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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.41, #4)
The role of zinc in the S100 proteins: insights from the X-ray structures by Olga V. Moroz; Keith S. Wilson; Igor B. Bronstein (pp. 761-772).
We here aim to summarise the present knowledge on zinc binding by S100 proteins. While the importance of modulation of the function of the S100 family of EF-hand proteins by calcium is well established, a substantial proportion is also regulated by zinc or copper. Indeed regulation by zinc in addition to calcium was suggested almost as soon as the first S100 protein was discovered and has been confirmed for many family members by numerous experiments. For the first, “His-Zn”, group, zinc-binding sites composed of three histidines and an aspartic acid were first proposed based on sequence comparisons and later confirmed by structural studies. A second, “Cys-Zn”, group lacks such well-defined zinc-binding motifs and for these cysteines were suggested as the main zinc ligands. There is no three-dimensional structure for a Cys-Zn S100 in the presence of zinc. However, analysis of their sequences together with their X-ray structures in the absence of zinc suggests the possibility of two zinc-binding sites: a conserved site with a degree of similarity to those of the His-Zn group and a less-defined site with a Cys interdimer-binding motif. Some S100 protein-mediated events, such as signalling in the extracellular space, where the levels of calcium are already high, are most unlikely to be calcium regulated. Therefore, a broader knowledge of the role of zinc in the functioning of the S100 proteins will add significantly to the understanding how they propagate their signals.
Keywords: S100 proteins; Zinc; Zinc-binding site; X-ray structure; Extracellular signalling
S100A6 binding protein and Siah-1 interacting protein (CacyBP/SIP): spotlight on properties and cellular function by Gabriela Schneider; Anna Filipek (pp. 773-780).
The CacyBP/SIP protein (S100A6 binding protein and Siah-1 interacting protein) was originally discovered in Ehrlich ascites tumor cells as a S100A6 (calcyclin) target (Filipek and Wojda in Biochem J 320:585–587, 1996; Filipek and Kuźnicki in J Neurochem 70(5):1793–1798, 1998) and later on as a Siah-1 interacting protein (Matsuzawa and Reed in Mol Cell 7(5):915–926, 2001). CacyBP/SIP binds several target proteins such as some calcium binding proteins of the S100 family (Filipek et al. in J Biol Chem 277(32):28848–28852, 2002), Skp1 (Matsuzawa and Reed in Mol Cell 7(5):915–926, 2001), tubulin (Schneider et al. in Biochim Biophys Acta 1773(11):1628–1636, 2007) and ERK1/2 (Kilanczyk et al. in Biochem Biophys Res Commun 380:54–59, 2009). Studies concerning distribution of CacyBP/SIP show that it is present in various tissues and that a particularly high level of CacyBP/SIP is observed in brain (Jastrzebska et al. in J Histochem Cytochem 48(9):1195–1202, 2000). Regarding the function of CacyBP/SIP, there are some reports suggesting its role in cellular processes such as ubiquitination, proliferation, differentiation, tumorigenesis, cytoskeletal rearrangement or regulation of transcription. This review describes the properties of CacyBP/SIP and summarizes all findings concerning its cellular function.
Keywords: CacyBP/SIP; S100A6 (calcyclin); Ubiquitination; Proliferation; Differentiation; Tumorigenesis
S100A6 mediates nuclear translocation of Sgt1: a heat shock-regulated protein by Wiktor Prus; Anna Filipek (pp. 781-787).
Sgt1 was originally identified in yeast as a suppressor of the Skp1 protein. Later, it was found that Sgt1 is present in plant and mammalian organisms and that it binds other ligands such as S100A6, a calcium-binding protein. In this work we show that in HEp-2 cells Sgt1 translocates to the nucleus due to heat shock. We also found that in HEp-2 cells with diminished level of S100A6, due to stable transfection with siRNA against S100A6, such translocation occurred at a much smaller scale in comparison with cells expressing a normal level of S100A6. Moreover, translocation of Sgt1 was observed in HEp-2 cells treated with thapsigargin instead of heat shock. In contrast thapsigargin was ineffective in cells with diminished level of S100A6. Thus, our results suggest that increase in intracellular concentration of Ca2+, transduced by S100A6, is necessary for nuclear translocation of the Sgt1 protein.
