor expression

Tissue factor (TF) is a core protein with an essential function in the coagulation cascade that maintains the homeostasis of the blood vessels. TF not only participates in neointima formation, but also causes the development of atherosclerosis. This study investigated the mechanism regulating TF expression in macrophages using Pam₃CSK₄, a TLR2 ligand. Pam₃CSK₄ induced TF expression in two types of macrophages (Raw264.7 and BMDM), but not in TLR2 KO mice derived BMDM. Pam₃CSK₄ induced TF expression was inhibited by pretreatment with pan‐JAK inhibitor or JAK2 inhibitor AG490. JAK2 knock‐down by siRNA inhibited Pam₃CSK₄ induced TF expression. Pam₃CSK₄ stimulated STAT3 phosphorylation (S727), while STAT3 knock‐down by siRNA reduced Pam₃CSK₄ induced TF expression. These results suggest that Pam₃CSK₄ induced TF expression is regulated by the JAK2–STAT3 signaling pathway. Pam₃CSK₄, unlike increased TF expression, significantly decreased RGS2 expression, while RGS2 overexpression decreased Pam₃CSK₄ induced TF expression. Inhibition of TF by RGS2 WT did not occur in mutants with flawed RGS domains. We also investigated the correlation between RGS2 and STAT3 phosphorylation. RGS2 knock‐down elevated Pam₃CSK₄ induced STAT3 phosphorylation, but RGS2 overexpression had the opposite effect on STAT3 phosphorylation. These results suggest that, while Pam₃CSK₄ induced TF expression is regulated by JAK2–STAT3 signaling, RGS2 is a negative regulator targeted to STAT3. J. Cell. Biochem. 114: 1315–1321, 2013. © 2012 Wiley Periodicals, Inc.     

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin‐induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label‐free LC‐MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO‐P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal‐regulated kinase (ERK) were identified to be key mediators of pro‐ and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO‐P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.     

A proteomics approach to study synergistic and antagonistic interactions of the fungal–bacterial consortium Fusarium oxysporum wild‐type MSA 35

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil‐borne plant pathogen, including non‐pathogenic F. oxysporum strains. In this study, F. oxysporum wild‐type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to γ‐proteobacteria, was analyzed by two complementary metaproteomic approaches (2‐DE combined with MALDI‐Tof/Tof MS and 1‐D PAGE combined with LC‐ESI‐MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter‐part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.     

Physiological adaptation of Corynebacterium glutamicum to benzoate as alternative carbon source – a membrane proteome‐centric view

The ability of microorganisms to assimilate aromatic substances as alternative carbon sources is the basis of biodegradation of natural as well as industrial aromatic compounds. In this study, Corynebacterium glutamicum was grown on benzoate as sole carbon and energy source. To extend the scarce knowledge about physiological adaptation processes occurring in this cell compartment, the membrane proteome was investigated under quantitative and qualitative aspects by applying shotgun proteomics to reach a comprehensive survey. Membrane proteins were relatively quantified using an internal standard metabolically labeled with ¹⁵N. Altogether, 40 proteins were found to change their abundance during growth on benzoate in comparison to glucose. A global adaptation was observed in the membrane of benzoate‐grown cells, characterized by increased abundance of proteins of the respiratory chain, by a starvation response, and by changes in sulfur metabolism involving the regulator McbR. Additional to the relative quantification, stable isotope‐labeled synthetic peptides were used for the absolute quantification of the two benzoate transporters of C. glutamicum, BenK and BenE. It was found that both transporters were expressed during growth on benzoate, suggesting that both contribute substantially to benzoate uptake.     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Inhibition of bromodomain‐containing protein 4 ameliorates oxidative stress–mediated apoptosis and cartilage matrix degeneration through activation of NF‐E2–related factor 2‐heme oxygenase‐1 signaling in rat chondrocytes

During the progression of osteoarthritis, dysregulation of extracellular matrix (ECM) anabolism, abnormal generation of reactive oxygen species, and proteolytic enzymes have been shown to accelerate the degradation process of cartilage. The purpose of the current study was to investigate the functional role of bromodomain‐containing protein 4 (BRD4) in hydrogen peroxide (H₂O₂)–stimulated chondrocyte injury and delineate the underlying molecular mechanisms. We observed that the expression BRD4 was markedly elevated in rat chondrocytes after H₂O₂ stimulation. Additionally, inhibition of BRD4 using small interfering RNA or JQ1 (a selective potent chemical inhibitor) led to repression of H₂O₂‐induced oxidative stress, as revealed by a decrease in the reactive oxygen species production accompanied by a decreased malondialdehyde content, along with increased activities of antioxidant markers superoxide dismutase, catalase, and glutathione peroxidase on exposure of chondrocytes to H₂O₂. Meanwhile, depletion of BRD4 led to repress the oxidative stress–induced apoptosis of chondrocytes triggered by H₂O₂ accompanied by an increase in the expression of anti‐apoptotic Bcl‐2 and a decrease in the expression of pro‐apoptotic Bax and caspase 3 as well as attenuated caspase 3 activity. Moreover, knockdown of BRD4 or treatment with JQ1 markedly attenuated ECM deposition, reflected in a marked upregulation of proteoglycans collagen type II and aggrecan as well as downregulation of ECM–degrading enzymes matrix metalloproteinase 13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5). More importantly, inhibition of BRD4‐activated NF‐E2–related factor 2 (Nrf2)–heme oxygenase‐1 signaling. Mechanistically, the protective effect of BRD4 inhibition on H₂O₂‐stimulated apoptosis and cartilage matrix degeneration was markedly abrogated by Nrf2 depletion. Altogether, we concluded that the protective effect of BRD4 inhibition against oxidative stress–mediated apoptosis and cartilage matrix degeneration occurred through Nrf2–heme oxygenase‐1 signaling, implying that BRD4 inhibition may be a more effective therapeutic strategy against osteoarthritis.     