Keywords: Sgt1; S100A6 (calcyclin); Heat shock proteins; Nuclear translocation
Novel S100A7 (psoriasin)/S100A15 (koebnerisin) subfamily: highly homologous but distinct in regulation and function by Ronald Wolf; Thomas Ruzicka; Stuart H. Yuspa (pp. 789-796).
S100A7 (psoriasin) and S100A15 (koebnerisin) were first identified in inflamed psoriatic skin. They are of major interest because of their putative functional roles in innate immunity, epidermal cell maturation, and epithelial tumorigenesis. Human S100A7 and S100A15 have lately evolved by gene duplications within the epidermal differentiation complex (chromosome 1q21) during primate evolution forming a novel S100 subfamily. Therefore, S100A7 and S100A15 are almost identical in sequence (>90%) and are difficult to discriminate. Despite their high homology, S100A7 and S100A15 are distinct in tissue distribution, regulation, and function, and thus, exemplary for the diversity within the S100 family. Their different properties are compelling reasons to discriminate S100A7 (psoriasin) and S100A15 (koebnerisin) in epithelial homeostasis, inflammation, and cancer.
Keywords: Calcium-binding protein; S100; Koebnerisin; Psoriasin; Evolution; Paralogs; RAGE; Innate immunity; Inflammation; Cancer
S100A11, a dual growth regulator of epidermal keratinocytes by Masakiyo Sakaguchi; Nam-ho Huh (pp. 797-807).
S100A11, a member of the family of S100 proteins, is a dimmer, each monomer of which has two EF-hands. Expression of S100A11 is ubiquitous in various tissues at different levels, with a high expression level in the skin. We have analyzed functions of S100A11 mainly in normal human keratinocytes (NHK) as a model cell system of human epithelial cells. High Ca2+ and transforming growth factor-β (TGF-β), two representative growth suppressors for NHK, need a common S100A11-mediated pathway in addition to unique pathways (NFAT1-mediated pathway for high Ca2+ and Smad-mediated pathway for TGF-β) for exhibiting a growth inhibitory effect. S100A11 has another action point for growth suppression in NHK. Annexin A1 (ANXA1) complexed with S100A11 efficiently binds to and inhibits cytosolic phospholipase A2 (cPLA2), the activity of which is needed for the growth of NHK. On exposure of NHK to epidermal growth factor (EGF), ANXA1 is cleaved at 12Trp, and this truncated ANXA1 loses binding capacity to S100A11, resulting in maintenance of an active state of cPLA2. On the other hand, we found that S100A11 is actively secreted by NHK. Extracellular S100A11 acts on NHK to enhance the production of EGF family proteins, resulting in growth stimulation. These findings indicate that S100A11 plays a dual role in growth regulation, being suppressive in cells and being promotive from outside of cells.
Keywords: S100 protein; EF-hand; Cell growth; Ca2+ ; TGF-β; EGF; Epithelial cell; Skin; Cancer; p21/WAF1; RAGE
Expression, purification and fluorine-18 radiolabeling of recombinant S100A4: a potential probe for molecular imaging of receptor for advanced glycation endproducts in vivo? by Susann Wolf; Cathleen Haase-Kohn; Jens Lenk; Susan Hoppmann; Ralf Bergmann; Joerg Steinbach; Jens Pietzsch (pp. 809-820).
Data concerning the pathophysiological role of extracellular S100A4, a member of the multigenic family of Ca2+-modulated S100 proteins, and its interaction with the receptor for advanced glycation endproducts (RAGE) or other putative receptors in tumorigenesis, metastasis, and inflammatory processes in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A4 as potential probe for molecular imaging and functional characterization of this interaction. Therefore, human S100A4 was cloned as GST fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100A4 was radiolabeled with the positron emitter fluorine-18 (18F) by conjugation with N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). The radioligand [18F]fluorobenzoyl-S100A4 (18F-S100A4) was used in cell binding experiments in RAGE-bearing human melanoma cells and endothelial cells in vitro, and in both biodistribution experiments and small animal positron emission tomography (PET) studies in normal rats in vivo. The cellular association and tissue-specific distribution of 18F-S100A4 in vitro and in vivo correlated well with the protein expression and anatomical localization of RAGE, e.g., in the vascular system and in lung. Compared to other S100 RAGE radioligands, the overall findings of this study indicate that extracellular S100A4 in vivo shows only a moderate interaction with RAGE and, furthermore, exhibits a substantially faster metabolic degradation. On the other hand, the approach allows the use of quantitative small animal PET and provides a novel probe to both delineate functional expression and differentiate multiligand interaction of RAGE under normal and pathophysiological conditions in rodent models of disease.
Keywords: Endothelial cells; Melanoma cells; Molecular imaging; Multiligand receptors; Protein radiolabeling; Small animal positron emission tomography (PET); S100 proteins
Inflammation-associated S100 proteins: new mechanisms that regulate function by Jesse Goyette; Carolyn L. Geczy (pp. 821-842).
This review focuses on new aspects of extracellular roles of the calgranulins. S100A8, S100A9 and S100A12 are constitutively expressed in neutrophils and induced in several cell types. The S100A8 and S100A9 genes are regulated by pro- and anti-inflammatory mediators and their functions may depend on cell type, mediators within a particular inflammatory milieu, receptors involved in their recognition and their post-translational modification. The S100A8 gene induction in macrophages is dependent on IL-10 and potentiated by immunosuppressive agents. S100A8 and S100A9 are oxidized by peroxide, hypochlorite and nitric oxide (NO). HOCl generates intra-chain sulfinamide bonds; stronger oxidation promotes cross-linked forms that are seen in human atheroma. S100A8 is >200-fold more sensitive to oxidative cross-linking than low-density lipoprotein and may reduce oxidative damage. S100A8 and S100A9 can be S-nitrosylated. S100A8–SNO suppresses mast cell activation and inflammation in the microcirculation and may act as an NO transporter to regulate vessel tone in inflammatory lesions. S100A12 activates mast cells and is a monocyte and mast cell chemoattractant; a G-protein-coupled mechanism may be involved. Structure–function studies are discussed in relation to conservation and divergence of functions in S100A8. S100A12 induces cytokines in mast cells, but not monocytes/macrophages. It forms complexes with Zn2+ and, by chelating Zn2+, S100A12 significantly inhibits MMPs. Zn2+ in S100A12 complexes co-localize with MMP-9 in foam cells in atheroma. In summary, S100A12 has pro-inflammatory properties that are likely to be stable in an oxidative environment, because it lacks Cys and Met residues. Conversely, S100A8 and S100A9 oxidation and S-nitrosylation may have important protective mechanisms in inflammation.
Keywords: S100; Calgranulins; S100A8; S100A9; S100A12; Inflammation; Interleukin 10
S100B: a multifunctional role in cardiovascular pathophysiology by James N. Tsoporis; Forough Mohammadzadeh; Thomas G. Parker (pp. 843-847).
S100B, a calcium-binding protein of the EF-hand type exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures, and high level expression of S100B in transgenic mice hearts and aortic smooth muscle cells inhibit cardiac hypertrophy and the associated phenotype, arterial smooth muscle proliferation, respectively, but demonstrate increased apoptosis following α1-adrenergic stimulation or myocardial infarction. Knocking out S100B, augmented hypertrophy, decreased apoptosis and preserved cardiac function following myocardial infarction. S100B induces apoptosis by an extracellular mechanism by interacting with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular, and extracellular, roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular disease.