Amyloid beta peptide (1–42)‐mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate‐reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1–42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O₂ pressure, in Abeta‐treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O₂ pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta‐treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post‐translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage. Copyright © 2015 John Wiley & Sons, Ltd.     

Stable forest–savanna mosaic in north‐western Tanzania: local‐scale evidence from δ13C signatures and 14C ages of soil fractions

Aim The spatio‐temporal dynamics of dry evergreen forest patches in the savanna biome of the Kagera region (north‐western Tanzania) are largely unknown owing to a lack of pollen and macrofossil evidence. Our aims were to reconstruct local‐scale shifts of the forest–savanna boundary in order to determine whether the forests have been expanding or retreating on a centennial and millennial time‐scale. Location The Kagera region of north‐western Tanzania, East Africa. Methods The vegetation reconstruction was based on analysing δ¹³C signatures in soils along a transect spanning both C₄ open savanna and C₃ forest vegetation. Furthermore, we fractionated soil organic matter (SOM) according to density and chemical stability to analyse δ¹³C values of soil fractions with distinct radiocarbon ages. Results We found sharp changes in δ¹³C signatures in bulk SOM from the forest to the savanna, within a few metres along the transect. The forest soil profiles carried a persistent C₃‐dominated signature. Radiocarbon dating of the oldest, most recalcitrant forest soil fraction yielded a mean age of 5500 cal. yr bp , demonstrating that the forest has existed since at least the mid‐Holocene. The savanna sites showed a typical C₄ isotopic signature in SOM of topsoils, but subsoils and more recalcitrant SOM fractions also contained signals of C₃ plants. The dense soil fraction (ρ > 1.6 g cm^?3) carrying a pure C₄ label had a mean age of c. 1200 cal. yr bp , indicating the minimum duration of the dominance of grass vegetation on the savanna site. At the forest edge, the older C₄ grass signature of SOM has steadily been replaced by the more negative δ¹³C fingerprint of the forest trees. As this replacement has occurred mainly in the 10‐m‐wide forest–savanna ecotone over the last c. 1200 years, the forest expansion must be very slow and is very likely less than 15 m century^?1. Main conclusions Our results suggest that forest patches in the Kagera savanna landscape are very stable vegetation formations which have persisted for millennia. During the last millennium, they have been expanding very slowly into the surrounding savanna at a rate of less than 15 m century^?1.     

Inhibition of histone deacetylases is the major pathway mediated by astaxanthin to antagonize LPS‐induced inflammatory responses in mammary epithelial cells

Mastitis is a major inflammatory response of the mammary gland due to various pathogenic invasions and is a serious disease that affects the production yield and health status of cows. Astaxanthin (AST), a xanthophyll carotenoid, is a secondary metabolite synthesized by microalgae and yeasts that has been reported to suppress various inflammatory responses. However, the protective effect of AST on lipopolysaccharide (LPS)‐induced mammary epithelial cells has not yet been reported. The present study results indicated that AST treatment markedly attenuated the oxidative stress markers and nitric oxide (NO) while improving the anti‐oxidant enzymes in LPS exposed cells. On the other hand, LPS‐exposed cells showed nuclear translocation of nuclear factor‐κB (NF‐κB) with the activation of inflammatory cytokines such as monocyte chemoattractant protein‐1, tumor necrosis factor‐α, interferon‐γ, and interleukin‐6 (IL‐6). In addition, mRNA expression analysis revealed that the histone deacetylase (HDAC) ‐1, ‐2, ‐3, ‐6, ‐7 and pentraxin 3 (PTX3) expressions were increased in the LPS group. Furthermore, the activity of HDAC was increased to 2‐fold with a significant reduction in the histone acetyltransferase activity in cells exposed to LPS. However, AST was able to inhibit the nuclear translocation of NF‐κB with attenuated HDAC activity. Intriguingly, HDAC inhibition studies demonstrated that the cytokines such as IL‐4, IL‐8, granulocyte‐mcrophage colony stimulating factor, C‐reactive protein, IL‐17A, and IL‐22 were significantly suppressed which were upregulated in LPS treatment; while AST was found acting by improving the anti‐inflammatory cytokine IL‐10, and thioredoxin reductase levels. Collectively, these findings provide novel insights into the role of HDACs in regulating cellular processes involved in the pathogenesis of LPS‐induced mastitis as well as the potential use of AST as a therapeutic in treatment for controlling disease progression.