Keywords: S100B; Myocyte hypertrophy; Apoptosis; Receptor for advanced glycation end products
S100A2 in cancerogenesis: a friend or a foe? by Susann Wolf; Cathleen Haase-Kohn; Jens Pietzsch (pp. 849-861).
Owing to the exceptional intracellular distribution and the heterogeneous expression pattern during transformation and metastasis in various tumors, the EF-hand calcium-binding protein S100A2 attracts increasing attention. Unlike the majority of S100 proteins, S100A2 expression is downregulated in many cancers and the loss in nuclear expression has been associated with poor prognosis. On the other hand, S100A2 is upregulated in some cancers. This mini review highlights the general characteristics of S100A2 and discusses recent findings on its putative functional implication as a suppressor or promoter in cancerogenesis.
Keywords: Calcium-binding protein; EF-hand; p53 Protein family; Receptor for advanced glycation endproducts (RAGE); Tumor promotor; Tumor suppressor
Evaluation of potential interactions between the metastasis-associated protein S100A4 and the tumor suppressor protein p53 by Gisle Berge; Gunhild M. Mælandsmo (pp. 863-873).
Metastasis is a complex cascade of events involving a finely tuned interplay between malignant cells and multiple host factors. The transition from benign tumor growth to malignancy is manifested by the ability of tumor cells to traverse tissue barriers and invade surrounding tissues. Among a multitude of factors playing a role, the small calcium-binding protein S100A4 has been found to add to the invasive and metastatic capacity of cancer cells. However, the exact molecular function or mechanism by which S100A4 exerts its putative metastasis-promoting effects has not been fully elucidated, and the protein is most likely involved in several aspects of tumor progression. Several studies have recently described a direct interaction and/or reciprocal influence between S100A4 and the tumor suppressor protein p53. This corresponds to reports linking p53 to other S100-family members, especially S100B. The consequences are intriguing, connecting the metastasis-promoting protein S100A4 to the large set of important p53-mediated functions, with broad potential importance in cancer development and metastasis. In this review we emphasize the studies involving p53 and S100A4, elucidating and comparing reported results and conclusions.
Coexpression and nuclear colocalization of metastasis-promoting protein S100A4 and p53 without mutual regulation in colorectal carcinoma by Gisle Berge; Daniela Elena Costea; Marianne Berg; Heidi Rasmussen; Ida Grotterød; Ragnhild A. Lothe; Gunhild M. Mælandsmo; Kjersti Flatmark (pp. 875-884).
Nuclear localization of the metastasis-associated protein S100A4 has been shown to correlate with advanced disease stage in primary colorectal carcinomas (CRC), but nuclear function and its relevance for the metastatic capacity of tumor cells is still unclear. Among several nuclear interacting protein partners suggested for S100A4, the tumor suppressor protein p53 has attracted particular interest, and previous studies suggest direct and indirect modes of interaction between the two proteins. The present study was undertaken to assess coexpression and potential interaction in CRC. TP53 mutational status and S100A4 expression were investigated in a selected series of primary CRC specimens (n = 40) and cell lines (n = 17) using DNA sequencing, western blot, and double immunostaining. Additionally, S100A4 and p53 were experimentally up- and down-regulated in vitro to assess reciprocal effects. For the first time, S100A4 and p53 coexpression was demonstrated in individual CRC cells, with nuclear colocalization as a particularly interesting feature. In contrast to previous studies, no correlation was observed between TP53 mutational status and S100A4 expression, and no evidence was obtained to support reciprocal regulation between the two molecules in the HCT116 isogenic cell line model. In conclusion, S100A4 and p53 were shown to be colocalized in individual nuclei of CRC cells, and it might be speculated whether the proteins interact in this subcellular compartment.
Keywords: Colorectal neoplasms; S100A4; p53; TP53 ; Metastasis
Transcriptional regulation and functional implication of S100P in cancer by Adriana Gibadulinova; Veronika Tothova; Jaromir Pastorek; Silvia Pastorekova (pp. 885-892).
S100P is an EF-hand calcium-binding protein that was originally identified in placenta and subsequently associated with cancer. It is a member of S100 family of proteins that function as extracellular and/or intracellular regulators of diverse cellular processes and participate in various human pathologies. S100P expression was detected in a spectrum of human tumor cell lines and tissues derived from breast, prostate, pancreas, lung and colon, where it was connected with malignant phenotype, hormone independence and resistance to chemotherapy. Overexpression of S100P was shown to promote tumorigenesis and metastasis in diverse cancer models. Functional studies of S100P indicate that its biological activities are exerted through extracellular signaling via RAGE receptor, resulting in increased proliferation and survival, or through intracellular interaction with ezrin, leading to increased cell migration and metastasis. Molecular mechanisms regulating expression of S100P in cancer cells are just emerging. Besides earlier described DNA methylation, recent studies implicate bone morphogenic protein and non-steroidal anti-inflammatory drugs in control of S100P expression during tumor progression. Functional analysis of S100P promoter identified SMAD, STAT/CREB and SP/KLF binding sites as key regulatory elements participating in transcriptional activation of S100P gene in cancer cells. Moreover, the most recent data reveal that expression of S100P is up-regulated by activation of glucocorticoid receptor suggesting that S100P could play a role in therapy resistance mediated by glucocorticoids in solid tumors. Elucidation of S100P regulation is an important step towards understanding biological significance of its tissue distribution and proposing strategies for targeted S100P modulation.
Keywords: S100P; Calcium-binding protein; Transcriptional regulation; Cancer
S100P: a novel therapeutic target for cancer by Thiruvengadam Arumugam; Craig D. Logsdon (pp. 893-899).
S100P expression is described in many different cancers, and its expression is associated with drug resistance, metastasis, and poor clinical outcome. S100P is member of the S100 family of small calcium-binding proteins that have been reported to have either intracellular or extracellular functions, or both. Extracellular S100P can bind with the receptor for advanced glycation end products (RAGE) and activate cellular signaling. Through RAGE, S100P has been shown to mediate tumor growth, drug resistance, and metastasis. S100P is specifically expressed in cancer cells in the adult. Therefore, S100P is a useful marker for differentiating cancer cells from normal cells, and can aid in the diagnosis of cancer by cytological examination. The expression of S100P in cancer cells has been related to hypomethylation of the gene. Multiple studies have confirmed the beneficial effects of blocking S100P/RAGE in cancer cells, and different blockers are being developed including small molecules and antagonist peptides. This review summarizes the role and significance of S100P in different cancers.
Keywords: S100P; RAGE; Calcium-binding protein; Cancer; Cromolyn
Taurine prevents fat deposition and ameliorates plasma lipid profile in monosodium glutamate-obese rats by Tarlliza Romanna Nardelli; Rosane Aparecida Ribeiro; Sandra Lucinei Balbo; Emerielle Cristine Vanzela; Everardo Magalhães Carneiro; Antonio Carlos Boschero; Maria Lúcia Bonfleur (pp. 901-908).
The aim of the present study was to evaluate the preventive effects of taurine (TAU) supplementation upon monosodium glutamate (MSG)-induced obesity. Rats treated during the first 5 days of life with MSG or saline were distributed into the following groups: control (CTL), CTL-treated with TAU (CTAU), MSG and MSG-supplemented with TAU (MTAU). CTAU and MTAU received 2.5% of TAU in their drinking water from 21 to 90 days of life. At the end of treatment, MSG and MTAU rats were hyperinsulinemic, glucose intolerant and insulin resistant, as judged by the HOMA index. MSG and MTAU rat islets secreted more insulin at 16.7 mM glucose compared to CTL. MSG rats also showed higher triglycerides (TG) and non-esterified fatty acids (NEFA) plasma levels, Lee Index, retroperitoneal and periepidydimal fat pads, compared with CTL, whereas plasma lipid concentrations and fat depots were lower in MTAU, compared with MSG rats. In addition, MSG rats had a higher liver TG content compared with CTL. TAU decreased liver TG content in both supplemented groups, but fat content only in MTAU rats. TAU supplementation did not change glucose homeostasis, insulin secretion and action, but reduced plasma and liver lipid levels in MSG rats.
Keywords: NEFA; MSG; Obesity; TG; Taurine supplementation
The role of tissue transglutaminase (TG2) in regulating the tumour progression of the mouse colon carcinoma CT26 by Panayiotis Kotsakis; Zhuo Wang; Russell John Collighan; Martin Griffin (pp. 909-921).
The multifunctional enzyme tissue transglutaminase (TG2) is reported to both mediate and inhibit tumour progression. To elucidate these different roles of TG2, we established a series of stable-transfected mouse colon carcinoma CT26 cells expressing a catalytically active (wild type) and a transamidating-inactive TG2 (Cys277Ser) mutant. Comparison of the TG2-transfected cells with the empty vector control indicated no differences in cell proliferation, apoptosis and susceptibility to doxorubicin, which correlated with no detectable changes in the activation of the transcription factor NF-κB. TG2-transfected cells showed increased expression of integrin β3, and were more adherent and less migratory on fibronectin than control cells. Direct interaction of TG2 with β3 integrins was demonstrated by immunoprecipitation, suggesting that TG2 acts as a coreceptor for fibronectin with β3 integrins. All cells expressed the same level of TGFβ receptors I and II, but only cells transfected with active TG2 had increased levels of TGFβ1 and matrix-deposited fibronectin, which could be inhibited by TG2 site-directed inhibitors. Moreover, only cells transfected with active TG2 were capable of inhibiting tumour growth when compared to the empty vector controls. We conclude that in this colon carcinoma model increased levels of active TG2 are unfavourable to tumour growth due to their role in activation of TGFβ1 and increased matrix deposition, which in turn favours increased cell adhesion and a lowered migratory and invasive behaviour.
Keywords: Tissue transglutaminase; Colon cancer; TGFβ1; β3 integrins; Metastasis
Proteomic analysis of apoptosis induction in human lung cancer cells by recombinant MVL by Yuqin Li; Bochao Zhang; Xiaoqin Wang; Huidan Yan; Gu Chen; Xuewu Zhang (pp. 923-932).
Lung cancer is still difficult to treat by current chemotherapeutic procedures. We recently found that MVL, an anti-HIV lectin from blue-green algae Microcystis viridis, also has antitumor activity. The objective of this study was to investigate apoptosis-inducing activity of recombinant MVL (R-MVL) and proteomic changes in A549 cells, and to identify the molecular pathways responsible for the anti-cancer action of R-MVL. We found that R-MVL induces A549 cells apoptosis in a dose-dependent manner by using MTT assay, fluorescent microscope (FM) and flow cytometry (FCM), and the IC50 was calculated to be 24.12 μg/ml. Subsequently, 7 altered proteins in R-MVL-treated A549 cells were identified, including upregulated aldehyde dehydrogenase 1 and β-actin, and five downregulated proteins: heat shock protein 90, heat shock 60, plastin 3, tropomyosin 3, and β-tubulin. Further bioinformatics analysis predicted the potential pathways for R-MVL to induce apoptosis of A549 cells. In conclusion, this is the first report to investigate anti-cancer activity of R-MVL and its mechanism of action by proteomics analysis. Our observations provide potential therapeutic targets for lung cancer inhibitor intervention and implicated the development of novel anti-cancer therapeutic strategies.
Keywords: Recombinant MVL; Lung cancer; Apoptosis; Proteomics; Pathways; Bioinformatics
A new approach to 3-substituted tetrahydro-β-carboline derivative via diethyl acetamidomalonate by Sambasivarao Kotha; Shilpi Misra; Shaikh M. Mobin (pp. 933-936).
Strategically a new approach for the synthesis tetrahydro-β-carboline unit with the aid of diethyl acetamidomalonate as a glycine equivalent has been described.
Keywords: Diethyl acetamidomalonate; Indole alkaloids; Tetrahydro-β-carboline; Unusual α-amino acid derivatives
Effects of d-kyotorphin on nociception and NADPH-d neurons in rat’s periaqueductal gray after immobilization stress by Elena B. Dzambazova; Boycho V. Landzhov; Adriana I. Bocheva; Anastasia A. Bozhilova-Pastirova (pp. 937-944).
d-kyotorphin (d-Kyo) is a synthetic analogue of the neuropeptide kyotorphin and produces naloxone reversible analgesia. Stress-induced analgesia (SIA) is an in-built mammalian pain-suppression response that occurs during or following exposure to a stressful stimulus. The periaqueductal gray (PAG) is implicated as a critical site for processing strategies for coping with different types of stress and pain and NO affects its activity. The objectives of the present study were twofold: (1) to examine the effects of d-Kyo (5 mg/kg) on acute immobilization SIA; (2) to investigate the effect of peptide on NO activity in rat PAG after the stress procedure mentioned above. All drugs were injected intraperitoneally in male Wistar rats. The nociception was measured by the paw pressure and hot plate tests. A histochemical procedure for nicotinamide adenine dinucleotide phosphate–diaphorase (NADPH-d)-reactive neurons was used as indirect marker of NO activity. Our results revealed that d-Kyo has modulating effects on acute immobilization stress-induced analgesia in rats may be by opioid and non-opioid systems. Although d-Kyo is incapable of crossing the blood–brain barrier it showed an increased number of NADPH-d reactive neurons in dorsolateral periaqueductal gray (dlPAG) in control but not in stressed groups. We may speculate that the effect of d-Kyo in the brain is due to structural and functional interaction between opioidergic and NO-ergic systems or d-Kyo appears itself as a stressor. Further studies are needed to clarify the exact mechanisms of its action.
Keywords: d-Kyotorphin; Immobilization; Stress-induced analgesia; PAG; NADPH-d reactive neurons
Evidences of a natively unfolded state for the human topoisomerase IB N-terminal domain by Oscar Vassallo; Silvia Castelli; Ilda D’Annessa; Blasco Morozzo della Rocca; Lorenzo Stella; Birgitta R. Knudsen; Alessandro Desideri (pp. 945-953).
The N-terminal domain of human topoisomerase IB has been expressed, purified and characterized by spectroscopic techniques. CD spectra as a function of concentration and pH indicate that the domain does not possess any defined secondary structure. The protein is probably in a natively unfolded state since its denaturation curve is indicative of a non-cooperative transition. Evidence of a partially folded structure comes from the fluorescence spectrum of ANS, whose intensity increases in presence of the domain. Indication of a partial structural arrangement of the domain comes also from the endogenous fluorescence of tryptophans that is centred at 350 nm in the native and shifts to 354 nm in the fully denaturated protein. Interestingly despite the poor structural degree, as also confirmed by a predictive approach, the domain efficiently binds DNA, suggesting that the absence of a defined 3D structure has a functional meaning that permits the domain to be available for the interaction with different molecular partners.
Keywords: Human topoisomerase IB; Natively unfolded; Fluorescence measurements; DNA binding; Hydrophobic site; Chemical denaturation
A first partial Aplysia californica proteome by Yanwei Sun; Francisco J. Monje; Daniela D. Pollak; Gert Lubec (pp. 955-968).
Aplysia proteins have not been studied systematically and it was therefore the aim of the study to carry out protein profiling in ganglia from Aplysia californica (AC). AC ganglia were extirpated, proteins extracted and run on 2DE with subsequent in-gel digestion, followed by identification of proteins by nano-LC–ESI–MS/MS on an ion trap. Proteins were identified based upon a public Aplysia EST database. Out of 408 picked spots, 276 spots were identified corresponding to 172 ESTs and 118 individual proteins. The range of sequence coverage was between 14 and 80% and the average amount of peptides used for the identification of proteins was 9 (from 3 to 24). Mean score for protein identification was 516. Comparison of protein levels between cerebral, pleural, pedal and abdominal ganglia revealed a series of significant differences including: signaling, metabolism, cytoskeleton and structural, redox, chaperone, replication/transcription and electron/proton transport proteins. The generation of a protein map complements transcriptional studies carried out in AC ganglia. The findings provide the basis for investigation into post-translational modifications, splice variants and assist in the generation of antibodies against AC proteins. Moreover, differences in protein expression between ganglia may be valuable for the design of future studies in neurobiology of AC.
Keywords: 2-DE; Aplysia californica ; Nano-LC–ESI–MS/MS; Protein map; Protein profiling; Ganglia
A role for PPARα in the regulation of arginine metabolism and nitric oxide synthesis by Najoua Guelzim; François Mariotti; Pascal G. P. Martin; Frédéric Lasserre; Thierry Pineau; Dominique Hermier (pp. 969-979).
The pleiotropic effects of PPARα may include the regulation of amino acid metabolism. Nitric oxide (NO) is a key player in vascular homeostasis. NO synthesis may be jeopardized by a differential channeling of arginine toward urea (via arginase) versus NO (via NO synthase, NOS). This was studied in wild-type (WT) and PPARα-null (KO) mice fed diets containing either saturated fatty acids (COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers of arginine metabolism were assayed in urine and plasma. mRNA levels of arginases and NOS were determined in liver. Whole-body NO synthesis and the conversion of systemic arginine into urea were assessed by using 15N2-guanido-arginine and measuring urinary 15NO3 and [15N]-urea. PPARα deficiency resulted in a markedly lower whole-body NO synthesis, whereas the conversion of systemic arginine into urea remained unaffected. PPARα deficiency also increased plasma arginine and decreased citrulline concentration in plasma. These changes could not be ascribed to a direct effect on hepatic target genes, since NOS mRNA levels were unaffected, and arginase mRNA levels decreased in KO mice. Despite the low level in the diet, the nature of the fatty acids modulated some effects of PPARα deficiency, including plasma arginine and urea, which increased more in KO mice fed the LIN diet than in those fed the COCO diet. In conclusion, PPARα is largely involved in normal whole-body NO synthesis. This warrants further study on the potential of PPARα activation to maintain NO synthesis in the initiation of the metabolic syndrome.
Keywords: Metabolic syndrome; Amino acids; α-Linolenic acid; n-3 PUFA; Urea
Synthetic strategy for side chain mono-N-alkylation of Fmoc-amino acids promoted by molecular sieves by Luca Monfregola; Stefania De Luca (pp. 981-990).
A new synthetic strategy to alkylate amino groups under mild conditions has been developed. It utilizes only 4 Å molecular sieves as base in order to promote the N-alkylation reaction, in presence of the appropriate alkyl halide. The methodology was validated by the simple and efficient side-chain N-alkylation of o-Ns-protected Fmoc-amino acid. One of them was introduced as building block into a peptide sequence, thus allowing the preparation of site-specific alkylated peptide molecules.
Keywords: N-alkylation; Molecular sieves; N-alkylated amino acids; Peptidomimetics
Increased in vivo release of neuropeptide S in the amygdala of freely moving rats after local depolarisation and emotional stress by Karl Ebner; Alesja Rjabokon; Hans-Christian Pape; Nicolas Singewald (pp. 991-996).
Intracerebral microdialysis in conjunction with a highly sensitive radioimmunoassay was used to study the in vivo release of neuropeptide S (NPS) within the amygdala of freely moving rats. NPS was consistently detected in basolateral amygdala dialysates and the release considerably enhanced in response to local depolarisation as well as exposure to forced swim stress. Thus, our data demonstrate for the first time emotional stress-induced release of NPS in the amygdala supporting a functional role of endogenous NPS in stress/anxiety-related phenomena.
Keywords: Neuropeptide S antibody; Anxiety; Depression; Stress-related disorders; Microdialysis; Radioimmunoassay